Osteoarthritis, Inflammation and Degradation

OSTEOARTHRITIS, INFLAMMATION AND
DEGRADATION: A CONTINUUM
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ISSN 0929-6743
Osteoarthritis, Inflammation and
Degradation: A Continuum
Edited by
Joseph A. Buckwalter
University of Iowa, Department of Orthopaedics and Rehabilitation,
Iowa City, USA
Martin Lotz
Division of Arthritis Research, The Scripps Research Institute, La Jolla, USA
and
Jean-François Stoltz
Groupe Ingénierie et Thérapie Cellulaire
UMR CNRS – UHP 7563 – Faculté de Médecine
Vandoeuvre Lès Nancy – France
and
Unité de Thérapie Cellulaire et Tissus
CHU NANCY – Vandoeuvre lès Nancy – France
Amsterdam • Berlin • Oxford • Tokyo • Washington, DC

© 2007 The authors and IOS Press.
All rights reserved. No part of this book may be reproduced, stored in a retrieval system,
or transmitted, in any form or by any means, without prior written permission from the publisher.
ISBN 978-1-58603-773-4
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Osteoarthritis, Inflammation and Degradation: A Continuum v
J. Buckwalter et al. (Eds.)
IOS Press, 2007
© 2007 The authors and IOS Press. All rights reserved.
Preface
Osteoarthritis is a major public health issue due to its impact in term of handicap.
Moreover, Ageing of the world population and outbreak of obesity in industrialized and
non-industrialized countries will dramatically increase its incidence in the next years.
Regarded as a multi-factorial disease, today mechanistic and inflammatory theories are
no more opposed but, on the contrary, are framed within the same continuum: os-
teoarthritis, inflammation and degeneration. In order to collect major information in a
benchmark book on the fundamental aspects of this disease, internationally well-known
authors, from multiple specialties, gathered to analyse, dissect and finally try to under-
stand the secrets of a disease which should no more be regarded as the common and
relentless result of ageing or of passive wear but much more as an active disease able to
benefit from the best targeted pharmacological (anti-cytokines, inhibitors of signalling
pathways, inhibitors of proteases, etc.) and non-pharmacological (cellular therapy, gene
therapy, cartilage engineering etc.) therapies, current and future.
Update of these therapies goes through a sharp knowledge of the different patho-
physiological mechanisms of osteoarthritis. Major new paradigms have emerged in this
field in the very last years. For example, it is noteworthy that cartilage used to be the
unique tissue involved in the OA process. But in this book, many chapters refer to
novel findings on the role of other tissues like bone or synovial tissue which should be
of critical importance for the degradative process of the OA joint. Another challenge
refers to the possibility in the future to evaluate the potential severity of the disease in a
single patient from the very early stage. The recent new advances in imaging and bio-
markers detailed in this volume suggest that we are not so far from this capacity. These
recent advances have been compiled in this monograph, that should captivate a large
audience, scientists and clinicians.
F. Berenbaum
Elected President of OARSI
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vii
Acknowledgments
Writing a monograph is a difficult and collective task. That is the reason why, on be-
half of Joseph Buckwalter, and Martin Lotz, I would like to thank all the authors who
have contributed to this work. A special acknowledgements to Drs Martine Burger and
Jean Gavaudan (MD, FCP) for their friendly and efficient help. I would not forget the
secretaries of my department, Isabelle and Estelle, who have followed up the adminis-
trative work and maintained contacts between the authors and the publisher.
J.F. Stoltz
Contributors
Aigner Thomas Blanco Franscisco J.

Osteoarticular and Molecular Pathology Osteoarticular and Aging Research Lab
Institute of Pathology Biomedical Research Center
University of Leipzig CH Universitario Juan Canalejo
Germany A Coruña
thomas.aigner@medizin.uni-leipzig.de Spain
Phone: 34-981-178272
Fax: 34-981-178273
Berenbaum Francis
francisco_blanco@canalejo.org
1 – Unité Mixte de Recherche CNRS
fblagar@canalejo.org
7079 Physiology and Physiopathology
Laboratory Buckwalter Joseph
University Paris Professor, Head and Steindler Chair
6, 7 quai St. Bernard, Bât A Department of Orthopaedics and
France Rehabilitation
2 – Department of Rheumatology University of Lowa
UFR Pierre et Marie Curie 01008-A JPP, 200 Hawkins Drive
Saint-Antoine hospital Iowa City, IA 52242
75012 Paris USA
France Phone: 319-356-3595
francis.berenbaum@sat.ap-hop.fr Fax: 319-356-8999
joseph-buckwalter@uiowa.edu
Bianchi Arnaud
Laboratoire de Physiopathologie et De Isla Natalia
Pharmacologie Articulaires (LPPA) Groupe d’Ingénierie Cellulaire et
UMR 7561 Tissulaire, LEMTA-UMR CNRS 7563
CNRS-Nancy Université Faculté de Médecine
Avenue de la forêt de Haye, BP 184 Université Henri Poincaré, 9 av. de Haye
54505 VANDŒUVRE-lès-NANCY 54505 Vandoeuvre les Nancy
France France
arnaud.bianchi@medecine.uhp-nancy.fr natalia.de-isla@medecine.uhp-nancy.fr
viii
Ding Lei New York, NY 10021

William E. Cornatzer Chair in USA
Biochemistry Tel.: 212-774-7564
Department of Biochemistry and Fax: 212-249-2373
Molecular Biology goldringm@hss.edu
University of North Dakota http://www.hss.edu/research-
School of Medicine and Health Sciences staff_goldring-mary.asp
Box 9037
501 N. Columbia Road Gomez Rodolfo
Grand Forks, ND 58202 Santiago University Clinical Hospital
USA NEIRID Lab, NeuroEndocrine
leiding@medicine.nodak.edu Interactions in Rheumatology and
Inflammatory Diseases
Dumas Dominique Research Area, Laboratory nº4
Groupe d’Ingénierie Cellulaire et Trav. Choupana sn
Tissulaire, Faculté de Médecine 15706 Santiago de Compostela
Université Henri Poincéré Spain
UMR CNRS 7563 LEMTA Ph. & Fax: 34+981+950905
54505 Vandoeuvre lès Nancy rodolfo.gomez.bahamonde@sergas.es
France
dominique.dumas@medecine.uhp- Gomez-Reino Juan J.
nancy.fr Santiago University Clinical Hospital
Division of Rheumatology
Gabay Odile Door 36. Trav. Choupana sn 15706
Unité Mixte de Recherche CNRS 7079 Santiago de Compostela
Physiology and Physiopathology Spain
Laboratory, University Paris Phone: 34+981+951036
6, 7 quai St. Bernard, Bât A juan.gomez-reino.carnota@sergas.es
France
odile.gabay@snv.jussieu.fr Gosset Marjolaine
UMR CNRS 7079 Physiology and
Galteau Marie-Madeleine Physiopathology Laboratory
Nancy Université University Paris 6, 7 quai St. Bernard
UHP, Laboratoire de Physiopathologie Bât A, 75 000 Paris
et Pharmacologie Articulaires France
UMR 7561 CNRS marjolaine.gosset@snv.jussieu.fr
Avenue de la forêt de Haye, BP 184
54505 Vandoeuvre lès nancy Gualillo Oreste
France Santiago University Clinical Hospital
marie-madeleine.galteau@pharma.uhp- NEIRID Lab, NeuroEndocrine
nancy.fr Interactions in Rheumatology and
Goldring Mary B. Research Area, Laboratory nº4
Laboratory for Cartolage Biology Trav. Choupana sn
Hospita for Special Surgery 15706 Santiago de Compostela
Weill College of Medicine of Spain
Cornell University Ph. & Fax: 34+981+950905
Caspary Research Building, Room 528 oreste.gualillo@sergas.es
535 E. 70th Street gualillo@usc.es
ix
Guo Danping Kirchmeyer Melanie

William E. Cornatzer Chair in Nancy université
Biochemistry UHP, Laboratoire de Physiopathologie
Department of Biochemistry and et Pharmacologie Articulaires
Molecular Biology UMR 7561 CNRS
University of North Dakota Avenue de la forêt de Haye, BP 184
School of Medicine and Health Sciences 54505 Vandoeuvre Lès Nancy
Box 9037 France
501 N. Columbia Road melanie.kirchmeyer@medecine.uhp-
Grand Forks, ND 58202 nancy.fr
USA
dguo@medicine.nodak.edu
Lago Francisca
Homandberg Gene A. Laboratory of Molecular and
Professor and Chair Cellular Cardiology
William E. Cornatzer Chair in Santiago University Clinical Hospital
Biochemistry Research Laboratory 1
Department of Biochemistry and Trav. Choupana sn
Molecular Biology 15706 Santiago de Compostela
University of North Dakota Spain
School of Medicine and Health Sciences Phone: 34+981+950902
Box 9037 Fax: 34+981+950905
501 N. Columbia Road frlago@usc.es
Grand Forks, ND 58202
USA Lago Rocio
Phone: 701-777-6422 Santiago University Clinical Hospital
Fax: 701-777-2382 NEIRID Lab
ghomandberg@medicine.nodak.edu NeuroEndocrine
Interactions in Rheumatology and
Jimenez Segio Inflammatory Diseases
Jefferson Institute of Molecular Medicine Research Area, Laboratory nº4
Thomas Jefferson University Trav. Choupana sn
Suite 509 15706 Santiago de Compostela
Bluemle Life Sciences Building Spain
233 South 10th Street Ph. & Fax: 34+981+950905
Philadelphia, PA, 19107 rocio.lago.cabaleiro@sergas.es
USA
sergio.jimenez@jefferson.edu
Lajeunesse Daniel
Jouzeau Jean-Yves Osteoarthritis Research Unit
Nancy université University of Montreal Hospital Centre
UHP, Laboratoire de Physiopathologie Notre-Dame Hospital
et Pharmacologie Articulaires 1560 Sherbrooke Street East
UMR 7561 CNRS Montreal, Quebec, H2L 4M1
Avenue de la forêt de Haye, BP 184 Canada
54505 Vandoeuvre Lès Nancy Tel.: 514-890-8000, ext. 28914
France Fax: 514-412-7583
jean-yves.jouzeau@pharma.uhp-nancy.fr daniel.lajeunesse@umontreal.ca
x
Lopez-Armada Maria J. Oliviero Francesca

Osteoarticular and Aging Research Lab Rheumatology Unit
Biomedical Research Center Department of clinical and Experimental
CH Universitario Juan Canalejo Medicine
A Coruña University of Padova
Spain Via Giustiniani 2
armada@canalejo.org 35128 Padova
Italy
Lotz Martin Phone: +39 049 8212190
Division of Arthritis Research Fax: +39 049 8212191
The Scripps Research Institute francesca.oliviero@unipd.it
Div MEM 161
10550 North Torrey Pines Road Otero Miguel
La Jolla, CA 92037 Santiago University Clinical Hospital
USA NEIRID Lab, NeuroEndocrine
mlotz@scripps.edu Interactions in Rheumatology and
Malemud Charles J. Research Area, Laboratory nº4
Charles J. Malemud, Ph.D. Trav. Choupana sn
Case Western Reserve University and 15706 Santiago de Compostela
University Hospitals Case Spain
Medical Center Ph. & Fax: 34+981+950905
Department of Medicine Present address is:
Division of Rheumatic Diseases Hospital for Special Surgery
2061 Cornell Road, Rm. 207 Caspary Research Building
Cleveland, Ohio 44106-5076 5th Floor, 535 E. 70th Street
USA New York, NY, 10021
cjm4@cwru.edu USA
Phone: 212-774-755
Martel-Pelletier Johanne Pedersen Douglas R.
Director, Osteoarthritis Research Unit University of Iowa
and Osteoarthritis Chair Department of Orthopaedics
University of Montreal Hospital and Rehabilitation
CHUM, Notre-Dame Hospital 200 Hawkins Drive
1560 Sherbrooke Street East Iowa City, IA 52242
Montreal, Quebec, H2L 4M1 USA
Canada
Tel.: 514-890-8000, ext. 26658 Pelletier Jean-Pierre
Fax: 514-412-7582 Head, Arthritis Centre
jm@martelpelletier.ca University of Montreal
Director, Osteoarthritis Researche
Martin James A. Unit and Osteoarthritis Chair
University of Iowa University of Montreal
Department of Orthopaedics CHUM, Notre-Dame hospital
and Rehabilitation 1560 Sherbrooke Street East
200 Hawkins Drive Montreal, Quebec, H2L 4M1
Iowa City, IA 52242 Canada
USA dr@jppelletier.ca
xi
Piera-Velazquez Sonsoles Sfriso Paolo

Jefferson Institute of Molecular Medicine Rheumatology Unit
Thomas Jefferson University Department of clinical and Experimental
Suite 509 Medicine
Bluemle Life Sciences Building University of Padova
233 South 10th Street Via Giustiniani 2, 35128 Padova
Philadelphia, PA, 19107 Italy
USA Phone: +39 049 8212190
Fax: +39 049 8212191
Punzi Leonardo paolo.sfriso@unipd.it
Rheumatology Unit
Department of clinical and Experimental Smith Robert Lane
Medicine Rehabilitation Research and
University of Padova Development Center
Via Giustiniani 2, 35128 Padova VA Palo Alto Health Care System
Italy Palo Alto, CA
Phone: +39 049 8212190 Department of Orthopaedic Surgery
Fax: +39 049 8212191 Stanford University School of Medicine
punzireu@unipd.it Stanford, CA
USA
smith@rrd.stanford.edu
Rego Ignacio
Osteoarticular and Aging Research Lab
Stoltz J.F.
Biomedical Research Center
Directeur du groupe d’Ingénierie
CH Universitario Juan Canalejo
Cellulaire et Tissulaire
A Coruña
LEMTA-UMR CNRS 7563
Spain
Faculté de Médecine
iregper@canalejo.org
Université Henri Poincaré
9 av. de Haye (Nancy Université-UHP)
Riquelme Bibiana 54505 Vandoeuvre Lès Nancy
Areas Física e Inmunología France
Facultad de Ciencias Bioquímicas y and
Farmacéuticas Chef de service
Universidad Nacional de Rosario Unité de Thérapie Cellulaire et Tissulaire
Suipacha 531, 2000 Rosario Brabois – 54 500 Vandoeuvre Lès Nancy
Argentina France
riquelme@ifir.edu.ar Tel.: +33 3 83 15 37 79
jf.stoltz@chu-nancy.fr
Sandell Linda
Department of Orthopaedic Surgery and Terkeltaub Robert, MD
Cell Biology Chief, VA Rheumatology Section
Washington University School of Professor of Medicine, UCSD
Medicine, MS 8233 111K, VAMC
660 S. Euclid Ave. 3350 La Jolla Village Drive
St. Louis, MO 63110 San Diego, CA 92161
USA USA
Tel.: 314-454-7800 Tel.: 858-642-3519
Fax: 314-454-5900 Fax: 858-552-7425
sandelll@wudosis.wustl.edu rterkeltaub@ucsd.edu
xii
Thedens Daniel R. Nijmegen

Department of Radiology The Netherlands
The University of Iowa City w.vandenberg@reuma.umcn.nl
IA 52242
USA Van Der Kraan Peter
dan-thedens@uiowa.edu Experimental Rheumatology & Advanced
Therapeutics
Van Den Berg Wim Nijmegen Centre For Molecular Life
Experimental Rheumatology & Advanced Sciences
Therapeutics Radboud University Medical Centre
Nijmegen Centre For Molecular Life Nijmegen
Sciences The Netherlands
Radboud University Medical Centre p.vanderkraan@reuma.umcn.nl
This monograph was published in appreciation of OARSI and of European Society on

Cell and Tissue Engineering and Therapy and under the patronage of Université Henri
Poincaré (Nancy université) with an unrestricted educational grant of Negma-Lerads
(Wockhardt international).
xiii
Contents
Preface v
F. Berenbaum
Acknowledgments and Contributors vii
J.F. Stoltz
Part I. Extra Cellular Stimuli
I. Inflammatory Factors Involved in Osteoarthritis 3

Johanne Martel-Pelletier and Jean-Pierre Pelletier
II. Mechanical Loading Effects on Articular Cartilage Matrix Metabolism
and Osteoarthritis 14
Robert Lane Smith
III. Aging, Inflammation, and Altered Chondrocyte Differentation in Articular
Cartilage Calcification and Osteoarthritis 31
Robert A. Terkeltaub
IV. Leptin, the Prototype of Adipokines: Molecules at the Crossroads
of Inflammation and Metabolism 43
Rodolfo Gómez, Rocío Lago, Francisca Lago, Juan J. Gómez-Reino,
Miguel Otero and Oreste Gualillo
V. The Role of Extracellular Matrix Fragments in the Autocrine Regulation
of Cartilage Metabolism 56
Gene A. Homandberg, Lei Ding and Danping Guo
VI. Pathophysiological Relevance of PPAR to Osteoarthritis: From the Control
of Inflammation to Cartilage Protection? 77
Arnaud Bianchi, Mélanie Kirchmeyer, Marie-Madeleine Galteau
and Jean-Yves Jouzeau
Part II. Signalling Mechanisms
VII. MAP Kinases 99

Charles J. Malemud
VIII. Transcriptional Control of Chondrocyte Gene Expression 118
Mary B. Goldring and Linda J. Sandell
IX. Gene Expression Profiling of Human Articular Chondrocytes
and Osteoarthritis 143
Sergio A. Jimenez and Sonsoles Piera-Velazquez
xiv
Part III. Effectors and Different Pathways
X. Prostaglandin E2 and Osteoarthritis: The Role of Cyclooxygenases,

Prostaglandin E Synthases and 15-Prostaglandin Dehydrogenases 163
Odile Gabay, Marjolaine Gosset and Francis Berenbaum
XI. NO and Other Radicals in the Pathogenesis of Osteoarthritis 182
Martin Lotz
XII. Mitochondria and Chondrocytes: Role in Osteoarthritis 192
Francisco J. Blanco, María J. López-Armada and Ignacio Rego
XIII. Subchondral Bone and Osteoarthritis Progression: A Very Significant Role 206
Johanne Martel-Pelletier, Daniel Lajeunesse and Jean-Pierre Pelletier
XIV. Osteoarthritis and Inflammation – Inflammatory Changes in Osteoarthritic
Synoviopathy 219
Thomas Aigner, Peter van der Kraan and Wim van den Berg
Part IV. Imaging and Clinical Applications
XV. Magnetic Resonance Imaging of Cartilage: New Imaging and Clinical

Approaches 239
Daniel R. Thedens, James A. Martin and Douglas R. Pedersen
XVI. Multimodality of Microscopy Imaging Applied to Cartilage Tissue
Engineering 254
D. Dumas, B. Riquelme, E. Werkmeister, N.D. Isla and J.F. Stoltz
XVII. Biomarkers of Matrix Fragments, Inflammation Markers in Osteoarthritis 267
Leonardo Punzi, Francesca Oliviero and Paolo Sfriso
XVIII. Cartilage Engineering 280
J.F. Stoltz, M. Lotz and J. Buckwalter
XIX. Therapeutics and Osteoarthritis 287
J. Buckwalter, M. Lotz and J.F. Stoltz
Author Index 299

Part I
Extra Cellular Stimuli
Osteoarthritis, Inflammation and Degradation: A Continuum 3
IOS Press, 2007
Inflammatory Factors Involved in

Osteoarthritis
Johanne MARTEL-PELLETIER ∗, PhD and Jean-Pierre PELLETIER, MD
Osteoarthritis Research Unit, University of Montreal Hospital Centre,
Notre-Dame Hospital, 1560 Sherbrooke Street East, Montreal, Quebec,
Canada, H2L 4M1
Abstract. Osteoarthritis (OA) is a disease that predominantly, but not solely, af-
fects the diarthrodial joints and results from an interaction between a number of
complex mechanical and biological processes. Knowledge of the etiopathogenesis
of OA has progressed significantly in the past few decades. A major characteristic
of OA is articular cartilage destruction, yet it has become obvious that synovial in-
flammation, although not a primary cause of the disease, is among the significant
structural changes that take place during its development. There is compelling evi-
dence suggesting that secreted inflammatory mediators impact on the matrix ho-
meostasis of articular tissue cells by altering their metabolism. Among these me-
diators that are responsible for the progression of the disease, evidence points to
the proinflammatory cytokine interleukin-1 beta (IL-1ß) as the most important fac-
tor responsible for the catabolic process in OA. New members of the IL-1 super-
family have recently been identified (ILF5-ILF10), some of which are suggested to
be of interest for the arthritic diseases. Other proinflammatory cytokines, such as
tumor necrosis factor (TNF)-α, IL-6, leukemia inhibitory factor (LIF), oncostatin
M (OSM), IL-17, IL-18, and IL-8, are also considered potential contributing fac-
tors in the pathogenesis of OA. However, the exact role and importance of each in
the OA process is not yet clearly established. In addition to cytokines, other in-
flammatory mediators also play a major role in the OA pathological process. These
include nitric oxide (NO), eicosanoids (prostaglandins and leukotriene), and a
newly identified cell membrane receptor family, the protease-activated receptors
(PARs), in which an important role for PAR-2 in chronic arthritis has been sug-
gested. All these topics will be discussed in this review and should help the reader
to better understand the most recent advances concerning the inflammatory factors
involved in the pathophysiology of OA.
Introduction
Osteoarthritis (OA) is a disease closely associated with the aging process and therefore
represents a growing public health cost, not only for the Western countries but world-
wide. Interestingly, arthritis is the second most expansive disease category in North
∗
Corresponding Author: Johanne Martel-Pelletier, PhD, Osteoarthritis Research Unit, University of Mont-
real Hospital Centre, Notre-Dame Hospital, 1560 Sherbrooke Street East, Montreal, Quebec, Canada,
H2L 4M1, Phone: 514-890-8000, ext. 26658, Fax: 514-412-7582, E-mail: jm@martelpelletier.ca.
4 J. Martel-Pelletier and J.-P. Pelletier / Inflammatory Factors Involved in Osteoarthritis
America, second only to cardiovascular disease and followed by cancer. OA is the most
prevalent arthritic disease and the one most seen and treated by rheumatologists and
general practitioners. Since there is, as yet, no cure for this disease, the economical
impact of OA on our health economy is an important concern in the context of an aging
population. Indeed, this disease affects 10 to 15 percent of the world’s population, and
its frequency increases with aging; its incidence is higher than 60 percent in the popula-
tion over 65 years of age.
The aetiology of OA is multifactorial, yet this disease is characterized by a number
of articular structural changes, including cartilage destruction and alterations in syno-
vial membrane and subchondral bone, which impair joint movement and cause pain.
Cartilage destruction is associated with, and it is believed that it may even be preceded
by, subchondral bone alterations. During the course of OA, intermittent flares, which
reflect the presence of an inflammatory process, appear at the synovial membrane.
There is a general consensus that synovial inflammation in OA, although not a primary
phenomenon in this disease, contributes to its progression.
This review focuses on bringing to the reader an understanding of which of the in-
flammatory factors participate in the complex interaction in OA tissues, and lead to the
progression of structural changes observed in this disease. This knowledge is essential
not only to a further understanding of the pathophysiology of the disease per se, but
also to the development of new therapeutic strategies that can modify the progression
of the disease.
Pathophysiology
It is now well established that in OA, the earliest histopathological alteration that oc-
curs in cartilage is a depletion of major matrix macromolecules including collagen and
aggrecan. Collagen is of particular importance as its breakdown results in the loss of
the structural integrity of the tissue. It appears that alterations of the collagen network,
as well as the aggrecan, result from an increased level of proteolytic enzymes synthe-
sized by chondrocytes.
A great deal of attention has been given to identifying the protease most likely re-
sponsible for the occurrence of matrix degradation. It is widely accepted that the metal-
loprotease (MMP) family comprises a major involvement in the disease process [1]. Of
this family, collagenases (enzymes responsible for collagen degradation) and aggreca-
nases (enzymes responsible for the aggrecan cleavage found in OA synovial fluid) have
been suggested to play major roles in the degradation of the extracellular matrix ob-
served in OA. The increase in the level of collagenases (collagenase-1 [MMP-1], colla-
genase-2 [MMP-8], and collagenase-3 [MMP-13]) found in human OA cartilage pro-
vide strong evidence to this effect. Although all three collagenases are active on colla-
gen fibrils, differences in their in situ roles and in mechanisms regulating their expres-
sions have been reported. Taking into account the specificity of each of these colla-
genases, neutralizing the synthesis/activity of collagenase-3 (MMP-13) seems to be the
most promising strategy to halt cartilage breakdown [2]. As for the aggrecanases, the
importance of two such enzymes has been reported: aggrecanase-1 (ADAMTS-4) and
aggrecanase-2 (ADAMTS-5). However, recent studies have demonstrated that
ADAMTS-5 appears to be the predominant enzyme involved in the OA degradative
process [3,4].
J. Martel-Pelletier and J.-P. Pelletier / Inflammatory Factors Involved in Osteoarthritis 5
Other enzymes from the serine-dependent protease family, such as the plasmino-
gen activator/plasmin system, are also likely to play a role, but, in cartilage, primarily
as activators of MMPs. Enzymes from the cysteine protease family, including cathepsin
members, also appear to be involved in articular tissue degradation.
The enzymatic alterations in cartilage matrix may explain the exhaustive degrada-
tion of this tissue but do not provide an explanation for the factors involved in the
upregulation of the expression and synthesis of enzymes in this disease. The following
hypothesis has been put forward to explain the pathological development of OA at the
clinical stage of the disease. The cartilage matrix breakdown, produced by proteolytic
enzymes, releases increased amounts of matrix macromolecule fragments and neoanti-
gens into the synovial fluid which, upon phagocytosis, promote inflammation in the
synovial membrane. In turn, the inflamed synovial membrane releases several media-
tors capable of creating a vicious cycle by inducing increased cartilage degradation and
subsequently triggering further inflammation.
Inflammatory Mediators
Proinflammatory Cytokines
Considerable evidence has accumulated to indicate that the proinflammatory cytokines

are crucial in mediating inflammation and tissue destruction in OA. It is claimed, and
substantiated by studies on animal models, that interleukin-1-ß (IL-1ß) is of pivotal
importance in OA cartilage destruction and considered to be the principal mover of the
enzyme system [5–7]. The catabolic effects of IL-1ß are multiple. This cytokine is able
to stimulate its own production, to increase the synthesis of enzymes (MMPs, plasmi-
nogen activator/plasmin), to inhibit the synthesis of the major physiological inhibitors
of these enzymes (TIMPs, PAI-1), to inhibit the synthesis of matrix constituents such
as collagen and proteoglycans, and to stimulate the synthesis and release of some eico-
sanoids including prostaglandins and leukotrienes. The action of this proinflammatory
cytokine on the enzyme process combined with the suppression of matrix synthesis
results in severe degradation of articular tissues and the appearance of conditions that
we know to be characteristic of OA.
This cytokine also plays important roles in normal physiology, including stimula-
tion of the turnover of extracellular matrix. Hence, control mechanisms exist to limit
the extent of cytokine activation and to avoid potential tissue injury. One of these con-
trol mechanisms, unique to the IL-1 system, is a physiological inhibitor of its recep-
tor known as the IL-1 receptor antagonist (IL-1Ra). IL-1Ra is a structural derivative of
IL-1 that binds to IL-1 receptors but does not activate target cells [8]. IL-1Ra blocks
the effects of IL-1 in the immediate cell environment by competing for binding to
cell-surface receptors. Although IL-1Ra was originally described as a secreted form
(sIL-1Ra), it is now known that IL-1Ra consists of a family of molecules. Three addi-
tional intracellular structural variants of IL-1Ra (icIL-1Ra1, 2, 3) have been identified
and are formed by alternate transcriptional splice mechanisms. These isoforms of
IL-1Ra do not possess leader sequences and are therefore synthesized in the cytoplasm
but are not usually secreted from cells. The icIL-1Ra1 also binds to IL-1 receptors with
equal avidity as the secreted form (sIL-1Ra), and can be secreted from cells under cer-
tain conditions. Its role is suggested to be the inhibition of IL-1 binding to extracellular
receptors in specific situations. icIL-1Ra2 has been described in cells only at the
mRNA level and may not normally exist as a protein. icIL-1Ra3 is a lower molecular
weight protein that is produced in large amounts by neutrophils and hepatocytes, and
binds poorly to IL-1 receptors.
In order to verify the effects of blocking IL-1β, in vivo studies were first con-
ducted in animal models of OA, in which IL-1Ra was administered by intra-articular
injection or by gene therapy. Data showed such in vivo treatment was therapeutically
beneficial [9–11]. IL-1Ra injection in patients with symptomatic knee OA also showed
significant improvement on symptoms [12]. However, the results of this pilot study
could not be confirmed in another recent Phase II double-blind study [13].
Other proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α), IL-6,
leukemia inhibitory factor (LIF), oncostatin M (OSM), IL-17 and IL-18, as well as
some chemokines such as IL-8, are also considered potential contributing factors in the
pathogenesis of OA. It has been shown that all of these cytokines are expressed in OA
tissues; however, the exact role and importance of each in the OA process is not yet
clearly established, and it is not known whether a functional hierarchy exists between
them.
TNF-α is a potent cytokine that exerts diverse effects by stimulating a variety of
cells. The best-studied aspect of TNF-α is its ability to promote inflammation. This
proinflammatory cytokine’s dominant role in rheumatoid arthritis is well illustrated by
the blocking of its activity in vivo in studies on animal models and, more recently, on
humans. This cytokine is also present in OA but at a severe stage of the disease.
Recent studies provide evidence that OSM, a member of the IL-6 family, plays a
role in the inflammatory response. However, because this cytokine is also involved in
physiological as well as pathological functions, its exact role is not known. Indeed,
although most of the in vitro findings point to the catabolic effects of OSM (14–17],
some in vivo studies suggest anabolic effects, whereby OSM promotes wound healing
and bone formation in addition to having anti-inflammatory effects (14–21]. Interest-
ingly, OSM is the only member of this cytokine family to cause proteolytic release of
proteoglycan and collagen from human articular cartilage. Although OSM upregu-
lates a spectrum of protease inhibitors, including TIMP and serine protease inhibitors
(α1-protease inhibitor, antichymotrypsin and PAI-1), its proinflammatory role in arthri-
tis may rely on the upregulation of the synthesis of some MMPs and PGE2. Also of
interest is the capacity of OSM to synergize the action of other inflammatory mediators
[17,22–24], including IL-1, TNF-α, IL-17 and lipopolysaccharide. More particularly, in
chondrocytes this striking synergistic effect appears to occur through the induction of
the expression of the collagenases, stromelysin-1, MT1-MMP and aggrecanases.
Among actions relevant to joint inflammation, OSM also induces IL-6. However, the
role of IL-6 in inflammation remains unclear, since IL-6 can induce the production of
TIMP-1, IL-1Ra and the soluble TNF receptor 55.
Among the other cytokines, it has been suggested that IL-17 and IL-18 play a role
in OA pathophysiology as they both share many properties with IL-1. However, on
articular tissue cells, these cytokines also demonstrate effects independent of IL-1ß.
Moreover, both seem to be involved in the early phase of the inflammatory process.
Local in vivo overexpression of IL-17 has been shown to promote destructive ar-
thritis [25]. In collagen-induced arthritis in mice, the blocking of endogenous IL-17
resulted in suppression of arthritis, including a clear suppression of joint damage [26].
It is interesting to note that this study also showed that neutralization of IL-1 had no
effect on IL-17-induced inflammation and joint damage, identifying the IL-1-
independent role of IL-17. Furthermore, IL-17 induced the expression of the receptor
activator of NF-κB ligand, the RANKL, in osteoblasts, a crucial factor in bone resorp-
tion [27]. In addition, in vivo studies using adenoviral overexpression of IL-17 in the
joint showed that this cytokine induced focal erosions in the bone. Consequently, this
factor could be of great relevance in OA, as subchondral bone remodelling appears to
be intimately involved in the early phase of the disease.
IL-18 is a pleiotropic cytokine belonging to the IL-1 family. Although this cyto-
kine is a critical factor in developing immune responses, in vivo data in experimental
animals indicate that the net effect of IL-18 in the development of arthritis is proin-
flammatory. In addition, IL-18 can induce other pro-inflammatory cytokines such as
IL-1, creating an amplifying loop. Its potential involvement in OA has been suggested
based on its enhanced presence in OA cartilage and synovial membrane [28,29].
Six new members of the IL-1 family were identified primarily through the use of
DNA database searches for IL-1 homologues, and named IL-1F5 to IL-1F10 [30]. Al-
though expression patterns and the biological functions of these new IL-1 family mem-
bers have not yet been well characterized, some of them could be of interest for the
arthritic diseases. IL-1F7 forms a complex with IL-18 binding protein, which might
bind to and sequester IL-18R [31]. IL-1F10 is a low nonagonistic ligand for IL-1R1
[32]. IL-1F9 as well as IL-1F6 and IL-1F8 activate NF-κB (one of the most important
inducers of inflammation), and IL-1F5 might be an IL-1F9 antagonist [33,34]. Finally,
IL-1F8 has been shown to exert proinflammatory effects in human joint cells [35].
Nitric Oxide and Eicosanoids
In addition to proinflammatory cytokines, other inflammatory mediators could also

play major roles in the OA process, the principals being nitric oxide (NO) and the eico-
sanoids, including prostaglandins and leukotrienes.
NO acts as a mediator in various physiological and pathophysiological processes in
the human body. NO generated by the inducible NO synthase (iNOS) has regulatory,
proinflammatory and destructive effects. OA cartilage produces a large amount of NO
(and reactive oxygen species), and high levels of nitrites/nitrates have been found in the
synovial fluid and serum of arthritis patients. NO has been shown to be involved in the
promotion of cartilage catabolism in OA through a number of mechanisms, including
the induction of synovial inflammation. It can inhibit the synthesis of cartilage matrix
macromolecules, such as aggrecans, enhance MMP activity, and reduce the synthesis
of IL-1Ra by chondrocytes. NO also plays a role in chondrocyte apoptosis and induces
cyclooxygenase (COX)-2/prostaglandin E2 (PGE2) synthesis. Also demonstrated was
that exogenous PGE2 could sensitize human OA chondrocytes to cell death induced by
NO [36,37]. In vivo findings in studies done using OA experimental animal models in
which oral administration of therapeutic dosages of a specific inhibitor of iNOS dem-
onstrated positive therapeutic benefits on the progression of lesions [37–39]. Collec-
tively, inducible NO acts by reducing the major anabolic processes and increasing the
catabolic processes, making it a complete factor favouring joint destruction. Moreover,
the present knowledge points to a possible therapeutic value for iNOS inhibitors in the
treatment of OA as chondroprotective, anti-inflammatory and analgesic compounds.
This molecule is therefore believed to be an attractive target in OA, because reducing
its excess production may not only slow the disease progression, but is also likely to
reduce the symptoms, making it able to reach two targets simultaneously.
Other major inflammatory factors involved in OA pathophysiology are the pros-

taglandins and leukotrienes. Prostaglandins are synthesized from arachidonic acid via
the actions of the COX enzymes, either constitutively or in response to cell-specific
trauma, stimuli, or signalling molecules. The most abundant prostanoid in the human
body is PGE2. Dependent upon context, PGE2 exerts a homeostatic or inflammatory
effect. Inhibition of PGE2 synthesis by non-steroidal anti-inflammatory drugs
(NSAIDs) has been an important anti-inflammatory strategy for more than a century. In
addition to exacerbating joint inflammation, PGE2 can also potentiate the effects of
other mediators of inflammation. It can affect cartilage remodelling directly or function
indirectly as an autocrine regulatory factor. PGE2 may also contribute to joint damage
by promoting MMP production, osteoclastic bone resorption, and angiogenesis.
COX (COX-1 and -2) activity had been considered the key step in prostaglandin
synthesis. Recently, other splice variants of COX-1 have been identified and named
COX-3 and PCOX-1 [40,41]. They were first identified in canine tissues. Recently the
presence of a COX-3 mRNA transcript was confirmed in human cells. The regulation
of COX-3 appears to be identical to that of COX-1, and one of the PCOX, the PCOX-
1a, was shown to lack the COX activity. Moreover, splice variants of COX-2 have also
been reported, but have failed to show enzymatic activity [42].
Metabolism of arachidonic acid by COX-1 or COX-2 yields to the unstable inter-
mediary PGH2, which can be further metabolized into PGE2, PGD2, PGF2α, PGI2
(prostacyclin) or tromboxane A2. The enzyme responsible for the isomerization of
PGH2 was not known until recent identification of PG synthase (PGS) as the terminal
enzyme responsible for prostanoid synthesis. In the case of PGE2, studies suggest the
presence of at least three distinct PGES, named cytosolic PGES (cPGES), microsomal
PGES-1 (mPGES-1), and mPGES-2 [43–46]. cPGES is constitutively and ubiquitously
expressed and is preferentially coupled with COX-1, promoting immediate production
of PGE2 [45–47]. COX-2 and mPGES-1 protein expression are concordantly induced
by IL-1ß, consistent with the hypothesis that mPGES-1 and COX-2 are co-regulated
and that stimulated PGE2 synthesis may depend on upregulation of both of these en-
zymes. mPGES-2, the most recently identified PGES, is constitutively expressed in
diverse tissues. While its role remains elusive, it has been found to be functionally
linked to both COX-1 and COX-2. However, and although mPGES-2 can couple with
COX-1 to produce PGE2 in response to acute inflammation and to COX-2 in response
to chronic inflammation, data have demonstrated a modest preference for coordination
with COX-2. Studies from mPGES-1-deficient mice and animal models of inflamma-
tory arthritis strongly suggest the role of mPGES-1 in inducible PGE2 production and
arthritis [48,49]; and accumulating evidence implicates mPGES-1 in the pathogenesis
of OA. Hence, mPGES-1 is localized in the superficial layers of human OA cartilage,
areas where IL-1ß is also found, which is consistent with a role for IL-1ß in stimulating
chondrocyte mPGES-1 [50,51]. Moreover, PGE2 secretion from OA chondrocytes cor-
relates well with mPGES-1 concentrations following stimulation [52]. Since the con-
troversy surrounding selective COX-2 inhibitors owing to an apparent increase in the
risk of cardiovascular disease and stroke, researchers have been looking at extensively
the potential utility of clinically targeting mPGES-1, yet no specific pharmacologic
inhibitors are currently available.
The use of NSAIDs or COX-2 selective inhibitors has shown that PGE2 inhibition
alone does not seem to delay the natural history of progressive OA. In recent years, it
has been shown that PGE2 synthesis is only one part of the arachidonic acid pathway.
The precursor, arachidonic acid, is a substrate that gives origin to many other lipid me-
diators, including leukotrienes. Leukotrienes themselves play a major role in the devel-
opment and persistence of the inflammatory process, and it is now clear that pros-
taglandins and leukotrienes have complementary effects. Leukotrienes are produced by
the enzyme 5-lipoxygenase (5-LOX). Leukotriene A4 (LTA4) is the first to be synthe-
sized and is then processed into LTB4 or LTC4, then LTD4 and LTE4, which are potent
chemotactic and inflammatory factors. Levels of LTB4 and LTC4 are increased in OA
synovial tissue [53,54]. Studies have also revealed that, on human OA synovial mem-
brane, LTB4 potently stimulates the release of proinflammatory cytokines such as IL-1ß
and TNF-α [54–58]. Thus, the failure of NSAIDs to impact OA progression could be
due to the fact that inhibiting only the COX pathways leads to a shunt to leukotriene
production in these tissues [59–63]. From this concept, it is hypothesized that blocking
production of both leukotrienes and prostaglandins could have a synergistic effect in
achieving optimal or a wider-spectrum of anti-inflammatory activity. Data on a disease
modifying OA drug (DMOAD) Phase III clinical trial of such a dual inhibitor of COX
and 5-LOX revealed that such a drug can significantly reduce the progression of knee
OA structural changes using quantitative magnetic resonance imaging (qMRI) [64].
More particularly, this was found for the cartilage volume. This drug was also equally
effective as a known NSAID, naproxen, at reducing OA symptoms.
Protease-Activated Receptors
Some members of the protease-activated receptors (PARs) have been recently shown to
be involved in inflammatory pathways, and, more specifically, an important role for
PAR-2 in chronic arthritis has been demonstrated. These receptors belong to a novel
family of seven-transmembrane G-protein-coupled receptors that are activated through
a unique process. Cleavage of their N-terminal domains by proteases unmasks a new
N-terminal sequence that acts as a tethered ligand, binding and activating the receptor
itself [65–67]. Once activated, this process is irreversible. To date, four members of
this family have been identified and designated PAR-1 to -4. They exhibit differential
tissue expression as well as selectivity in activation. The enzymes activating PARs
belong to the serine protease family; thrombin activates PAR-1, -3, and -4, trypsin
PAR-1, -2 and -4, tryptase and membrane-type serine protease-1 PAR-2, and cathepsin
G as well as plasmin triggers PAR-4 activation [68,70]. PARs, particularly PAR-2,
have been reported to be involved in multiple cellular responses related to tissue injury
and repair, angiogenesis, nociception and neurogenic inflammation, and recently in-
flammatory conditions including those in rheumatoid arthritis and, more recently, in
OA. In that regard, an important role for PAR-2 in chronic arthritis has been shown by
using a PAR-2 gene knockout mouse in which inflammation was significantly delayed
with the adjuvant-induced arthritis model [71,72]. PAR-2 expression has recently been
found in human chondrocytes and synovial fibroblasts [73–75], and was modulated by
the proinflammatory cytokines IL-1ß and TNF-α as well as the growth factors bFGF
and TGF-ß. TGF-ß, however, differentially regulates normal and OA human chondro-
cytes [74]. The inflammatory function of this receptor has been found to be associated,
depending on the cell systems, to NO, COX-2, PGE2, and MMPs. It is also suggested
that activation of PAR-1 and -2 induce the production of the proinflammatory cyto-
kines IL-1ß, IL-6, IL-8, IL-18 and TNF-α. Considering the pro-algesic and
-inflammatory effects of PAR-2, this receptor might constitute a novel alternative
therapeutic target for OA.
Conclusion
OA is mediated by a multitude of complex autocrine and paracrine anabolic and cata-

bolic factors that act upon diverse cells from articular tissues. Various pathways result
in alterations in transcription factors that transduce signals intracellularly. The end re-
sult of these pathways is the production of proinflammatory cytokines in which multi-
ple lines of research have established the central role of IL-1ß, as well as other inflam-
matory mediators such as other cytokines and the factors NO, prostaglandins, leukot-
rienes and PARs. Therapies targeted at any of these steps, both upstream and down-
stream may prove to be beneficial.
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14 Osteoarthritis, Inflammation and Degradation: A Continuum
IOS Press, 2007
II
Mechanical Loading Effects on Articular

Cartilage Matrix Metabolism
and Osteoarthritis
Robert Lane SMITH, PhD
Rehabilitation Research and Development Center
VA Palo Alto Health Care System, Palo Alto, CA
Department of Orthopaedic Surgery
Stanford University School of Medicine
Stanford, CA
Abstract. This chapter examines the effects of mechanical loading on cartilage

metabolism in explant culture and as isolated chondrocytes in high density
monolayer and agarose/scaffold cultures. The mechanical loading effects are de-
fined in terms of the two stress states that arise within cartilage, shear stress and
hydrostatic pressure. The paper examines possible signaling pathways that could
contribute to transduction of changes in the physical environment of articular
chondrocyte to modulation of chondrocyte gene and protein expression.
Introduction
Osteoarthritis (OA) is a disabling disease that is estimated to impact 1 in 3 individuals

over the age of sixty [1]. The precise etiology of the disease remains unclear in part
because the onset of OA and progression of the disease rest with a multiplicity of
physical and biological factors [2]. This chapter will explore the overlap between me-
chanical load-mediated events and chondrocyte-based metabolic responses that may
contribute to the progressive joint destruction characteristic of OA.
Early manifestations of OA are generally silent and have only recently become
amenable to discovery through improvements in imaging methods, arthroscopic visu-
alization and analysis of gait abnormalities [3–5]. New imaging techniques can now
reveal localized thinning of the cartilage extracellular matrix. Arthroscopic examina-
tion can pinpoint localized defects and detect surface irregularities [6]. Analysis of gait
can establish how weight transfer with activity may generate harmful joint loading pat-
terns [7]. As OA continues, patient disability ultimately results from destruction of the
articular cartilage causing severely painful joint motion as bone meets bone [8,9].
There are two classifications of OA. In primary disease, although the exact cause
remains unclear, onset of cartilage destruction appears to be associated with some ele-
ment of abnormal joint biomechanics. In secondary disease, injury, infection, heredi-
tary factors, developmental processes and metabolic or neurological disorders influence
R.L. Smith / Mechanical Loading Effects on Articular Cartilage Matrix Metabolism and OA 15
joint tissue metabolism and can initiate cartilage breakdown [10]. While the onset of
OA and joint loading appear linked, the search for molecular signaling pathways regu-
lating articular chondrocyte cartilage matrix metabolism continues to be a subject of
intense investigation [11]. Understanding the basis of mechanical signaling in cartilage
is central for discovery of therapeutic agents to prevent progression of OA and devel-
opment of methods for cartilage repair and/or regeneration.
1. Mechanical Loading and Cartilage Homeostasis
In normal joints, the combined effects of mechanical loading and multiple interacting
physiological mediators sustain chondrocyte viability and promote cartilage matrix
homeostasis [12,13]. In normal adult cartilage, chondrocyte proliferation and anabolic
metabolism remain relatively quiescent so that extracellular matrix longevity and func-
tionality is preserved [14]. Stabilization of the balance between matrix synthesis and
degradation by the chondrocytes is critical for prevention of joint degeneration [15].
Defining the cartilage catabolic response to injurious stimuli confirmed that matrix
metabolism must remain under stringent control by genetic and epigenetic mechanisms
for joints to remain healthy [16].
From a mechanical perspective, activities of daily living generate compressive
loads across the diarthroidal joints due to surface sliding, rolling, spinning and fluid
exudation [17,18]. The motion at the joint surfaces creates two types of fundamental
stresses, shear stress and hydrostatic stress (pressure) within the cartilage extracellular
matrix [19]. The specific type of motion and degree of loading will generate varying
levels of shear and hydrostatic stress within the cartilage. Our in vitro studies demon-
strate that both types of stress differentially influence chondrocyte metabolism [20–28].
Articular chondrocytes exposed to shear stress increase expression of the proinflamma-
tory mediators, nitric oxide, prostaglandin E2 (PGE2) and interleukin-6, and decrease
expression of the cartilage matrix macromolecules [20–24]. In contrast, applying hy-
drostatic pressure increases cartilage matrix gene expression and enhance matrix pro-
duction [25–28]. In other experimental models where shear stress predominates, an
increase in proinflammatory cytokines and other factors is associated with inhibition of
hormonal and growth factor effects on cartilage metabolism [29–31].
In spite of clinical and experimental data implicating mechanical effects in OA, a
precise mechanism is lacking regarding ways in which day to day diarthroidal joint
loading contributes to cartilage degeneration. This is in spite of human clinical observa-
tions implicating ligamentous injury, limb alignment, obesity, trauma and genetics as
risk factors for OA [32–36]. Each of these clinical conditions alters the profile of joint
loads and each is recognized as a potential cause of progressive loss of cartilage. While
normal joint loads are essential for articular cartilage matrix homeostasis, the strong
association of OA with dense connective tissue injuries that disrupt joint mechanics
points to a prominent role for inappropriate stress states to induce cartilage degenera-
tion.
2. Articular Cartilage: Biochemistry and Biomechanics
In diarthroidal joints, articular cartilage distributes loads that reach up to 7 times body
weight as a result of specialized matrix macromolecules that provide compressive resil-
16 R.L. Smith / Mechanical Loading Effects on Articular Cartilage Matrix Metabolism and OA
ience and tensile strength. In an intact cartilage matrix, joint motion remains essentially
pain free. This aspect of tissue functionality is due primarily to three matrix constitu-
ents, water, aggrecans, and type II collagen [37,38]. Aggrecans provide resistance to
compression due to the hydrophilic and electronegative properties of covalently at-
tached glycosaminoglycans [39]. Type II collagen provides tensile strength through an
organized network of cross-linked fibrils. Hyaluronan together with a limited comple-
ment of glycoproteins and low molecular weight proteoglycans ensure matrix organiza-
tion [40]. The cartilage matrix constituents originate from chondrocytes distributed in
zones extending from the flattened cells of the superficial layer to the rounded cells
within deeper layers adjacent to the subchondral bone interface [41].
Mathematical models of joint loading resolve two principal stresses, shear and hy-
drostatic, that arise within cartilage when compressive forces are applied [19,42,43].
Major shifts in the relative proportion and/or distribution of these stresses in cartilage
might be a major factor regulation of chondrocyte metabolism. The mechanical
changes associated with OA risk factors, such as laxity or excessive loads, may selec-
tively alter the level and distribution of shear stress in the cartilage while not influenc-
ing pressure. At present, the precise levels of shear stress that can incite cartilage deg-
radation in vivo are unknown. There is, however, evidence linking the incidence of
localized inflammation in OA joints to changes in load distribution [44]. It is likely that
continuous exposure to inappropriate stresses contributes to a catabolic phenotype in
chondrocytes. Cartilage catabolism is accompanied by release of proinflammatory cy-
tokines and degradative enzymes into the joint space and into the serum [45]. The
range of chondrocyte-derived inflammatory factors will be examined below with re-
spect to vitro studies of cartilage degradation induced by mechanical loading.
3. Cartilage Explants: Mechanical Loading and Metabolism
Effects of mechanical loading on articular cartilage metabolism have been investigated

in vitro with cartilage explants and with isolated chondrocytes. Explant cultures pro-
vide a quasi-tissue systems approach to examine how mechanical loads alter cartilage
metabolism since the morphology and zonal distribution of the cells remain organized
in a three-dimensional matrix. Under defined culture conditions, chondrocyte viability
persists in all zones of full-thickness explants and aggrecan and collagen content re-
main stable throughout a three-week culture period [46]. In addition, dynamic and
equilibrium mechanical properties also remain stable. With significant variation in the
loading conditions, cartilage explant cultures can exhibit distinct metabolic behavior
depending on the type, duration and magnitude of the physical stimulus. For instance,
injurious compression alters the physical properties of cartilage due to onset of chon-
drocyte-mediated turnover and release of proteoglycans from the matrix [47,48]. Simi-
larly, disruption of the normal joint architecture results in degradation of the flow-
independent viscoelastic and equilibrium properties of articular cartilage [49,50].
The response of cartilage explants to mechanical loading induces production of
multiple chemical and biological molecules that coincide with early and late metabolic
changes in chondrocytes. When dynamic compression is applied to cartilage explants,
an initial up-regulation of aggrecan and type II collagen expression is subsequently
followed by decreased synthesis patterns for both matrix macromolecules [51,52]. The
chondrocyte response remains subject in part to the distribution of stress states within
the matrix. In full-thickness explants retaining a thin layer of bone, cyclic loading in
unconfined compression produces a weakening of the collagen network and an increase

in hydraulic permeability [53]. In explant cultures not exposed to loading, less denatu-
ration of type II collagen occurs, a finding consistent with low turnover and consistent
with less tissue remodeling. The exposure to cyclic compression also increased release
of fragments of the major matrix proteins into the culture medium when compared to
unloaded cultures. The observations imply that, on some level, cyclic loads destabilize
matrix homeostasis depending on loading conditions.
In a study where static compression is applied to the explant cultures, aggrecan and
type II collagen synthesis rates were generally decreased from the outset of loading
[54]. The mRNA signal levels for aggrecan and type II collagen exhibited a transient
increase at 0.5 hours but then decreased between 4 and 24 hours after compression was
applied. The protein levels decreased ahead of the mRNA level suggesting that matrix
biosynthesis involves pathways other than mRNA expression. In studies where the
compressive loading of cartilage explants consisted of injurious levels of peak stresses
(>20 MPa), chondrocyte apoptosis was maximally induced by 24 hours after ending the
loading protocol [55]. At high peak stresses, the equilibrium and dynamic stiffness of
the explants decreased with the severity of the load in uniaxial confined compression.
When cartilage mechanical properties were assessed in radially unconfined compres-
sion, the equilibrium and dynamic stiffness values were decreased a peak stresses in the
range of 7 to 12 MPa [55]. The change in mechanical properties was accompanied by
degradation of the collagen fibrillar network, loss of glycosaminoglycans and increased
tissue swelling.
One hypothesis for the effects of compressive loading on the cartilage explants is
that increased shear stresses act directly on the cells as they undergo a distortional
change in shape. Examination of chondrocytes within a compressed cartilage matrix
show that both the cell body and the nucleus undergo changes in shape that are depend-
ent on the stress levels being applied [56,57]. The assumption has to be advanced that
chondrocytes in explants become subject to distortion because of the cut edges of the
tissue samples where some fluid loss can occur, particularly in unconfined compressive
loading. The extent to which the same degree of deformation might occur in the intact
joint surface cartilage remains unclear.
4. Isolated Chondrocytes: Mechanical Loading and Metabolism
Under in vitro loading conditions, the metabolic responses observed for cartilage ex-
plants under compression appear to represent patterns where injury leads to decreased
matrix biosynthesis and loss of matrix macromolecules. The results obtained from stud-
ies of compressive loading of cartilage explants is mirrored to a significant degree by
studies examining the response of isolated articular chondrocytes to physical stimula-
tion. In these types of in vitro experiments, the chondrocytes are typically subjected to
mechanical loading in the absence of an assembled extracellular matrix.
A number of model systems have been used to assess strain dependent effects on
chondrocyte metabolism at the level of gene and protein expression. One system used
to study mechanical effects relies on preservation of the articular chondrocyte pheno-
type by placing the cells in primary high density monolayer culture [58]. The high den-
sity cultures are defined by having the cells present in concentrations of 1×10 6/cm2
without the cells ever having been passaged following isolated by collagenase diges-
tion. In our use of this model, low levels of shear stress applied to articular chondro-
cytes through the application of fluid motion by a cone viscometer activated release of
proinflammatory mediators and inhibited matrix protein synthesis [20–24].
Exposure of human OA chondrocytes to a continuously applied shear stress
(1.64 Pa) increased nitric oxide (NO) release by 1.8-, 2.4-, and 3.5-fold at 2, 6, and
24 h, respectively, into the culture medium. NO is a short-lived but highly soluble and
diffusible signaling factor that is implicated in the regulation of cartilage matrix protein
gene expression. Shear stress also significantly increased gene expression of the induc-
ible form of nitric oxide synthase. Exposure of chondrocytes to shear stress for 2, 6,
and 24 h inhibited type II collagen mRNA signal levels by 27%, 18% and 20% after a
constant post-shear incubation period of 24 h. Aggrecan mRNA signal levels were in-
hibited by 30%, 32% and 41% under identical conditions. Addition of an NO antago-
nist increased type II collagen mRNA signal levels by an average of 1.8-fold (137% of
the un-sheared control) and reestablished the aggrecan mRNA signal levels by an aver-
age of 1.4-fold after shear stress (92% of the un-sheared control) (ANOVA p < 0.05).
Applying shear stress to human OA chondrocytes also results in an increase in
apoptosis as evidenced by presentation of membrane phosphatidylserine and increased
nucleosomal degradation. As might be expected, expression of the anti-apoptotic fac-
tor, bcl-2, was decreased by shear stress and addition of the nitric oxide antagonists,
L-N(5)-(1-iminoethyl) ornithine and N-omega-nitro-L-arginine methyl ester
(L-NAME), reduced shear stress induced nucleosomal degradation by 62% and 74%,
respectively. Inhibition of shear stress induced nitric oxide release by L-NAME coin-
cided with a 2.7-fold increase of bcl-2, when compared to chondrocytes exposed to
shear stress in the absence of L-NAME. These results support the fact that shifts in
mechanical stresses in cartilage, even at low levels, may induce factors such as nitric
oxide that can then lead to joint degeneration through effects on chondrocyte viability.
A number of other model systems in which chondrocytes placed in agarose have
been used to determine the effects of varying levels of compression on matrix metabo-
lism [59–66]. Chondrocytes isolated from fetal, young and aged bovine cartilage and
cultured in agarose gels showed that fetal and young chondrocytes were similar with
respect to cell proliferation and proteoglycan accumulation whereas aged chondrocytes
exhibited diminished capacity with respect to both activities [59]. Collagen accumula-
tion was also reduced in the aged chondrocytes by approximately 55% when compared
to younger cells. The differences in metabolism of the chondrocytes resulted in the
production of an extracellular matrix that exhibited a significant reduction in stiffness
when compared to the matrix produced by younger chondrocytes [59]. Other studies of
chondrocytes placed in agarose demonstrate that the cells maintain fidelity with respect
to the extracellular matrix synthesis representative of the zone of articular cartilage
from which the cells originated. Superficial zone cells from the upper 15 to 20% region
of the cartilage responded differently when compared to the deeper tissue chondrocytes
[60]. In agarose culture, superficial cell proliferation was stimulated by dynamic strain
whereas the deeper cells remained unchanged. The deeper cells did exhibit an increase
of glycosaminoglycan synthesis under dynamic strain at a frequency of 1 Hz with am-
plitude of 15%. These data imply cell specific responsiveness to compressive strain.
The reactivity of articular chondrocytes to dynamic compression is also apparent
through studies on matrix biosynthesis in an agarose disk model [61]. Although dy-
namic compression stimulates extracellular synthesis, the deposition of the matrix mac-
romolecules and the physical properties of the matrix varied depending on whether the
cells were in the center or in the periphery of the disk. These data imply that cell-matrix
interactions may have equal importance to matrix-Independent cell deformation and
transport limitations for maintenance of an organized load-bearing matrix. Studies on

cell seeding density together with comparison studies of different types of support scaf-
folds suggest that direct mechanical effects occur through complex and overlapping
signaling mechanisms [62,63].
Dynamic compression also has functional effects on chondrocyte metabolism and
has been shown to counteract effects of interleukin-1beta induced release of nitric ox-
ide and prostaglandin E2 [64–66]. The question remains as to the mechanism by which
dynamic loading modulates cell metabolism. Recent studies show that ion channel in-
hibitors can differentially influence the chondrocyte responses to mechanical stimuli in
such a way that either glycosaminoglycan synthesis or protein synthesis may show al-
tered rates depending on the type of channel being blocked [67]. The fact that the dif-
ferences occur while the type and level of mechanical loading is held constant rein-
forces the hypothesis that redundant multi-level regulatory mechanisms must be active
in regulation of cartilage homeostasis.
Efforts to quantify the relationship of intracellular cytoskeletal components with
cell-surface integrin-mediated extracellular matrix attachments are just beginning.
However, initial studies provide evidence that the cell-matrix transition points are dy-
namic and under continual time-dependent reorganization in response to the application
of mechanical loading [68]. The cytoskeletal data are compatible with early cell culture
studies and finite element modeling that attempted to discern the behavior of the articu-
lar chondrocyte under uniaxial compressive loading. One early study showed that the
cells decreased in cross-sectional area depending on the level of compressive strain
[69]. The authors suggested that the chondrocyte may alter its intracellular composition
by cellular processes to compensate for the compressive load. Given our increasing
understanding of intracellular signaling pathways, these observations suggest that
changes in cell morphology may provide both signaling mechanisms as well as protec-
tion under excessively high compressive loads.
Compression induced changes in chondrocyte morphology vary with local tissue
strains [70] and have been shown to coincide with differential expression of collagen
types [71] and release of latent degradative enzymes, such as procollagenase [72,73].
Thus, cellular deformation may be a primary determinant in the regulation of cartilage
metabolism by mechanical loading. The question remains to what extent that sensitivity
to shear stress in normal and OA chondrocytes underlies proinflammatory mediator
expression that subsequently leads to matrix degeneration through the induction of
catabolic processes in cartilage and synovial tissue.
The second major stress type that occurs within diarthroidal joints is hydrostatic
pressure. Within the matrix the levels of hydrostatic pressure are proportional to the
contact stresses that are generated with joint surface loads. A number of studies show
that contact stresses in the major joints can reach levels up to 20 to 25 MPa depending
on the activity [74,75]. These contact stress are accompanied by the generation of hy-
drostatic pressure in cartilage in the range of 2 to 15 MPa [74,75]. In vitro, levels of
hydrostatic pressure at 5 to 15 MPa increase matrix synthesis as quantified by incorpo-
ration of radiolabeled sulfate and proline in adult bovine articular cartilage explants
[76]. Organ culture experiments demonstrate that sites of proteoglycan production co-
incide with regions of pure hydrostatic pressure [77].
Our in vitro studies show that physiological levels of hydrostatic pressure results in
anabolic patterns of chondrocyte gene expression with a counteracting effect on cata-
bolic factors [25–28]. Normal bovine articular chondrocytes exposed to 10 MPa of
intermittent hydrostatic pressure at a frequency of 1 Hz for periods of 2, 4, 8, 12, and
24 hrs showed that type II collagen mRNA signal levels exhibited a biphasic pattern in
response to continuously applied loading. There was an initial increase of approxi-
mately five-fold at 4 and 8 hrs that subsequently decreased by 24 hrs. In contrast, ag-
grecan mRNA signal increased progressively up to three-fold throughout the loading
period. In separate experiments, chondrocytes were exposed to intermittent hydrostatic
pressure for a period of 4 hrs per day for 4 days. Changing the loading profile to 4 hrs
per day for 4 days increased the mRNA signal levels for type II collagen nine-fold and
for aggrecan twenty-fold when compared to unloaded cultures. These data confirmed
that mechanical loading in vitro with time-specific protocols produced changes in
chondrocyte metabolism consistent with predictions from mathematical models.
In separate studies, aggrecan mRNA signal levels increased 1.3- and 1.5-fold at
5 and 10 MPa, respectively, relative to beta-actin mRNA, when exposed to intermittent
hydrostatic pressure for 4 hrs/day for 1 day. Extending the loading period to 4 hrs/day
for 4 days (4×4) increased aggrecan mRNA signal levels by 1.4-, 1.8- and 1.9-fold at
loads of 1, 5 and 10 MPa, respectively. Type II collagen mRNA signal levels were in-
creased at loads of 5 and 10 MPa with the 4×4 loading regimen. Western blotting con-
firmed that IHP increased aggrecan and type II collagen in the chondrocyte extracts.
Other effects of intermittent hydrostatic pressure (IHP) include reversal of inhibi-
tory effects of bacterial antigen (LPS) on chondrocyte metabolism. Intermittent hydro-
static pressure applied to LPS-activated chondrocytes decreased both nitric oxide syn-
thase mRNA signal levels and nitric oxide released into the culture medium. Exposure
of LPS-activated chondrocytes to IHP also increased type II collagen and aggrecan
mRNA signal levels by 1.7-fold, when compared to chondrocytes activated by LPS and
maintained without loading. Applying IHP decreased the signal levels for monocyte
chemotactic factor-1 and matrix metalloproteinase-2 following LPS activation by 45%
and 15%, respectively. These data confirmed that IHP counteracts effects of inflamma-
tory agents, such as bacterial LPS, on chondrocytes.
Application of intermittent hydrostatic pressure (IHP) reduced the levels of NO in-
duced by shear but did not alter release NO from chondrocytes not exposed to shear
stress. NO induced by shear stress or by addition of an NO donor (sodium nitroprus-
side) decreased mRNA signal levels for the cartilage matrix proteins, aggrecan, and
type II collagen. IHP blocked the inhibitory effects of the NO donor on matrix gene
expression but failed to alter the inhibitory effects of shear stress on matrix mRNA
levels. This data suggest that shear stress inhibits matrix synthesis through multiple
mediators and/or overlapping pathways with hydrostatic pressure possibly acting
through different pathways. Interestingly, in alginate cultures of bovine chondrocytes
hydrostatic pressure decreased expression of MMP-13 and type I collagen and upregu-
lated expression of TIMP-1 [78]. Cyclic tension in this system downregulated TIMP-1
while increasing expression of expression of hypertrophic chondrocyte markers, con-
sistent with differential responsiveness of chondrocytes to stress states.
The precise mechanism by which hydrostatic pressure modulates chondrocyte me-
tabolism remains unclear. A recent study implicates changes in calcium ion distribu-
tion, both intracellular and extracellular, as a signaling mechanism for hydrostatic load-
ing [79]. A five minute exposure of chondrocytes on cultured on cover slips to constant
hydrostatic pressure at a level of 0.5 MPa increased free calcium ion levels by two-fold
for cells isolated from middle zone of cartilage whereas cells of the superficial and
deep zone exhibited a 1.5-fold increase in free calcium ion. Effects of inhibitors of cal-
cium ion flux showed that a calcium channel blocker (verapamil) did not block the ef-
fect of hydrostatic pressure on the chondrocytes whereas a stretch-activated channel
blocker and an intracellular storage blocker partially reduced the effects of hydrostatic
pressure on calcium ion distribution. These data were interpreted as suggesting the in-
volvement of inositol 1,4,5-triphosphate (IP3)-mediated calcium increase, similar to the
processes active in bone cells.
5. Cartilage Metabolism and Matrix Degradation
During joint development and through adult life, cartilage metabolism is continually
influenced by biological and mechanical factors [80,81]. In early cartilage develop-
ment, the growth hormone/IGF-1 dependent pathway is a primary stimulus for chon-
drocyte proliferation and matrix synthesis [82]. With skeletal maturation, the IGF-1
effect on chondrocyte metabolism through specific receptors is modulated in part
through fluctuating levels of IGF-1 binding proteins that are present in the serum
[83,84]. In OA, articular chondrocytes exhibit a refractory response to IGF-I, that ap-
pears independent of functional plasma membrane receptors [85]. OA joints also ex-
hibit increased levels of proinflammatory mediators that alter chondrocyte metabolism
[86]. These inflammatory mediators include nitric oxide, interleukin-1, interleukin-6
and tumor necrosis factor-alpha. Interleukin-1alpha and interleukin-1beta (IL-1) inhibit
matrix synthesis [87,88] and together with tumor necrosis factor alpha [89,90] induce
matrix degrading enzyme expression by chondrocytes [91–93]. Other inflammatory
mediators including nitric oxide and IL-6 are associated with induction of degradative
enzymes [94] and inhibition of aggrecan synthesis [95], respectively.
Experimental animal models that produce symptoms consistent with OA in include
immobilization [96], increased instability (excision of anterior cruciate ligament, 97),
excessive impact or blunt traumatic injury [98,99]. Other causes OA have been ad-
vanced such as a stiffening of subchondral surfaces so that load distribution to bone
becomes limited [100]. The chondrocyte responses to varying levels of the two types of
cartilage stress states may provide the metabolic link to all of these risk factors. For
example, increased shear stress may act as an inducer of the matrix metalloproteinases
(MMPs) associated with arthritis [101]. Cartilage damaged by trauma exhibit increased
release of collagenase (MMP-1) and stromelysin (MMP-3), either of which may
degrade proteoglycans [102–105]. Collagenases may then initiate degradation of type II
collagen after activation [106,107]. Stromelysin is activated by plasmin and subse-
quently activates other MMPs [108]. Collagenase and stromelysin together may also
destabilize the matrix by degrading minor components, such as link protein and other
glycoproteins [109,110]. Three unique collagenases, the fibroblast collagenase
(MMP-1), the neutrophil collagenase (MMP-8) and the collagenase 3 (MMP-13) are
proposal as integral elements in matrix degradation [111].
In naturally occurring OA in dogs, MMP-9 was the matrix metalloproteinase that
correlated with onset and progression of disease [112]. Under conditions of a shear
stress of 16 dyn/cm2 (1.6 Pa), MMP-9 expression was increased in rabbit chondrocytes.
In this model, transfection of the chondrocytes with a dominant negative mutant of c-
Jun NH2-terminal kinase [113] attenuated the MMP-9 expression. Our studies of OA
cartilage demonstrated that expression of the MMP-9 was highest in areas of the carti-
lage that exhibited the greatest amount of fibrillation [114,115]. Of interest, even
the most normal appearing cartilage in the OA joint showed significant elevation of
MMP-9 expression. This data fits with the proposed role of active neutral matrix metal-
loproteinases in the pathogenesis of arthritis [116–118]. Degradation products from
metalloproteinases are elevated during periods of active joint destruction [119–121].

The enzyme activities include aggrecanases that degrade the cartilage proteoglycans
[122,123]. Core protein peptides corresponding to an aggrecanase cleavage site, be-
tween Glu373 and Ala374 are elevated in human joint fluid [124,125]. Multiple enzymes
must be active since neoepitopes of aggrecan globular domain G1 generated by MMP
and aggrecanase activity are present in OA cartilage [126].
6. Chondrocyte Mechanotransduction
Transmittal of the mechanical forces to chondrocytes localized within an extracellular

matrix is assumed to involve interactions between proteins of cell plasma membrane
and matrix components [127–129]. Modes for transfer of the mechanical stimulus for
regulation of transcriptional and translational processes depend on signaling pathways
linked to ion channels, membrane receptors, and cytoskeletal proteins [130]. Selective
integrin subunits present in the plasma membrane are involved in when chondrocytes
respond to fluid shear, compressive loading and stretch [131,132]. The interactions
between the chondrocytes and matrix then activate intracellular second messenger
pathways that orchestrate multiple metabolic reactions to changes in mechanical envi-
ronment [133].
In setting a hierarchical pattern by which extracellular stimuli are converted into
cellular responses, the mitogen-activated protein kinases (MAPKs) emerge as a pri-
mary effectors for positive and negative changes in cellular metabolism [134–136].
Downstream effects of the MAPKs follow receptor-mediated phosphorylation of the
MAPK-kinase kinase (MEK kinases) [137]. Activation of MEK kinases leads to phos-
phorylation of the MEKS or MKKs that then phosphorylate the MAPKs. The MAPKs
are divided into three families, the extracellular regulated kinases (ERK-1, -2), the
stress-activated protein kinases (SAPK) or c-jun NH2-terminal kinases (JNK) and the
p38 MAPKs [138]. The p38 MAPKs originate from four genes and exhibit specificity
for upstream activators and downstream substrates.
Mechanical loads alter chondrocyte metabolism through activation of specific in-
tracellular kinase pathways depending on the type of load applied [139–142]. Com-
pressive loading induces a phased phosphorylation of the extracellular regulated
kinases 1 and 2 (ERK 1/2) where ERK2 exhibits a persistent phosphorylated state for
up to 24 hours [143]. In embryonic limb bud, compressive loading increases collagen
and aggrecan expression while decreasing IL-1beta expression [124]. Other kinases,
p38 mitogen-activated kinase (p38) and a member of c-Jun N-terminal kinase (JNK)
pathway (SEK1), exhibited more transient responses (10 minutes to 1 hour) [144]. In
endothelial cells, kinase activation induces transcription genes encoding monocyte
chemotactic protein-1 (MCP-1) and c-Fos [145].
Mechanical signaling events that activate or suppress gene expression involve
translocation of signaling molecules into the nucleus for activation of the transcription
factor proteins that bind to the promoter sequences of gene as enhancers or suppressors
of mRNA synthesis [146]. The p38 MAPKs play crucial roles in the regulation of gene
expression either by activating transcription factors or by stabilizing RNA and protein
turnover [147]. Transcription factors act through interactions with binding motifs lo-
cated in the upstream 5’-flanking regulatory DNA sequences [148] and in combination
with co-activators and initiation factors that bind to RNA polymerase II [149]. In grow-
ing cartilage, type II collagen expression depends on the transcription factor, SOX-9,
but in adult tissue type II collagen expression may be less reliant on this factor [150].
Hydrostatic pressure increases transcription factor activation [151] and shear stress is
implicated in the translocation of the STAT proteins [152]. Stimulation of chondro-
cytes by proinflammatory mediators, such as IL-1, involves activation of nuclear factor
kappa B (NF-kappaB), NF-IL6 (CREB), AP-1 [153]. Similarly, MMP expression is
controlled by the transcription factors, AP-1, NF-kappaB, Sp1, and STAT [154]. The
extent to which specific levels of shear stress also act through nuclear protein activation
remains of interest and will be focus of one aspect of this proposal.
7. Summary
The available data from in vitro studies of cartilage explants and isolated chondrocytes
together with clinical experience leads one to a conclusion that at some critical level
shear stress is deleterious to decades-long preservation of articular cartilage. A range of
studies show that the cause is linked to activation of proinflammatory mediator release
from the articular chondrocytes. However, a full understanding of how the threshold
for this mechanical stimulus to incite articular cartilage degeneration is set remains
lacking. A challenge for the future is establish at a molecular level how the balance
between the two different types of mechanical stresses that arise in the cartilage can
preserve matrix stability over 8 to 9 decades in some individuals. Such knowledge will
contribute to three important long term goals. First, understanding the role of mechani-
cal loading on cartilage matrix production will contribute to fundamental information
to improve the success of cartilage repair by tissue engineering. Second, understanding
the precise role of mechanical loading on cartilage repair will advance surgical proce-
dures that could forestall the need for total joint arthroplasty. Third, the information
may contribute to the development of new classes of drugs to prevent progression of
early OA.
Acknowledgements
This work was supported by a Department of Veterans Affairs, RR&D Merit Review
Proposal A2128-RC and a Research Career Scientist Award (RLS).
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IOS Press, 2007
III
Aging, Inflammation, and Altered

Chondrocyte Differentation in Articular
Cartilage Calcification and Osteoarthritis
Robert A. TERKELTAUB, MD
Section Chief of Rheumatology Allergy-Immunology, VA Medical Center
Professor of Medicine, University of California San Diego Division of Rheumatology,
3350 La Jolla Village Drive, San Diego, CA 92161
Phone: 858-642-3519, Fax: 858-552-7425, E-mail: rterkeltaub@ucsd.edu
Abstract. Extracellular matrix calcification with calcium pyrophosphate dihydrate

(CPPD) and/or hydroxyapatite crystals commonly develops in osteoarthritic (OA)
cartilage. Primary forms of articular cartilage CPPD crystal deposition and less
commonly hydroxyapatite deposition can present as degenerative joint disease.
Moreover, CPPD and hydroxyapatite crystals can traffic from cartilage to syno-
vium and induce cytotoxic, catabolic, and inflammatory responses of chondrocytes
and synovial lining cells, and promote synovial proliferation. Such changes have
the potential to not only contribute to low-grade synovitis and inflammatory symp-
toms in OA but also to accelerate the progression of OA. In addition, CPPD and
hydroxyapatite crystals are commonly found in joints with advanced OA. How-
ever, it is not clear that CPPD and hydroxyapatite crystal deposition actually
worsen the course of primary OA. Instead, it appears that CPPD and hydroxyapa-
tite crystal deposition in OA articular cartilage reflect aging, inflammation, altered
IGF-I and TGFβ responsiveness, chondrocyte hypertrophic differentiation,
changes in the closely linked metabolism of ATP, PPi, and Pi, and possibly local
changes in PTHrP expression and systemic changes in PTH. As such, OA patho-
genesis richly informs us on mechanisms that drive articular cartilage calcification.
Conversely, the presence of cartilage calcification informs us about pathogenesis
and progression factors in subsets of affected subjects with OA.
Keywords. PTH, PTHrP, NPP1, ANKH, PPi, transglutaminase, chondrocyte hy-

pertrophy, chondrocalcinosis, TGFβ, IGF-I, Cartilage Intermediate Layer Protein
Introduction
Scope of the Problem of Joint Cartilage Calcification
Extracellular matrix calcification commonly develops in osteoarthritic (OA) cartilage.

CPPD and basic calcium phosphate (BCP) crystals (principally hydroxyapatite) are by
far the commonest forms of calcium-containing crystals in articular cartilages [1–4].
CPPD and hydroxyapatite crystals can traffic from cartilage to synovium and directly
turn on cells and activate complement to induce cytotoxic, catabolic, and inflammatory
32 R.A. Terkeltaub / Articular Cartilage Calcification and Osteoarthritis
responses of chondrocytes and synovial lining cells [5–7]. In addition, both CPPD and
HA crystal ingestion by synovial fibroblasts promotes proliferation, mediated partly by
calcium release from crystal dissolution as well as by mitogen activated protein kinase
(MAPK) signaling [5,6]. As such, CPPD and hydroxyapatite crystal deposition have
the potential to not only contribute to low-grade synovitis and inflammatory symptoms
such as pain and stiffness in OA but also to accelerate the progression of OA.
There is a particularly high frequency of positive knee joint synovial fluids for
CPPD and/or BCP crystals in aged patients with advanced degenerative arthritis at the
time of joint arthroplasty [1]. The presence of BCP crystals in affected joints correlates
directly with OA severity [1,2]. However, the apparent sources of joint fluid BCP crys-
tals in advanced OA include not only perichondrocytic crystal deposits (especially in
the superficial zone) but also bone shards embedded in cartilage and bony debris ex-
posed by cartilage erosion in established OA. Primary degenerative arthropathy due to
intra-articular BCP crystal deposition occurs [6]. However, secondary forms of BCP
crystal deposition arthropathy appears to be much more common, particularly in asso-
ciation with periarticular disease such as mechanical instability of the shoulder joint
due to chronic rotator cuff tear [8].
CPPD crystals are commonly found in meniscal fibrocartilage and less commonly
in hyaline articular cartilage of the knee in association with OA and aging [9]. De-
creased hydration and unknown distinctions in extracellular matrix composition may
account for preferential localization of CPPD crystals to meniscal fibrocartilage [9].
Monoclinic CPPD crystals have a greater inflammatory potential than triclinic CPPD
crystals [10], one of the likely reasons that clinical manifestations of CPPD deposition
are so variable. Cartilage CPPD crystal deposition can be asymptomatic or can mimic
OA, gout, rheumatoid arthritis, or neuropathic joint disease [11,12]. Cartilage degen-
erative changes in CPPD deposition disease can be observed in not only typical joints
affected by primary OA such as the knee but also in atypical sites for primary OA
joints such as the glenohumeral, wrist, and metacarpophalangeal joints [12]. Local dis-
turbances in PPi metabolism occur in OA cartilage, whereas heterogeneous but sys-
temic disturbance in PPi metabolism clinically manifested primarily in the joint may
underlie the distinct distribution of arthropathy in idiopathic/sporadic, familial, and
secondary metabolic disease-associated CPPD deposition [13,14].
CPPD Crystal Deposition and Prognosis in OA
The presence of CPPD crystals in primary knee OA was initially suggested to be a pre-
dictive factor for more frequent knee replacement surgery [15]. In addition, higher
mean radiographic scores correlated with the presence of calcium-containing crystals in
OA in a recent study of patients at the time of total joint arthroplasty [1]. However,
degenerative cartilage disease associated with sporadic CPPD crystal deposition dis-
ease may be less destructive than that observed in primary OA. For example, prospec-
tive analysis of CPPD deposition disease of the knee suggested that radiographic wors-
ening of degenerative arthritis was slow [16]. Typically, changes in radiographic extent
of chondrocalcinosis are observed over time [16]. There is no clear correlation between
the extent of calcification and progression of CPPD deposition arthropathy.
In the recent Boston OA Knee Study (BOKS) and in the Health, Aging and Body
Composition (Health ABC) Study [17], Neogi et al. prospectively evaluated the rela-
tionship between chondrocalcinosis and the progression of knee OA longitudinally
R.A. Terkeltaub / Articular Cartilage Calcification and Osteoarthritis 33
Figure 1. Mechanistic convergence of OA and cartilage matrix calcification. The figure schematizes funda-
mental disease processes within cartilage that ultimately promote both OA and matrix calcification, as re-
viewed in the text.
using MRI. In BOKS, knees with chondrocalcinosis had a decreased risk of cartilage
loss compared with knees without chondrocalcinosis and there was no difference in
risk in Health ABC. Stratification by the presence of intact or damaged knee menisci
produced comparable results within each cohort. In the setting of OA, the processes
leading to matrix calcification had been regarded to reflect passive secondary conse-
quences of advanced cartilage pathology. Moreover, joint inflammation induced by
deposited calcific crystals was thought to be determine the primary significance of
chondrocalcinosis for the progression of OA. The findings of Neogi et al. [17], and
recent advances in understanding the pathogeneses of OA and chondrocalcinosis sug-
gest quite different ways of looking at the significance of chondrocalcinosis in OA. For
example, the dysregulated cartilage matrix repair that generates cartilage calcification
may be more effective at slowing cartilage tissue failure than other forms of cartilage
repair in OA.
Below, I discuss the argument that cartilage matrix calcification and OA reflect the
convergence of heretogeneous processes that actively drive stereotypical patterns of
cartilage injury and repair (Fig. 1). Processes given particular attention in this review
are (i) certain low grade inflammatory alterations in cartilage that promote chondrocal-
cinosis by modulating chondrocyte maturation to hypertrophy, (ii) chondrocyte re-

sponses to PTH and PTHrP, (iii) dysregulated ATP and PPi metabolism in cartilage
aging, and (iv) imbalance in chondrocyte responses to the growth factors TGFβ and
IGF-I.
Altered Chondrocyte Differentiation and Matrix Calcification in OA:

Chondrocyte Hypertrophy
Unlike growth plate cartilage, articular cartilage is specialized to resist matrix calcifica-
tion. However, the physiologic regulated changes in chondrocyte differentiation and
viability characteristic of growth plate chondrocytes, including proliferation, hypertro-
phy, and apoptosis, as well as changes in mitochondrial function and Pi transport, can
be partially recapitulated in articular cartilage chondrocytes as a feature of the pathol-
ogy of OA [18–25]. The development of grossly enlarged chodrocytes that express
hypertrophic differentiation markers such as type X collagen, vascular endothelial
growth factor (VEGF), and the transglutaminases (TGs) TG2 and FXIIIA is well-
recognized in OA cartilage [18–21,26–28]. The orderly and sequential transitions of
growth plate chondrocytes from resting to proliferating, proliferating to prehypertro-
phic, and prehypertrophic to terminal hypertrophic differentiation are tightly regulated
by multiple mediators with opposing and, in some cases, direct mutually antagonistic
effects [29–32]. Figure 2 though far from a complete listing, illustrates major aligned
forces promoting and suppressing chondrocyte maturation to hypertrophy in the growth
plate. Some of these factors are already implicated in OA pathogenesis, and inflamma-
tory mediators such as S100A11, CXCL8, TNFα, and the role of RAGE and TG2, in
chondrocyte hypertrophy in OA cartilage receive intensive discussion below.
Chondrocyte hypertrophy is a differentiation state specialized for calcification, in
part by alteration of extracellular matrix composition, the enhanced release of matrix
vesicles to promote mineral seeding, increased generation of PPi and Pi, and increased
TG2 and FXIIIA expression (Fig. 3) [14,21,26,27,33]. VEGF release by hypertrophic
chondrocytes has the potential to promote synovial angiogenesis [19] and thereby con-
tribute to the synovitis that is observed to a variable degree in the OA joint. The in-
creased susceptibility of hypertrophic chondrocytes to apoptotic death also is partly
significant because of the pro-mineralizing effects of chondrocyte apoptosis [23,35,36].
Inflammatory Mediators in Extracellular Matrix Modification for Calcification
Alteration of the articular cartilage extracellular matrix by hypertrophic chondrocytes

that is a fundamental factor in preparing the matrix for calcification also has ramifica-
tions for the biomechanical and signaling properties of the matrix in OA and aging. For
example, stabilization of pericellular matrix proteins via transamidation-catalyzed
cross-linking by TG2 and FXIIIA may not only promote calcification [37] but also
stabilize the matrix. Collagen II is down-regulated, but collagen X, MMP-13 and
ADAMts5 are up-regulated in hypertrophic chondrocytes. Taken together, it is con-
ceivable that the dysregulated matrix repair response of hypertrophic chondrocytes in
OA may at least be superior as a tissue repair mechanism to that exerted by non-viable
chondrocytes.
Figure 2. Aligned forces promoting and suppressing chondrocyte maturation to hypertrophy in the growth
plate, sone of which are also active in OA cartilage. The Figure, though far from a complete listing, depicts
major aligned forces promoting and suppressing chondrocyte maturation to hypertrophy in the growth plate.
Some of these factors are already implicated in OA pathogenesis, as discussed in the text. Inflammatory
mediators such as S100A11, CXCL1, CXCL8, TNFα, and the role of RAGE and TG2, in chondrocyte hyper-
trophy in OA cartilage receive intensive discussion in the text.
Two distinct TGs, TG2 and Factor XIIIA are expressed in temporal and spatial as-
sociation with chondrocyte hypertrophy and matrix calcification in the growth plate
and are up-regulated in hypertrophic cells in the superficial and deep zones of knee OA
articular cartilage and the central (chondrocytic) zone of OA menisci [26,28,38]. IL-1β,
TNFα, the chemokines CXCL1 and CXCL8, nitric oxide (NO) and the potent oxidant
peroxynitrite induce increased chondrocyte TG catalytic activity [26,28,38]. OA sever-
ity-related, donor age-dependent, and particularly marked age-dependent IL-1-induced
increases in TG activity occur in chondrocytes from human knee menisci [28]. In-
creased Factor XIIIA and TG2 activities directly induce calcification by chondrocytes
[28]. TG2 also promotes activation from latency of TGFβ [39].
We recently discovered that inflammation-induced TG2 release from chondrocytes
appears to be a central amplification factor for both OA and cartilage matrix calcifica-
Figure 3. Functional implications of chondrocyte hypertrophy in calcification and the course of OA. The
Figure depicts that chondrocyte hypertrophy is a differentiation state specialized for calcification, in part by
alteration of extracellular matrix composition (with attendant dysregulation of matrix repair), as well as by
the enhanced release of matrix vesicles to promote mineral seeding, increased generation of PPi and Pi, and
increased TG2 and FXIIIA expression. Furthermore, VEGF release by hypertrophic chondrocytes has the
potential to promote angiogenesis in the synovium, and may thereby promote synovitis that is observed to a
variable degree in the OA joint. The increased susceptibility of hypertrophic chondrocytes to apoptotic death
also is partly significant because of the pro-mineralizing effects of chondrocyte apoptosis.
tion [26–28,38]. Specifically, we treated TG2 knockout mouse chondrocytes with

IL-1β, CXCL1, the all-trans form of retinoic acid (ATRA) (which promotes endo-
chondral chondrocyte hypertrophy and pathologic calcification), and with C-type natri-
uretic peptide (CNP) [26]. IL-1β and ATRA induced TG transamidation activity and
calcification in wild-type but not in TG2-/- mouse knee chondrocytes. In addition,
CXCL1 and ATRA induced multiple features of hypertrophic differentiation, and TG2
was required for these effects. TG2-/- chondrocytes lost the capacity for ATRA-induced
expression of Runx2 (cbfa1), a transcription factor necessary for ATRA-induced chon-
drocyte hypertrophy. In contrast, the essential physiologic growth plate chondrogenic
differentiation mediator CNP, which did not modulate TG activity, comparably pro-
moted Runx2 expression and hypertrophy in normal and TG2-deficient chondrocytes.
Thus, distinct TG2-independent and TG2-dependent mechanisms promote Runx2
expression, articular chondrocyte hypertrophy, and calcification. Up-regulated TG2
release alone is sufficient to promote chondrocyte hypertrophic differentiation, and
TG2 GTP binding, rather than transamidation-catalyzed crosslinking, is essential [27].
TG2 acts as a molecular switch to induce chondrocyte hypertrophy in a beta1 integrin-
mediated manner, associated with rapid phosphorylation of p38 kinase dependent on

TG2 being in the GTP-bound conformational state [27]. Though TG2 transamidation
activity is not required for TG2 to induce chondrocyte hypertrophy, TG2 transamida-
tion activity still likely plays a role in modulation of chondrocyte differentiation and
function. The capacity of TG2 to directly and rapidly drive chondrocyte hypertrophy
when applied to cartilage in organ culture is particularly compelling [27]. It suggests
that chondrocytes in OA articular cartilage could rapidly bypass the ordered physiol-
ogic progression from resting to proliferative through to hypertrophic differentiation
that spatially and temporally controls growth plate differentiation.
Mechanisms complementing the effects of TG2 on chondrocyte differentiation
driven by inflammation include expression of the Pi co-transporter Pit-1 and sodium-
dependent Pi uptake mediated by CXCR1 signaling essential for CXCL8 to induce
chondrocyte hypertrophy [25]. Of additional importance is the multiligand receptor for
advanced glycation end products (RAGE), which mediates several chronic vascular and
neurologic degenerative diseases accompanied by low-grade inflammation [40]. RAGE
ligands include S100/calgranulins, a class of small, calcium-binding polypeptides, sev-
eral of which are expressed by chondrocytes [40–42]. Normal human knee cartilages
demonstrate constitutive RAGE and S100A11 expression, and both RAGE and
S100A11 expression are up-regulated in OA cartilages [40,42]. CXCL8 and TNFα
induce S100A11 expression and release in cultured chondrocytes [40]. Moreover,
S100A11 induces chondrocyte hypertrophy in vitro [40] and the chondrocyte-expressed
calgranulin S100A4 stimulates MMP-13 expression [41]. CXCL1-induced and TNFα-
induced but not ATRA-induced chondrocyte hypertrophy are dependent on RAGE,
MAPK kinase 3, and p38 MAPK signaling [40]. Taken together, inflammation-
associated chondrocyte hypertrophy driven by several cytokines and calcgraulins, TG2,
Pi transport, and RAGE signaling can contribute to both chondrocalcinosis and pro-
gression of OA.
Role of PTH, PTHrP, and the Calcium Sensing Receptor (CaR)
PTHrP is a central regulator of spatial and temporal aspects of chondrocyte develop-

ment, as well as extent of matrix calcification, in endochondral development, and
PTHrP also modulates articular chondrocyte function [22,43]. In normal articular carti-
lage, PTH/PTHrP receptors are expressed by chondrocytes in all zones [22]. PTHrP
expression up-regulation in OA cartilage is robust through all zones, but PTH/PTHrP
receptor expression becomes limited principally to the superficial zone in OA [22].
Signaling via the PTH/PTHrP receptor stimulates chondrocyte proliferation, but PTHrP
also restrains the progression between prehypertrophic to hypertrophic differentiation
in chondrocytes. CPPD crystal deposition disease is some subjects (discussed below)
may be partly driven by excess PTH functioning to drive increased chondrocyte prolif-
eration and stimulating chondrocytes to enter an differentiation cascade like that of
endochondral maturation. However, hypercalcemia also likely plays a role, as CaR
expression is up-regulated in the Hartley guinea pig medial tibial plateau cartilage as
early spontaneous knee OA develops. CaR-mediated calcium sensing is known to drive
PTHrP release [44], and PTHrP content becomes significantly increased in the medial
tibial plateau cartilage as OA develops and progresses in this model. In cultured chon-
drocytes, CaR-mediated extracellular calcium-sensing, stimulated by the calcimimetic
NPS R-467, induces PTHrP and MMP-13 expression and suppresses expression of
tissue inhibitor of metalloproteinase (TIMP)-3, effects shared by elevated extracellular

calcium [44]. Moreover, extracellular calcium-sensing appears essential for PTHrP and
interleukin-1 to induce MMP-13 and for PTHrP to suppress TIMP-3 expression.
Potential Translational Relevance of PTH in Chondrocalcinosis
Though chondrocalcinosis is highly prevalent in elders in Western countries, there have

been scarce data on chondrocalcinosis prevalence in other racial or ethnic populations
that might provide clues to pathogenesis. Potential inhibitors of chondrocalcinosis
prevalence include high oral calcium intake that suppresses PTH production by the
parathyroid. There have been reports of “hard” drinking water in China mediated by
high calcium content, and primary hyperparathyroidism is rare in mainland China [45].
A recent study by Zhang et al. recruited a random sample of aged Beijing residents
aged > 60 years and assessed radiographic chondrocalcinosis, with comparisons made
to Whites in the American Framingham OA Study. In addition, identical methods were
employed to collect samples of tap water from Beijing and Framingham and measure
levels of calcium and magnesium. Chinese had a much lower prevalence of knee chon-
drocalcinosis, and wrist chondrocalcinosis was rare in Chinese elderly [46]. Calcium
levels in tap water in Beijing were 12–20 fold higher than that in Framingham, whereas
no difference was found in levels of magnesium (an inhibitor of CPPD crystal growth).
Despite the finding that radiographically detectable knee and wrist chondrocalcinosis
were far less common in older Chinese in Beijing than in White counterparts in Fram-
ingham, there is an excess of knee osteoarthritis in Beijing [47]. These findings suggest
the possibility that chondrocalcinosis is more of an environmentally mediated finding
than previously recognized. Given the current lack of effective, rational therapies to
prevent or lessen idiopathic CPPD crystal deposition, further study of the potential pro-
phylactic and therapeutic benefits of dietary calcium supplementation on chondrocalci-
nosis would be compelling.
Dysregulated Chondrocyte ATP Metabolism and PPi Generation in OA and

Chondrocalcinosis
NO is a central mediator of the pathogenesis of OA, and NO suppresses mitochondrial

respiration-mediated ATP generation [48] and stimulates apoptosis in chondrocytes
[49]. Treating chondrocytes with mitochondrial ATP synthesis inhibitors, and with
sodium nitroprusside (a donor of NO and the mitochondrial complex IV inhibitor cya-
nide) recapitulate several features of OA chondrocytes in vitro, including decreased
matrix synthesis and increased potential for calcification [48,50]. We observed steadily
increasing ATP depletion with aging in knee articular chondrocytes in the Hartley
guinea pig model of spontaneous OA, in association with up-regulation of ATP-
scavenging nucleotide pyrophophosphatase phosphodiesterase (NPP) activity and in-
creasing extracellular PPi, comparable to known changes in NPP and PPi in human OA
and aging joints [51]. Importantly, NPP1 (formerly known as PC-1) is a major genera-
tor of extracellular PPi in chondrocytes via ATP hydrolysis [52].
PPi potently suppresses hydroxyapatite crystal deposition and propagation, and
maintenance of a relatively high extracellular PPi concentrations by chondrocytes is a
vital physiologic mechanism to prevent articular cartilages from calcifying [53]. Para-
doxically, aging hyaline articular cartilage and meniscal fibrocartilages create excesses
of extracellular PPi in the extracellular matrix. A substantial fraction of extracellular
PPi, including that generated by NPP1 [54], appears to be transported from the cell in-
terior via the multiple-pass transmembrane protein ANK and its human homologue
ANKH, a known physiologic suppressor of cartilage calcification [55]. Chondrocyte
ANKH expression is robustly up-regulated in human knee OA cartilages [54]. More-
over, certain ANKH mutants are associated with autosomal dominant inherited degen-
erative arthropathy intimately linked with premature-onset CPPD crystal deposition
disease, and with autosomal recessive inherited late-onset CPPD crystal deposition
disease [reviewed in reference 56]. An ANKH 5'-untranslated region single nucleotide
polymorphism also has been linked to increased ANKH mRNA levels and idiopathic
chondocalcinosis of aging [57]. Excess extracellular PPi promotes chondrocyte hyper-
trophy [58], apoptosis [35], and MMP-13 expression [54] as well as CPPD crystal
deposition.
Dysregulated Responsiveness to the Chondrocyte Growth Factors TGFβ and

IGF-I and Cartilage Calcification
Levels of active TGFβ are increased in the OA joint. TGFβ markedly elevates ex-
tracellular PPi in chondrocytes [59], mediated partly by induction of NPP1 expression
and translocation to the plasma membrane [60]. The capacity of TGFβ to drive up ex-
tracellular PPi increases with cartilage aging, whereas TGFβ-induced chondrocyte
growth responses decrease [61]. Normally, IGF-I inhibits the capacity of TGFβ to raise
chondrocyte extracellular PPi [59]. Thus, decreased IGF-I responsiveness characteristic
of chondrocytes in OA may contribute to chronic elevation of PPi generation by carti-
lage. The pericellular and interterritorial matrix protein Cartilage Intermediate Layer
Protein-1 (CILP-1) is one of the inhibitors of IGF-I that is up-regulated in OA cartilage,
and CILP-1 promotes increased extracellular PPi in cultured chondrocytes [62].
Summary
This review has discussed fundamental mechanisms that converge to actively drive
both OA and matrix calcification (Fig. 1). However, it is noteworthy that calcification
is not a universally detected feature in joints with OA [1–5]. Conversely, cartilage crys-
tal deposition may occur without advanced cartilage degeneration, particularly in idio-
pathic CPPD deposition disease of aging. Mechanistic convergence and divergence of
OA and cartilage matrix calcification may inform as to predominant operative patho-
genic pathways. As discussed above, sustained chondrocyte hypertrophy without pro-
gression to apoptosis would be expected to drive both cartilage reparative and matrix
calcification responses, and favor CPPD deposition over further progression of OA. In
contrast, robust MMP and aggrecanase activation without substantial chondrocyte hy-
pertrophy and derangements in ATP and PPi metabolism would be predicted to favor
development of OA without matrix calcification.
BCP crystal deposition in cartilage is not easy to detect by standard clinical imag-
ing and synovial fluid analysis tools. Moreover, conventional radiography and synovial
fluid analyses have limits in sensitivities to detect CPPD crystal deposition. It is possi-
ble that more systematic evaluation of joints for evidence and forms of cartilage calci-
fication, done prior to end-stage disease, can provide information to weigh operative
pathogenic factors, the phenotype of cartilage repair, and prognosis in degenerative
arthritis. Similarly, clinical measurements of one or more of the intra-articular media-
tors discussed here (such as TG2, S100A11, ANKH, PTHrP, PPi, NPP1, and CILP-1)
might provide useful data for these purposes. How practical and cost-effective such
measures are may impact on development of rationally targeted and more effective
early treatment strategies for both OA and chondrocalcinosis.
Acknowledgements
Dr. Terkeltaub’s research on OA is supported by the Department of Veterans Affairs

and NIH.
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IOS Press, 2007
IV
Leptin, the Prototype of Adipokines:

Molecules at the Crossroads
of Inflammation and Metabolism
Rodolfo GÓMEZ a, Rocío LAGO a, Francisca LAGO b, Juan J. GÓMEZ-REINO a,
Miguel OTERO a and Oreste GUALILLO a,∗
a
Santiago University Clinical Hospital, Research Laboratory 4 (NEIRID LAB,
Laboratory of Neuro Endocrine Interactions in Rheumatology and Inflammatory
Diseases), Santiago de Compostela, Spain
b
Santiago University Clinical Hospital, Research Laboratory 1 (Molecular and
Cellular Cardiology), Santiago de Compostela, Spain
Abstract. The prevalence of obesity and obesity-related diseases focussed an in-

creasing interest, over the last 10 years, on white adipose tissue and its derived
bioactive peptides, being the discovery of leptin in 1994 the trigger of the renais-
sance of the studies about adipose tissue.
Leptin was initially depicted as the most important anorexigenic factor with
neuroendocrine actions, but it has been later shown to significantly modulate im-
mune and inflammatory processes. Leptin is a dual molecule: apart from its previ-
ously envisaged metabolic activities, increasing evidence frames leptin as a novel
pro-inflammatory adipokine and, at present it might be easily considered one of
the relevant links among immune system, inflammatory response and neuroendo-
crine system. Leptin regulates and participates both in immune homeostasis and
inflammatory processes by acting as a modulator of cell activity and playing an ac-
tive role in articular degenerative inflammatory diseases such as osteoarthritis and
rheumatoid arthritis, but also in a host of autoimmune inflammatory conditions
such as encephalomyelitis, type-1 diabetes, and bowel inflammation. This review
will be focussed more on the adipokine facet of leptin, even though its role as
metabolic hormone will be also addressed. In addition, the role od other relevant
adipokines in inflammation will be covered.
White Adipose Tissue: A Surprising Biochemical Factory
Historically, white adipose tissue (WAT) has been viewed as a passive depository of
energy and as a protective mechanism for heat loss. For the time being, this vision is
still proper but incomplete, and rather insufficient to understand the actual complex
functions of WAT. Adipocytes are able to produce and secrete a wide number of mole-
∗
Corresponding Author: Dr. Oreste Gualillo, Santiago University Clinical Hospital, Research Laboratory 4
(NEIRID LAB, Laboratory of Neuro Endocrine Interactions in Rheumatology and Inflammatory Diseases).
Calle Choupana s/n, 15706, Santiago de Compostela, Spain, Phone & Fax: 00+34+981+950905,
E-mail: gualillo@usc.es, oreste.gualillo@sergas.es.
44 R. Gómez et al. / Leptin, the Prototype of Adipokines
cules, including classical cytokines such as IL-1, IL-6 and TNF-alpha but also novel
factors such as adiponectin, resistin, visfatin, vaspin, apelin or leptin among others, the
so-called adipokines [1–3]. Thus, WAT is now considered as a true endocrine organ,
and probably the largest endocrine organ in the body, whose main actions include regu-
lation of energy homeostasis, metabolism, and immune and inflammatory processes
regulation [1–5]. There is a general consensus about the significant contribution of ex-
cessive fat accumulation and the so called “low grade inflammatory state”. This cluster
of dysfunctions is characterized prevalently by dyslipidemia, insulin resistance, altera-
tion of coagulation cascade and it is associated with increased risk of type 2 diabetes,
cardiovascular complications and autoimmune inflammatory diseases. Several con-
cerns support the basis of obesity as a pro-inflammatory condition; indeed, most of the
pro-inflammatory cytokines, as well as acute phase molecules, are elevated in obese
subjects. For completeness, it is dutiful to mention that WAT in obesity is able to syn-
thesize also anti-inflammatory factors such as IL1-RA, may be as a sort of adaptive
response. At any rate, it is reasonable to hipothesize that in obesity the balance between
pro-and anti-inflammatory factors is severly altered or at least compromised and that
white adipose tissue plays a lead role in the synthesis and secretion of several media-
tors of inflammatory response which, through a bidirectional way, are involved in the
perpetuation of obesity itself and contribute to the mechanisms responsible for the de-
velopment of the chronic disease associated with obesity.
Leptin and Its Receptors
The key event that marked the new glance of WAT as relevant endocrine tissue was the
identification of leptin by Zhang et al in 1994 [6]. Leptin is a 16 kDa peptide, encoded
by the ob gene and it is mainly produced by adipocytes [6,7], although other organs
produce leptin in significant amounts. Leptin expression is prevalently regulated by
R. Gómez et al. / Leptin, the Prototype of Adipokines 45
food intake [8], hormones, and cytokines. Leptin levels are directly correlated with
insulin [9,10] and negatively correlated with glucocorticoid levels [11,12].
Inflammatory mediators, such as interleukin (IL)-1, tumour necrosis factor (TNF)-
alpha or leukemia inhibitory factor (LIF), increase leptin synthesis [13–16]. Further-
more, it has been demonstrated a gender-related leptin regulation, based on the obser-
vation that testicular steroids inhibits leptin expression [17] whereas ovarian sex ster-
oids, as well as prolactin, increase it [18,19]. Leptin exerts its main action at a central
hypothalamic level by increasing energy expenditure and decreasing food intake [8,20].
Leptin has been involved as a modulatory agent in a variety of physiological processes
such as the regulation of hypothalamic-pituitary-adrenal axis [21–23], maturation of
reproductive system [24–26], hematopoiesis [27] and foetal development [28,29].
Leptin exerts its actions by binding to its specific receptors. Encoded by the diabe-
tes (db) gene, the Ob-R mRNA generates by alternative splicing six different receptor
isoforms, but only the long-functional isoform, Ob-Rb, is the functional.
Ob-R belongs to the class I cytokine receptors super-family, which typically con-
tains a cytokine receptor homologous domain in the extra-cellular region and includes
receptors for IL-6, LIF or gp 130 [30–32]. Leptin receptor long form (Ob-Rb) trans-
duces its signal trough a classic JAK/STAT pathway. Furthermore, Ob-Rb is able to
transduce signals by using alternative signalling pathways which involves the
SHC/GRB2 pathway as well the IRS-2, PI-3 kinase, MAPK, AMPK pathway and
Erk-1/2 SHP-2-dependent activation [33]. It is noteworthy to mention that structural
integrity of long form receptor is essential for mediating biological leptin response.
Actually, mutations of the STAT binding site of the receptor disrupt the signal trasduc-
tion pathway resulting in a phenotype characterised by impaired thermoregulation, obe-
sity and hyperphagia. In addition, the integrity of several tyrosine domains residues are
critical for leptin-mediated homeostatic action, but are not for mediating the permissive
role of leptin in reproductive function.
Leptin and Immunity
Leptin and its cognate receptor roles in immune-regulation were somehow enlightened
by the discovering of thymus atrophy in db/db mice. Since that observation, many re-
sults clearly pointed out a wide range of direct leptin’s effects on immune responses
[2,34,35–37].
On innate immunity, leptin induces activation of monocytes and increases phago-
cytosis by macrophages [38–40]. In rodent macrophages, elevated doses of leptin up-
regulates LPS induced-production of different pro-inflammatory cytokines up-
regulating inflammatory immune responses [41]. Furthermore, leptin is able to directly
modulate the activity and function of neutrophils acting via its receptors activation
[38,42,43], or by an indirect way mediated by monocyte- produced TNF in humans
[44]. On natural killer (NK) cells, leptin plays an important role in its development,
activation and function and it also increases its cytotoxic ability in a dose-dependent
way [45–47]. Consistent with these observations, leptin deficiency increases suscepti-
bility to infectious and inflammatory stimuli and it is linked with a marked dysregula-
tion of cytokine production.
Regarding leptin actions on adaptive immune responses, it has been proved that
leptin induces T-cell activation and proliferation, and protects T lymphocytes from
induced-apoptosis [48]; furthermore, leptin is able to polarize T-cell differentiation
towards a TH1 response [48], affecting the pattern of cytokines production [36].
Leptin and Endothelial Function
Emerging evidences link leptin to cardiovascular disease as an important mediator of

inflammatory response at endothelial levels. Indeed, in endothelial cells leptin induces
oxidative stress by promoting accumulation of reactive oxigen species and activating
endothelial nitric oxide synthase, up-regulates endothelin-1 production, potentiates
platelet aggregation, angiogenesis, adhesion molecules expression and expression of
monocyte chemo-attractant proteins [49–51].
Leptin: A Novel Pro-Inflammatory Adipokine
Several studies demonstrated that numerous inflammatory stimuli modulate both leptin
gene expression and circulating leptin levels [13–16]. Serum leptin levels are strongly
increased in experimental models of acute inflammation [15], whereas this increase in
plasma leptin is not always observed in humans [52]. Modulation of leptin levels dur-
ing acute inflammatory stimuli suggests that this adipokine is participating in the de-
velopment of inflammatory processes. This hypothesis has been tested and was con-
firmed by many observations in different autoimmune-inflammatory models, in which
leptin has been postulated to play a relevant role. It was observed that leptin-deficient
ob/ob mice were resistant, or at least, less susceptible to the development of different
inflammatory diseases. Thus, ob/ob mice were resistant to antigen induced arthritis
[53], experimental hepatitis [37,45], colitis [54] or autoimmune encephalomyelitis [55].
In the above mentioned experimental models, leptin deficiency caused a marked reduc-
tion in the severity of inflammation, whereas exogenous leptin administration restored
the normal secretion pattern of many of the inflammatory modulators and also restored
disease susceptibility, making it comparable to wild-type mice.
Taken together, all these data suggest that leptin plays a main role in the develop-
ment of the inflammatory response by acting in a pro-inflammatory fashion in a similar
way to that observed with other classical and well recognized pro-inflammatory cyto-
kines.
Leptin: A Disrupter for Articular Cartilage
The normal joint is a specialized structure consisting on multiple connective tissue

elements organized in a manner that permits stability and movement of the skeleton.
Among the multiple connective tissues integrated in normal joint, the articular cartilage
is, probably, the most affected during rheumatic diseases.
Essentially, this connective tissue is composed by an extracellular matrix and only
one cell type, the chondrocyte, which is the responsible of its synthesis and homeosta-
sis [56–59]. In a normal situation, chondrocytes are in a delicate balance between ma-
trix synthesis and destruction. Under inflammatory conditions, this balance becomes
altered and matrix destruction overcomes synthesis, resulting in a complete joint carti-
lage loss of structure [56–58].
It was found that chondrocytes expressed Ob-Rb, the functional leptin receptor iso-
form [59]. So, it is conceivable that leptin could be acting on joint cartilage through
these cells.
Obesity is a well known risk factor for knee OA. Indeed, the prevalence of knee
OA is increased in obese subjects, and conversely, weight reduction decreases this risk.
Generally, the influence of obesity to cartilage degradation has been related to an ab-
normal biomechanical loading. However, it has also been reported that obesity is re-
lated to the development of non-weight bearing hip and hand OA [60,61].
So that, it is reasonably conceivable that non mechanical factors but rather obesity-
associated proteic factors such as leptin, as well as other adipokines, are likely involved
in the development of articular degenerative inflammatory diseases.
Two recent studies demonstrated a clear detrimental effect of leptin on articular
cartilage. Namely, it has been demonstrated that leptin induces, in a synergistic way
with interferon-gamma (IFN-gamma) and interleukin-1 (IL-1), nitric oxide (NO) pro-
duction via nitric oxide synthase (NOS) type II in chondrocytes [62,63]. In both cases,
NOS II synergistic induction is signalled through a common transduction pathway
which involves several kinases including Jak-2, PI-3K, MEK-1 and p38K.
Nitric oxide has been shown to have a negative effect on chondrocytes physiology
and, thereby, over cartilage structures. Indeed, it has been demonstrated that NO in-
creases chondrocyte apoptosis, leads to phenotype loss and induces matrix metallopro-
teases synthesis [64] by causing complete cartilage degradation.
In addition, it has been shown that normal chondrocytes synthesizes leptin and that
leptin synthesis is increased in chondrocytes from osteoarthritis patients [65]. Further-
more, it has been demonstrated that leptin expression is increased in articular rat joints
injected with exogenous leptin [65], which implies a positive feedback regulation. So,
in terms of expression, leptin mimics classic cytokine behaviour under inflammatory
conditions. It has also been found that circulating leptin flows from blood to synovial
fluid during rheumatoid arthritis [66], as many other classic cytokines do.
Furthermore, it has been recently shown that circulating leptin levels, as well as
other plasma levels of other adipokines such as adiponectin and visfatin, are signifi-
cantly increased in patients affected with RA, independently from the amount of white
adipose tissue [67] and positively correlates with the C-reactive protein levels.
Adiponectin
Adiponectin was discovered more or less in the same period of leptin, but this adi-
pokine did not receive initially the same acclaim of leptin; really, its relevance as
a potential protective adipokine in obesity and obesity-related disorders was acknowl-
edged only few years later. Adiponectin, also called gelatin binding protein-28 (GBP-
28), adipose most abundant gene transcript 1(apM1), adipocyte complement related
protein (Acrp) 30 or AdipoQ, is a 244 aa adipose tissue specific protein that has struc-
tural homology to collagen VIII and X and complement factor C1q. Adiponectin circu-
lates in the blood in large amounts and constitutes about the 0.01% of total plasma pro-
teins. Adiponectin is present in serum as oligomeric isoforms constituted prevalently
by trimers, hexamers but also by high molecular weight isoforms (12–18 mer) [68].
Adiponectin exerts its biological action by mean of two recently described receptors
which are expressed prevalently in liver (Receptor 2) and skeletal muscle (Receptor 1)
and transduce signals by the activation of AMPkinase, PPAR-alpha and presumably
some other unknown signalling pathways leading to an increase of fatty acid oxidation
and reducing liver glucose synthesis [69]. Most of the biologic actions mediated by
adiponectin receptors involve the activation of AMP kinase as early step of intracellu-
lar signal transduction upon receptor activation, in spite of of a classic G-protein cou-
pling and cAMP induction, a quite unusual feature for a 7-transmembrane domain re-
ceptor. AMP kinase activation, induced by adiponectin, stimulates phosphorylation of
acetyl coenzyme A carboxylase, glucose uptake and fatty acid oxidation in miocytes,
whereas in liver induces a clear reduction of gluconoegenesis by limiting the synthesis
of specific enzymes.
Adiponectin and Inflammation
Adiponectin has been described as a potent antiatherogenic factor and a plethora of

actions have been described at endothelial and vascular level [70]. Indeed, adiponectin
inhibits monocytes adhesion to endothelial cells, reduces the synthesis of adhesion
molecules and tumor necrosis factor as well as decreases nuclear factor k beta levels
[71]. Adiponectin expression is inhibited by pro-inflammatory cytokines such as IL-6
[72] and TNF-alpha in cultured adipocytes [73].
There is a general consensus about a putative protective role of adiponectin from
inflammatory state, at least at endothelial-vascular level. Indeed, circulating adi-
ponectin levels are inversely proportional to obesity and therefore tend to be low in
morbid obese subjects. On the contrary, adiponectin levels increase with weight loss
and with use of insulin sensitizing drugs [74]. Low levels of adiponectin have been
linked to inflammatory atherosclerosis in humans [75]. In addition, animal models have
shown that low adiponectin levels increase smooth cell proliferation in response to in-
jury, increase free fatty acid levels and cause insulin resistance [76]. In addition, the
pro-diabetic and pro-atherogenic effects of low adiponectin levels, seen in the meta-
bolic syndrome, provide a clear link between inflammation and obesity. However, a
recent report by Ehling et al, in contrast to the previously envisaged adiponectin’s pro-
tective role in endocrinological and vascular diseases, suggests that adiponectin is in-
volved in key pathways of inflammation and matrix degradation in the human joint.
The effects of adiponectin in human synovial fibroblasts appear to be highly selective
by inducing two of the main mediators of rheumatoid arthritis pathophysiology, IL-6
and matrix metalloproteinase-1, via a p38 MAPK pathway [77]. More recently, it has
been proposed that cartilage is also a target tissue for adiponectin. Indeed, chondro-
cytes express functional adiponectin receptors whose activation lead to the induction of
nitric oxide synthase type II by a signalling pathway that involve PI3 Kinase (unpub-
lished data from our group). Moreover, adiponectin-challenged chondrocytes are able
to increase IL6, TNF-α and MCP-1 synthesis whereas, intriguingly, were unable to
modify prostaglandin E2 and leukotriene B4 release. Taken together, all these recent
results bind more closely the interactions between adiponectin and articular inflamma-
tory diseases, and suggest that adiponectin is a novel key element in the maintenance of
cartilage homeostasis which might be considered as a potential therapeutical target in
joint degenerative diseases. It is noteworthy, and somewhat unexpected, that plasma
adiponectin in patients with rheumatoid arthritis are higher than those observed in
healthy controls [67], the reasons for which are not evident, although increased levels
of adiponectin are observed in the synovial fluid of these patients compared with those
seen in patients with osteoarthritis [78]. The increased levels of adiponectin, in patients
with rheumatoid arthritis, suggest a compensatory mechanism under catabolic or ana-
bolic imbalance. So, it is conceivable that an increase in adiponectin level represents an
attempt to antagonise the anorexigenic and well-known pro-inflammatory effect of
leptin, suggesting that these two adipokines may act in parallel as opposing metabolic
counterparts.
Resistin
Resistin is a dimeric protein that received its name from the initial observation that it
induced insulin resistance in mice. Resistin belongs to the FIZZ family (found in in-
flammatory in zone) also known as RELMs (Resistin like molecules). The first identi-
fied protein of the family was FIZZ1 (also known as RELM-alpha), a protein that is up
regulated in the asthmatic lung in bronchoalveolar fluid of mice with experimentally-
induced asthma [79]. The other homologue FIZZ2 (also known as RELM-beta) was
next identified in the proliferating epithelia of intestinal crypt [80]. Finally, the third
homologue, FIZZ3 (also known as resistin) was later identified in adipocytes as well as
in other cell types such as macrophages. To date, many aspects of resistin biology re-
main controversial and studies in humans only demonstrate a weak relationship be-
tween obesity and diabetes, so its role as a mediator of insulin resistance is at present
questionable.
Resistin and Inflammation
Emerging concepts suggest a role of resistin in inflammatory conditions in humans

since a robust expression of resistin is present in monocytes. Some pro-inflammatory
cytokines such as TNF-alpha, IL-6 and lipopolysaccharide are able to regulate resistin
gene expression. It is noteworthy to stress that resistin regulation in response to pro-
inflammatory stimuli is tissue-dependent. Recent studies have shown the modulation of
pro-inflammatory cytokines synthesis by this molecule. Resistin is able to upregulated
IL-6 and TNF-alpha in blood mononuclear cells via NF-kB pathway [81]. Conversely,
LPS was reported to induce resistin gene expression in primary human and murine
macrophage via a cascade involving the secretion of pro-inflammatory cytokines [82].
Finally, resistin has been proposed to be involved in the pathogenesis of rheumatoid
arthritis in humans. In a rodent experimental model, local injection of resistin is able to
induce an arthritis-like syndrom. Indeed, following resistin local joint administration,
mice showed leukocyte infiltration of synovial tissues which were associated with hy-
pertrophy of synovial layer and pannus formation [81]. Resistin has been found in the
plasma and the synovial fluid of RA patients and in some studies, synovial fluids from
RA patients showed higher levels of resistin compared with the serum compartment
[83,84]. These findings provide evidence for a specific local dysregulation of adipoki-
nes in the joint space and suggest that circulating levels of adipokines do not represent
the situation in the joint. Anyway, the high synovial fluid levels of certain adipokines
compared to serum may be due to the increased permeability of inflamed synovial
membrane. However, plasma resistin levels were not different between RA patients and
healthy controls [67,81]. So, the role of resistin is merely apparent, but the precise
mechanism of regulation at joint levels needs to be analyzed in depth.
Visfatin
Visfatin is an insulin mimetic novel adipokine which was discovered by Fukuhara et al,
[85] using a differential display technique to identify genes specifically expressed in
abdominal fat. Visfatin was found to be identical to PBEF (pre-B colony enhancing
factor), a growth factor for B lymphocytes precursors previously known to be synthe-
sized in liver, skeletal muscle and bone marrow and to be up-modulated in models of
acute lung injury and sepsis. Visfatin circulating levels strongly correlates with WAT
accumulation and its mRNA expression is dependent from adipocyte differentiation. It
is noteworthy that in obesity circulating visfatin levels increases during the develop-
ment of obesity. Visfatin synthesis is regulated by several factors including glucocorti-
coids, TNF-alpha, IL-6. Visfatin is produced also by endotoxin-challenged neutrophils
and inhibits neutrophils apoptosis through a mechanism mediated by caspase 3 and 8
[86]. In humans, visfatin levels correlates with BMI but not with visceral fat mass or
waist-to-hip ratio [87]. In addition, visfatin levels in type II diabetes subjects are higher
than normoglycemic counterpart [88]. Interestingly, circulating visfatin is also higher
in patients with rheumatoid arthritis than in healthy controls [67]. It is currently unclear
what would be visfatin physiological role or relevance in the context of rheumatoid
arthritis. Visfatin might be part of a compensatory mechanism that facilitates the accu-
mulation of fat in the intra-abdominal depot, a feedback mechanism preventing the
deleterious effects of the rheumatoid cachexia. Moreover, at present it cannot rule out
that an increase in visfatin levels may be related to the modulation of inflammatory or
immune response or may simply be an epiphenomenon.
Conclusion
Obesity and the associated metabolic diseases are the most common and detrimental
illness affecting more than 50% of the adult western population. These conditions are
associated with a chronic inflammatory response characterized by abnormal cytokine
production, increased acute-phase reactants, and activation of inflammatory signaling
pathways [68]. This association is not inconsequential and is constrained to either obe-
sity itself or closely linked diseases such as insulin resistance, type 2 diabetes, and car-
diovascular disease. A very intriguing characteristic of the inflammatory response that
emerges in the presence of obesity is that it appears to be triggered, and to reside pre-
dominantly, in adipose tissue, although other metabolically critical sites may also be
involved during the course of the disease. Leptin, as the prototype of white adipose
tissue-produced adipokine, is much more than the initially envisaged anti-obese hor-
mone. Actually, besides its classical role as metabolic hormone, leptin shares many
common points, including structural and functional features, with most of the classical
pro-inflammatory cytokines. Anyhow, leptin might be considered as a promising thera-
peutic target in some pathology where this adipokine is thought to promote inflamma-
tory diseases. For instance, soluble receptors, which control the amount of bioavailable
leptin, could be used to counteract pro-inflammatory leptin’s action. A similar thera-
peutic strategy is at present used to antagonize the effect of TNF-alpha in RA. Other
potential therapeutic intervention can be achieved by the use of monoclonal humanized
antibodies or by leptin mutants with receptor blocker properties, which might be able to
bind OB receptor but not to activate it.
In conclusion, there are increasing evidences that argue for a role of leptin and
other adipokines in the pathogenesis of inflammatory disease, supporting the notions
that a pharmacological strategy based on the modulation of adipokines synthesis or
action could have attractive therapeutic advantages.
Acknowledgements
Some of the research described in this review has been supported by Spanish Ministry
of Health, Fondo de Investigación Sanitaria, Instituto de Salud Carlos III (PI 05/0525,
PI030115, PI050419 and G03/152), and Xunta de Galicia. Oreste Gualillo and
Francisca Lago are recipient of a contract under the “Programme of stabilization of
researchers” co-funded by the Spanish Ministry of Health/Instituto de Salud Carlos III
and Xunta de Galicia/SERGAS. Miguel Otero is a recipient of a post-graduate fellow-
ship funded by Fundación Caixa Galicia. Rocío Lago (FI05/01019) and Rodolfo
Gómez (PI05/0525) are recipients of pre-doctoral fellowships funded by Instituto de
Salud Carlos III. It is reasonable that not all published data were discussed in this re-
view. So, we really apologize to those whose works were not mentioned.
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IOS Press, 2007
The Role of Extracellular Matrix

Fragments in the Autocrine Regulation
of Cartilage Metabolism
Gene A. HOMANDBERG, Lei DING and Danping GUO
Department of Biochemistry and Molecular Biology, The University of North
Dakota School of Medicine and Health Sciences, Grand Forks, ND 58203
Abstract. The ability of degradation products of the extracellular matrix (ECM) to

regulate cartilage homeostasis has now been well documented. There are now nu-
merous observations that different types of products derived from the damaged
matrix can provide additional signals that can amplify catabolic processes that
serve either to clear tissue components for repair or to initiate reparative signals.
These fragments include fibronectin fragments (Fn-f), collagen fragments (Col-f)
and hyaluronan fragments (HA-f) and likely link protein fragments (LP-f). Active
fragments of other ECM components may be found in the future. ECM fragments
can arise during cartilage degeneration with enhanced levels of proteinases and
normal rates of matrix synthesis. Ironically and theoretically, fragments might also
arise from enhanced synthesis of their native precursors but only with basal levels
of proteinases and this might lead to enhanced proteinases. Further, certain types
of fragments might arise from synovial tissue. The linkage between catabolic and
anabolic pathways in cartilage is amply illustrated by the properties of Fn-fs in that
the damage pathways initiated by Fn-fs also initiate anabolic pathways of at-
tempted repair. Observations with Fn-fs show that lower concentrations that initi-
ate the lowest levels of matrix metalloproteinases (MMPs) can initiate anabolic
processes while higher concentrations also enhance catabolic protease driven
pathways that swamp out the anabolic pathway. Anabolism might be enhanced
through post-translational events such as proteolytic activation of ECM bound
growth factors although other explanations are possible. Thus, fragment systems
may be operative not only during damage, but also during normal metabolism and
in either case, may shift metabolism in either direction, depending on the concen-
tration of the fragments. Regulation of the fragment pathways may be through na-
tive ligands, since the ECM fragments are likely inhibitors of the native ligands
and vice versa. These ECM fragment pathways may define a global pathway in
which: (1) one type of fragment, such as a Fn-f, can bind either Fn or type II col-
lage and affect not only Fn integrins but also collagen integrins and (2) one type of
fragment may bind one type of integrin proximal to another type and affect in-
tegrin complexes or clusters. The signaling pathway of Fn-fs suggest that they bind
to receptors and disrupt receptor clusters and this may allow internalization of re-
ceptors and initiate new pathways involving MAP kinases, Nf-kB activation and
ultimately cytokine and MMP upregulation. It will be important to continue to
compare the Fn-f, Col-f and HA-f pathways to determine if there is a single global
mechanism that might be subject to therapeutic intervention. There are still some
basic questions that need to be addressed such as whether these fragments initiate
cartilage degeneration or simply amplify ongoing processes or where they are po-
sitioned in the early stages of i.e. osteoarthritis and whether or not partial vs com-
G.A. Homandberg et al. / The Role of Extracellular Matrix Fragments 57
plete inhibition of these pathways would be beneficial in a degradative state. More

information of the mechanisms is needed especially far upstream at the level of
membrane receptors, where re-distribution of integrins may be the key initiating
event in the pathway.
Keywords. Extracellular matrix, fibronectin fragments, collagen fragments, hyalu-

ronan fragments, integrins
Introduction
The ability of degradation products of the extracellular matrix (ECM) to regulate carti-
lage homeostasis is an ideal means of regulation of the health of cartilage tissue, since
the damaged ECM, because of its upstream position, could provide direct signals to
communicate with chondrocytes and initiate pathways required for tissue maintenance.
Some observations of ECM fragment pathways suggest that the ECM fragments should
not be considered only catabolic but may be able to regulate the balance between ca-
tabolism and anabolism, based on the relative levels of the fragments. This should not
be surprising because tissue damage is known to initiate reparative pathways. This re-
view will attempt to compare and consolidate observations of matrix fragment systems,
including fibronectin fragments (Fn-fs), type II collagen fragments (Col-f), hyaluronan
fragments (HA-f) and link protein fragments (LP-f) in order to address their physiol-
ogic relevance, their potential common mechanism of action, if any, and to conclude
with an understanding of their central role. More recent reviews on Fn-fs are [1,2] and
for HA-fs [3,4].
ECM Fragments and Their Precursors Are Elevated in Synovial Fluids and
Cartilage in RA and OA
The cartilage matrix consists of various macromolecules with the ability to contribute
degradation products that could theoretically perturb the ECM. These include fi-
bronectin (Fn), type II collagen, various types of proteoglycan, hyaluronan (HA), link
protein, COMP and minor amounts of other types of ECM ligands. Naturally, these
components are degraded through normal turnover, and thus, a certain level of frag-
mentation would be expected under normal conditions, but in cartilage degeneration the
level of fragmentation likely increases for all. The increases might arise either from
basal levels of native ligand with a background of enhanced proteinases in a frank cata-
bolic state or ironically, from elevated levels of native ligand against a background of
basal levels of proteinases that might occur in an anabolic repair response or from both.
The latter situation suggests that ECM fragments might also represent a secondary ef-
fect of attempted repair responses as the tissue responds to mild insults. Thus, it is con-
ceivable, that if the repair response is not tightly regulated, full flown catabolic path-
ways may result.
Fn and Fn-fs Are Elevated in OA
While levels of some proteins decrease in OA, the precursor of Fn-fs, native Fn, is ele-
vated in the cartilage matrix in human OA cartilage [5–7] as well as in canine [8,9] and
58 G.A. Homandberg et al. / The Role of Extracellular Matrix Fragments
rabbit [9] in vivo models of OA damage. This increase in Fn may be due to both en-
hanced synthesis and enhanced retention of Fn as shown by Burton-Wurster and Lust
[7]. How long Fn levels remain elevated during the course of the disease is not known.
It is likely that at some point, Fn synthesis may decrease due to metabolic overload on
chondrocytes. Fn levels also increase in synovial fluids in patients with OA and rheu-
matoid arthritis [RA] [10]. The increase may be up to 3 to 4 fold; the average concen-
tration of Fn in healthy donors increases from 171 μg/ml to 721 and to 568 in RA and
OA synovial fluids, respectively [11].
Since initiation of OA bears a strong biomechanical component, it is important to
point out that some of the enhanced Fn in OA which likely gives rise to elevated Fn-fs,
might be due to altered biomechanical forces. For example, Fn synthesis is increased in
canine articular cartilage explants after cyclic impact [12] and in bovine articular carti-
lage explants after intermittent cyclic loading [13]. Compressive loading and unloading
have also been shown to affect Fn synthesis [14]. It is also conceivable that enhanced
Fn synthesis may also occur indirectly through the effects of altered biomechanical
forces on activation and liberation of growth factors stored in cartilage tissue.
With the increases in Fn levels in cartilage and synovial fluids in RA and OA, it
would be expected that Fn-f levels would increase also. Griffiths et al. [15] found Fn-fs
from 24-kDa to 200-kDa in RA, OA, traumatic arthritis and septic arthritis synovial
fluids. These Fn-fs represented a major portion of the total Fn in most cases. Elevated
Fn-fs in RA synovial fluids has also been demonstrated [16]. We reported that over half
of the total Fn in OA synovial fluids was degraded, resulting in μM concentrations of
Fn-fs which ranged in mass from 30-kDa to about 200-kDa [17]. We later confirmed
the presence of several amino-terminal Fn-fs of 30 to 230-kDa in extracts of human OA
cartilage using an amino-terminal specific antibody [18]. Very recently, others have
confirmed our results [19]. It is important to emphasize here that Fn-fs have been re-
ported in many diverse pathologic body fluids and tissues as well and may also play
roles in tissue damage/repair in other tissues [20–27].
The Fn-fs found in synovial fluid might be derived from cartilage, synovium and
plasma Fn. Some indication has been provided by studies of differentially spliced Fn
isoforms that have been found to be relatively tissue specific. However, it appears that
synovial fluid contains more than one form. The isoforms in OA synovial fluids include
the synovial fluid Fn isoform, the ED-a[+] isoform [28] which is present at only low
levels in cartilage of OA patients [29]. In contrast, the population synthesized within
cartilage tissue is significantly different than in other tissues and includes relatively
high levels of an ED-b[+] form and of a cartilage specific form, [V+C]- which lacks
several segments found in the isoforms of other tissues, as reviewed [30]. Interestingly,
the ED-b[+] isoform increases throughout the cartilage matrix in a canine model of OA
[31] and this isoform is upregulated at the message level in human OA cartilage [29].
Thus, Fn-fs in synovial fluid could theoretically be derived not only from plasma Fn
but also from cartilage and synovial fluid isoforms. Since the isoforms have a con-
served sequence in the amino-terminus, we cannot deduce, in terms of amino-terminal
Fn-f, the source of synovial fluid Fn-fs. To obviate this difficulty, we tested activities
of Fn-f solutions from OA synovial fluid or those generated from bovine synovial fluid,
bovine cartilage or bovine plasma and found them to all be fully and equally active in
cartilage chondrolysis [18]. Thus, the Fn-f activities we have reported are not depend-
ent on the tissue source nor the Fn isoform. Further, we have estimated that cartilage, if
moderately degraded, could contribute Fn-fs to a level found in human OA synovial
fluids, based on our measurements of Fn content in both synovial fluids and cartilage
tissue [18]. We have also demonstrated that cartilage damage itself can lead to genera-
tion of Fn-fs by demonstraing that IL-1 treated or MMP-3 treated cartilage causes re-
lease of enhanced levels of Fn-fs into the culture media [18]. Thus, Fn-f activity as
measured by us is independent of the source of the Fn-f isoform. Further, the Fn-f we
have focused our studies on is an amino-terminal 29-kDa Fn-f which we have identi-
fied in human OA cartilage [18] and which is shared by all Fn isoforms as is the
50-kDa gelatin-binding Fn-f, also studied by us. Our 140-kDa Fn-f likely differs from
sequences found in other Fn isoforms since it contains the alternately spliced regions.
While we have no proof, we think it highly likely that the 140-kDa Fn-f we study, as
isolated from plasma Fn, is as active as its counterpart from cartilage or synovial fluid.
A very important question is whether or not Fn-fs could initiate early events in OA
or rather arrive on the scene later and amplify the catabolic insult. While there is
not yet a clear answer, it should be noted that in an experimental OA animal model,
MMP-3 was upregulated in the synovium and subsequently upregulated in cartilage at
later stages, contributing further to progression of cartilage lesions [32]. Thus, we could
propose that in OA, synovial MMP-3 and other MMPs might be released which act on
synovial fluid Fn to generate Fn-fs that might then penetrate cartilage tissue to initate
cartilage damage. Thus, Fn-fs could theoretically be an early effector of cartilage dam-
age.
Collagen Fragments Are Elevated in OA
The major type of collagen in cartilage, type II collagen, is thought to be elevated in

late stage OA [33] and this could lead to elevated type II collagen fragments. Detailed
analysis shows that type II collagen synthesis is elevated in OA lesions while the con-
tent decreases [34]. Whether or not this correlates with increases in collagen fragments
has not been shown. However, it is clear that Col-fs can be found in OA models. For
example, Col-fs at a level of 6 µg/ml have been found in synovial fluid of rabbits treated
surgically to induce OA [35]. Further, up to 20% of all collagen in human OA cartilage
can be partially degraded; suggesting that up to 40 mg of Col-fs per gm may reside or be
released from heavily damaged cartilage tissue [36]. While different types of Col-fs from
type II collagen can be generated in cartilage degeneration, many observations show that
telopeptides become enriched, namely the N and C-telopeptides, the peptides released by
MMP-3 attack on the crosslinking regions of type II collagen. N-telopeptides have been
shown to be markers of cancer while C-telopeptides are associated with both the preva-
lence and the progression of radiographic OA at the knee and hip [37]. Interestingly, the
C-telopeptide is elevated in urine from OA patients [38], correlates with high cartilage
turnover in OA patients [39], increases after joint injury [40] and is elevated in synovial
fluid in the rabbit meniscectomy model of OA [41]. Further, C-telopeptides are associ-
ated with both the prevalence and the progression of radiographic OA at the knee and hip
[42]. It is likely that other types of collagens might also contribute to enhanced levels of
collagen fragments that may have chondrolytic activities, however, the major contribution
would be expected to be from type II collagen. Thus, it is highly likely that Col-fs such as
the N and C telo peptides become enhanced in cartilage degeneration. As will be dis-
cussed later, this pool would contribute markedly to cartilage degradation which leads to
a question similar to that posed for Fn-fs: Do the Col-fs arise early in OA and help in the
early initiating events or simply amplify ongoing processes or both?
HA-f Are Elevated in OA
It is well known that hyaluronan (HA) is also elevated in OA and inflammatory condi-
tions [43]. For example, serum HA and synovium HA synthesis increase in the ACLT
canine model [44] and cytokines such as IL-1α have been shown shown to increase HA
production in bovine articular chondrocytes [45,46]. The increased production of HA and
the inflammatory response should lead to HA fragmentation as shown [47]. Some of the
fragmentation may be due to hyaluronidases or free radicals [4]. It has also been shown
that CD44, an HA binding receptor, and MMPs can be induced by hyaluronidase treat-
ment of articular chondrocytes [48]. Thus, there is a strong suggestion of enhanced HA
fragments (HA-f) in cartilage degeneration and as will be discussed below these HA-f
should also contribute to either early events in cartilage degradation or amplify ongoing
processes.
Might Other ECM Fragments Be Elevated and Have Activities?
During cartilage degeneration, it would be expected that fragments of other ECM mac-
romolecules would also be elevated. It has been shown that a 16-residue synthetic link
protein peptide, derived from the amino-terminus of link protein, a component of pro-
teoglycan aggregates, stimulates proteoglycan synthesis in human articular cartilage
[49–51]. The peptide was shown to decrease release of IL-1 and to enhance mRNA
levels of aggrecan and link protein mRNA [51]. Since link protein can be cleaved near
the amino-terminus by enzymes such as MMP-3, such amino-terminal peptides could
enhance reparative processes during matrix damage. It should be noted that at low con-
centrations of Fn-fs, PG synthesis is also stimulated [52,53]. This invites the question
or whether or not link peptides might be catabolic at high concentrations. Unfortunately,
little is known of the mechanism of action of these LP-f or peptides. It would be inter-
esting to speculate that they may physically perturb the matrix and enhance release of
trapped growth factors or indirectly perturb growth factor receptors through ECM in-
teractions.
Fn-f Were the First to Be Studied as Catabolic Mediators
Based on the discussion above, it is clear that ECM fragments are elevated in OA and
cartilage degeneration, but what are the consequences? Our work with Fn-fs suggests
that these ECM fragments play crucial roles in cartilage metabolism. Our laboratory
was the first to report that Fn-fs perturb normal signaling through Fn receptors or in-
tegrins in chondrocytes to upregulate catabolic processes as well as modulate anabolic
processes. We demonstrated that these Fn-fs enhance cartilage damage in vitro [54],
penetrate cartilage tissue and bind to the pericellular matrix [55], elevate MMP expres-
sion, temporarily suppress proteoglycan (PG) synthesis [56] and enhance rates of PG loss
from cartilage tissue in explant cultures [52–54]. The Fn-fs cause the release of half of
the total PG from cultured cartilage explants in 10% serum within a few days at con-
centrations of Fn-f at or below measured concentrations of Fn-fs in OA synovial fluid
[55,57]. Interestingly, native Fn is inactive [54] leading to the now accepted notion that
the activities of the Fn-fs are liberated from Fn upon proteolysis. The damaging activi-
Figure 1. Correlation of Cartilage PG Content (A) With MMP-3 Release (B) and Cytokine/Factor Release
(C) With High Fn-f. Cartilage explants were treated with 100 nM 29-kDa Fn-f continuously with media
changes every other day. In A, cartilage PG content was measured in explants; in B, MMP-3 (stromelysin)
was measured by ELISA and in C, cytokines and growth factors in the media were measured by ELISA.
ties of the Fn-fs require mRNA as well as de novo protein synthesis and metabolic en-
ergy, suggesting that the Fn-fs are not acting as proteinases [54]. Not only do the Fn-fs
enhance proteinase activity, but they also temporarily suppress PG and general protein
synthesis, by up to 50% [52,57]. However, the effects of the Fn-fs are not totally re-
versible. Upon removal of the Fn-fs from cartilage cultures, the PG synthesis rates in-
crease to values up to 140% of control values, however the PG content does not return
to normal levels [52,53]. Thus, the in vitro model is an example of “attempted but fail-
ing repair” of cartilage. This linkage of damage to repair will be discussed in more de-
tail later.
We have studied the effects of Fn-fs on cartilage in both serum-free and serum
conditions. Serum-free conditions were initially used to determine rates constants of
PG degradation and release into the media [54–56] since these conditions allowed a
greater proteolytic response. However, later studies required use of longer term cultures
and demonstration of activity in more physiologic conditions, that of serum cultures.
The serum slows the rate of PG degradation by several-fold and allows an anabolic
response of the cartilage to the damage and provides information on steady state
metabolism of PG, rather than simply kinetics of degradation. With these conditions we
discovered a very interesting dose response effect of the Fn-fs. A 0.1–1 μM
concentration causes a 50% decrease in PG content (Fig. 1A) and marked upregulation
of MMP-3 (Fig. 1B) with maximal effects by day 7. These events correlate with
enhanced release of IGF-I, TGF-β, TNF-α, IL-1β, IL-1α and IL-6 (Fig. 1C) [additional
data in refs 58,59]. However, the lowest concentration of 1 nM Fn-f immediately
enhances the PG content and PG synthesis rates (Fig. 2). Further, an intermediate
10 nM concentration shows a lag period for PG depletion (Fig. 2A) and both 10 and
100 nM concentrations, although first suppressing PG synthesis, allow a slow return to
normal levels and eventually supernormal levels (Fig. 2C) [52,53].
Figure 2. Biphasic Effects of Fn-f on PG Content (A) and PG Synthesis (B). Cartilage explants were treated
with 1, 10 or 100 nM 29-kDa Fn-f continuously with media changes every other day. At intervals, cartilage
was subjected to papain digests and measurement of PG content by DMB assay (left). Similar cultures were
subjected to 35S labeling to determine rates of sulfated proteoglycan synthesis (right).
We have proposed that the curves shown in Fig. 1 are due to the summation of
both the catabolic and anabolic effects of the Fn-fs. Enhanced anabolic effects occur
with lower concentrations of Fn-fs. As the concentration of Fn-f is increased, the
catabolic effects predominate. However, after the catabolic phase subsides, the anabolic
once again predominates, leading to the supernormal PG synthesis rates and PG
contents. We have also suggested that this illustrates that the Fn-fs are true regulators
of metabolism. Thus, the 1 nM concentration may reflect repair of mildly damaged
cartilage and the higher concentrations may reflect the kinetics of and the linkage of
severe damage and attempted repair. These studies also suggested to us the great value
of examination of explant cultures over extended periods of weeks. Of course, we
would not have discovered these anabolic effects in monolayer cultures or in short term
cartilage cultures.
In terms of proteinases responsible for the cartilage damage, we have found that
several MMPs are upregulated at the protein level including, but likely not limited to,
MMP-1, MMP-2, MMP-3 and MMP-9. MMP-3 is a major proteinase in the activity [60];
antibodies to MMP-3 slow damage to Fn-f treated bovine cartilage explants [56].
However, the Fn-fs also increase cleavage of the aggrecanase epitope [61,62]. We have
also shown that MMP-3 can degrade intact Fn into small Fn-fs [18]. Thus, MMP-3
induced in OA may generate more Fn-fs, which in turn amplify MMP-3 expression in a
positive feed-back loop. Others have also shown that Fn-fs elevate MMP-13 in human
cartilage [63]. We have confirmed effects on MMP-13 protein in bovine explants and
find that it is elevated in parallel with MMP-3 (unpublished).
It is important to note that high concentrations of Fn-fs have many other activities
that can be described as catabolic. We also found that Fn-f enhance levels of NO and
upregulate iNOS [64] and upregulate activities and levels of IGF-I binding proteins
[65]. Subsequently, others showed that Fn-fs upregulate NO production in RA cartilage
through the CD44 receptor [66]. Others have shown that a Fn-f containing an alter-
nately spliced domain enhances MMP gene expression [67]. Others have also shown
that a gelatin-binding Fn-f induces type II collagen degradation by collagenase [68].
Also, a 45-kDa collagen-binding fragment was shown to upregulate MMP-13 and ag-
grecanase activity [69]. A 120-kDa Fn-f was shown to upregulate MMP-1, MMP-3 and
uPA in fibrocartilaginous cells [70]. It has also been shown that Fn-f upregulate the
Toll-like receptor 2 in human articular chondrocytes [71]. Fn-fs, as with IL-1, enhance
expression of CD44 in bovine chondrocytes [72]. The above listing may not be totally
comprehensive, but it does illustrate the broad effects of Fn-fs.
Interestingly, Fn-fs also enhance catabolic effects on other types of joint relevant
tissues. Fn-fs have also been shown to modulate expression of proteinases and
inhibitors in human periodontal ligament cells [73] and to trigger anoikis of human
primary ligament cells by suppressing p53 and c-myc [74]. There has been much
interest in the role of Fn-f in spine degeneration. Oegema et al. showed elevated Fn-fs
in degenerated human intervertebral disc [75]. Subsequently, others showed that Fn-fs
stimulated disc degeneration [76,77]. More recently, we showed that Fn-f did not
simply enhance degeneration as a generality. When different types of disc cells were
examined there were differential effects from enhanced anabolism to enhanced
catabolism [78].
The above catabolic activities of Fn-fs could easily be explained if the Fn-fs acted
through catabolic cytokines since cytokines are involved in many of these procsses. We
showed that Fn-fs enhance protein levels of IL-1α, IL-1β, IL-6 and TNF-α in human
cartilage and proved their active role by demonstrating that neutralizing antibodies to these
cytokines decrease Fn-f activities [58,59]. Later, it was shown by Northern blotting that
Fn-fs upregulate IL-1 [67] and shown by microarray analysis that Fn-f upregulate IL-6 and
IL-8 [79]. Others have shown that Fn-fs induce TNF-α in basophilic leukemia cells [80] or
in cultured mesangial cells [81]. Thus, it may be a generality that Fn-fs can enhance
cytokine pathways in many different types of cells.
While we tested the roles of IL-1, IL-6 and TNF-α by use of blocking antibodies, many
investigators have tested the role of cytokines in Fn-f pathways by use of IRAP to block
IL-1 activities. IRAP decreases induction of MMP-3 and MMP-13 by a heparin-binding
Fn-f [82] or partially blocks NO release from human chondrocytes treated with an
N-terminal 29-kDa Fn-f [83] or blocks MMP-1 induced release by an extra domain A Fn
peptide [67] or decreases MMP-3 upregulation by an RGDS peptide found in the Fn cell-
binding domain [84] or blocks activation of MMP-13 in human chondrocytes by a 120-kD
cell binding Fn-f [63]. However, there are reports that IRAP failed to block upregulation of
MMP-3 and MMP-13 in chondrocytes by a 45-kDa gelatin binding Fn-f [69] or failed to
block upregulation of MMP-13 by a 120-kDa Fn-f added to human chondrocytes [63].
These differences may reflect differences in potencies of IRAP, or different cell culture
systems or different species of cartilage. It should be noted that in studies where IRAP is
tested, the ability of IRAP to block exogenous IL-1 is often used as a control. Yet, this may
not be a suitable control since it is likely much more difficult to block IL-1, which after
being upregulated may be more highly concentrated around the cell, than exogenously
added IL-1 which becomes diluted upon addition and thus easier to block. Further, it would
not be expected that blocking of IL-1 would totally decrease MMP upregulation since
TNF-α activity is also enhanced by Fn-fs as we have shown [58,59].
Our recent data with bovine cartilage are consistent with an MMP upregulation
pattern in which early MMP enhancement is driven by MAP kinases and a later phase
beyond 48 hours is augmented through cytokines (unpublished). We have found that
IRAP does not totally block Fn-f upregulated MMPs after a 24 hour treatment but is
more effective at 48 hours and beyond. We have also found that MAP kinase, PYK2, or
PKC inhibitiors can decrease a major portion of the MMPs within the first 24 hours
during a time in which we cannot detect significant levels of IL-1 or TNF-α. Thus, it is
likely that there is an early cytokine independent pathway as well as a later cytokine
driven pathway. Since the most relevant way of studying matrix damage is with
explants in long term culture beyond a few days, it is likely that cytokines do play a
major role in more physiologically relevant conditions beyond 24 hours.
The generality of the activities of the Fn-fs has been supported in many ways. We
have found that similar cathepsin D and thrombin generated Fn-fs from rabbit plasma
Fn, bovine plasma Fn, rat plasma Fn or guinea pig plasma Fn (unpublished) are equally
active on bovine cartilage (unpublished). Thus, as a generality, Fn-fs from one species
can cause damage to cartilage of another species. Further, the types of proteinases used
to generate the Fn-fs may not be important, since regardless of the specific bonds
cleaved, similar domains are generated. We have shown that MMP-3 generated human
plasma Fn-f mixtures are as active as cathepsin D and thrombin generated Fn-fs on a
molar basis [18]. Further, Fn-fs made from Fn from bovine plasma, bovine synovial
fluid or bovine cartilage are equally active [18]. We have also shown that removal of
Fn-fs from OA synovial fluid decreases the cartilage damaging activity of the resultant
fluids [18], demonstrating that OA derived Fn-fs are active.
Because of the suitability of the Fn-f cartilage damage model as an OA model, we
have tested various agents for their abilities to block the action of Fn-fs. One particular
aspect of the model is that Fn-f damaged cartilage does not spontaneously restore PG
after the Fn-fs are removed [52]. Thus, this model has given us the capability of testing
agents that might be useful in cartilage repair. We have tested synthetic peptide analogs
of the cell binding sequence found in Fn [85], anti-oxidants such as N-acetylcysteine,
[86,87] and the growth factor, IGF-1, and found that all were partially to fully effective
in not only decreasing Fn-f mediated cartilage damage but also promoting restoration
of PG in pre-damaged cartilage [88]. In other studies, another growth factor, OP-1, [89]
as well as high molecular weight HA [62,90], were tested and also found to both block
damage and promote repair. Others have subsequently shown that OP-1 blocks Fn-f
mediated MMP-13 upregulation in human cartilage [91]. Since these agents all have
different modes of action and some are anabolic and some are anti-catabolic, our data
suggest that either attenuation of the catabolic pathways or compensation for the
catabolic stress can enhance repair. The data also suggest that agents that block damage
also have potential in cartilage repair, perhaps by altering or compensating for
pathways of cartilage destruction that continue in the absence of the damage mediator.
The Fn-f model has also allowed us to compare the metabolism of human ankle
and knee cartilages [92] to address the question of whether or not the lesser susceptibil-
ity of ankle joints vs knee joints to OA, which has been supported by numerous ca-
daveric, radiographic and clinical studies, might have a biochemical basis. We found
that addition of Fn-fs to cultured human knee cartilage decreases the PG content com-
parable to that of bovine cartilage. However, ankle cartilage in most cases is not af-
fected. While MMPs are upregulated in both types of tissue, the knee tissue is more
sensitive to Fn-f mediated PG synthesis suppression, suggesting that the difference in
cartilage damage between the tissues is due more to effects on matrix synthesis than to
upregulation of MMPs.
The physiologic significance of the chondrolytic properties of the Fn-fs was sup-
ported by our demonstration that that Fn-fs cause a loss of up to 50% of the articular
cartilage PG when injected into rabbit knee joints [93]. More recent work shows that
this degree of damage can occur within 2 days of injection, that MMP-3 levels plateau
within this time and that the Fn-fs temporarily suppress PG synthesis and expose the
NITEGE epitope of aggrecan, just as in the in vitro model [94]. PG synthesis, although
initially suppressed, slowly increases to supernormal levels, also suggesting a super-
normal anabolic response, just as in the in vitro model. This anabolic response leads to
restoration of PG within 2 weeks in adolescent rabbits, while in skeletally mature rab-

bits, PG restoration requires months [96]. Thus, this model has potential in studies of
aging. One of the most striking observations has been that the non-injected knee carti-
lage shows evidence of a systemic effect. When a low dose of Fn-fs was injected into
the right knee, the noninjected knee showed enhanced PG synthesis rates and PG con-
tent. If a higher concentration were injected, the non injected knee showed a rapid de-
crease in PG synthesis rates and a decline in PG content to the same extent as the in-
jected knee. Much more work will be required to ascertain the mechanism for this sys-
temic effect.
Col-fs Also Have Catabolic Activities
Col-fs have also been shown to have potent cartilage damaging activity. These activities
should be considered in light of other reports of activities of collagen peptides. Various
types of collagen peptides when added to other types of cells have several activities
related to inflammation or tissue damage. For example, CNBr peptides of type II collagen
stimulate IL-1β release from human monocytes [97] or stimulate collagenase production
by synovial cells [98] or modulate processing of MMP-8 by gelatinase [99] or alter types
II and IX collagen turnover in bovine articular explants [100]. Perhaps one of the best
known Col-f systems is that of endostatin, the C terminal fragment of collagen XVIII, an
inhibitor of angiogenesis, that interacts with the Fn receptor, alpha5beta1, and induces
clustering and disassembles actin stress fibers and FCs through activation of c-src [101].
Thus, as with Fn-fs, Col-fs likely enhance catabolic states in various types of tissue.
The first reports of activities of Col-fs toward cartilage tissue showed that bacterial
collagenase digests of type II collagen from bovine articular cartilage generated
fragments of <10 kDa that upregulated MMPs and decreased cartilage PG from explants
[102]. Since these digests were enriched with N and C telopeptides, synthetic telo
peptides were subsequently studied and found to bind annexin V, a nonintegrin receptor
[103]. More recently Col-fs and synthetic N and C telopeptides were shown to increase
MMP-2, 3, 9 and 13 message levels [104]. Others have recently shown that peptides of
type II collagen can induce the cleavage of type II collagen and aggrecan in articular
cartilage [105]. In order to determine whether Fn-fs and Col-fs might share mechanistic
features we have begun to compare the two types of fragments. Our unpublished work
with collagenase generated type II Col-fs as well as synthetic N and C telopeptides
suggest that they have only slightly weaker potencies as Fn-f, when compared at
10–100 μg/ml levels, and can upregulate MMP-1, MMP-3 and MMP-13 protein levels
and decrease cartilage PG from explants to a slightly lower degree than Fn-fs
(unpublished). We have also found that Fn-fs enhance phosphorylation of MAP kinase
p38 and the transcription factor, Nf-kB (unpublished). The earlier discussion of the
presence of elevated Col-fs in OA and in OA models certainly suggests the relevance
of this system.
HA-fs Have Catabolic Activities
It has also been shown that treatment of isolated cultured chondrocytes with HA hex-
asacharides causes loss of PG, suppression of PG synthesis, reduction in the degree of
aggrecan aggregation and enhances gelatinase activity [106]. Anti-sense oligonucleo-
tides to CD44 have been shown to cause some of the effects of the hexasaccharides,
consistent with the possibility that hexasaccharides may enhance chondrolysis by
down-regulation of the CD44 receptor [107]. This HA receptor appears to play a major
role in chondrocyte matrix assembly at the cell surface [108], as well as in matrix ca-
tabolism [109] and mediates anchoring of PGs bound to filaments with the chondrocyte
cell surface [110,111]. Further, hyaluronidase treatment of rabbit and bovine articular
cartilage results in production of low mass HA-fs and upregulates expression of CD44
and MMP-1, -3 and -9 while addition of purified 100 kDa HA has no effect. In the
same study, the catabolic activities of the HA-fs were blocked by anti-CD44 antibody
[112]. More recently, HA hexasaccharides were shown to enhance activity of multiple
transcription factors in chondrocytes involved in matrix remodeling and turnover while
high mass HA blocked the activity [113] and HA oligosaccharides were shown to in-
duce MMP 13 via transcriptional activation of NfkappaB and p38 MAP kinase in ar-
ticular chondrocytes [114]. HA fragments also activate nitric oxide synthase and the
production of nitric oxide by articular chondrocytes [115] as do Fn-fs [64]. Lastly, it
has also been shown that HA fragments induce chemokine gene expression in macro-
phages [116] through NF-kB [117]. Thus, HA-f appear to have overlapping activities of
Fn-fs and there are similarities with Col-fs, where comparison is possible.
Could ECM Fragments Assist in Tissue Remodelling?
One of the most interesting aspects observed with Fn-fs is that they can be both cata-
bolic and anabolic. This brings into question whether HA-f and Col-f also have ana-
bolic activities. Studies of Fn-fs revealed very interesting effects on anabolism; several
observations suggested that Fn-fs can also enhance matrix synthesis (See Fig. 2). We
found that: (1) in serum cultures, about half of the PG was lost from cartilage within
7 days but subsequently the PG content remained constant rather than continued to de-
crease; (2) after an early damage phase of maximal PG depletion from the matrix, PG
synthesis rates that were initially suppressed, slowly increased to levels above untreated
controls; (3) treatment with lower concentrations of Fn-fs, down to 1 nM, did not cause
extensive PG loss from cartilage, but actually enhanced both PG synthesis rates and PG
content, often up to 140% of levels of untreated controls [52]; and (4) pretreatment of
cartilage with a low concentration of Fn-f markedly blocked the effect of subsequently
adjusting the Fn-f concentration to a higher level that normally would cause extensive
matrix damage [53]. It is interesting to speculate that the effects of low concentrations
of Fn-fs are similar to what is observed in early OA in that PG synthesis is elevated as
well [118 and references therein]. Further, the effects of higher concentrations of Fn-fs
may be compared to severe OA, where PG synthesis suppression continues [118,119].
In order to explain this anabolic effect, we assayed for the presence of IGF-1 in the
media and found enhanced levels [58,59]. Further, addition of exogenous IGF-1 did
decrease Fn-f mediated cartilage damaging activity [88] and IGF-1 receptor decoy
blocked the anabolic effects of Fn-f [65]. Since metabolic inhibitors had no effect on
release of IGF-1 and we could not detect elevated levels of IGF-1 protein in the matrix
[58,59], we reasoned that Fn-fs did not upregulate growth factor synthesis, but some-
how enhanced the release and activation.
The power of the anabolic effect was demonstrated by two different means. In the
first approach (Fig. 3A), we tested the effects of a 2 day pretreatment with 1 nM Fn-f,
followed by treatment with a catabolic damaging concentration of 100 nM and com-
Figure 3. Anabolic Effects of Fn-f – In A, cartilage was treated with 1 nM Fn- for days 0–28 (1 nM), or with
100 nM at days 7–28 (100 nM) or with 1nM for 2 days (–2) and then with 100 nM at days 0–28 (1>100). PG
content was measured every 7 days. In B, cartilage was cultured with 100 nM Fn-f for 4 days prior to start of
experiment (4d pre) or with Fn-f at days 7–28 Fn-f (7–28) or with Fn-f 4 days prior to start of experiment and
then with 100 nM Fn-f for days 7–28 (4d pre).
pared the effects on PG content to those of 1 nM treatment or 100 nM treatment with-

out pretreaments. Note in the figure that this pretreatment made cartilage more refrac-
tory to the catabolic insult as observed with a nearly two week delay in major damage.
In the second approach, we tested effects of a high concentration but short pretreatment
of 4 days. This short treatment was too short to maximally upregulate MMPs but was
sufficient to trigger an anabolic effect. As seen in Fig. 3B, this pretreatment markedly
slowed PG depletion as compared with the no pretreatment control.
This ability of Fn-f to make cartilage refractory to further damage was also studied
by determing the minimal length of time required for Fn-f to cause maximal damage.
We found that a single 7 day exposure caused as much damage as a continual exposure
for up to 28 days and that a less than 3 day initiated only the anabolic response [53].
We then compared the effects of multiple treatments that summed up to a 7 day expo-
sure and found that even if a second or third exposure added up to 7 days, maximal
damage could not be inflicted. Thus, damage required a continual exposure to Fn-f.
Otherwise, an anabolic response could occur and make tissue refractory.
We further proposed that light to moderate proteolysis of matrix PG, too light to
cause a decrease in steady state PG levels, causes the release of IGF-1 and TGF-β.
TGF-β is known to be trapped in the matrix [120] and IGF-1 has been shown to be
trapped in cartilage tissue [121,122]. Since proteinases have been shown to release
TGF-β in other cells [123] and proteinases, including MMP-3, can degrade IGF-1 bind-
ing proteins [124] and release IGF-1, the Fn-fs at low or high concentrations may help
to mobilize growth factors. To support this proposal, we demonstrated that addition of
MMP-3 to cultured cartilage results in the initial suppression of PG synthesis and ele-
vated IGF-1 in the media and after a few days, enhanced PG synthesis, just as do the
Fn-fs [65]. Thus, proteases induced during the damage phase might account for a link-
age between cartilage damage and attempted repair responses. While we have no evi-
dence of a role for protease induced activation of IGF-1 in the Fn-f system, we do
know that one of the IGF-1 binding proteins, BP4, decreases in the presence of the
Fn-fs [65]. Other work with the Fn-f model has suggested an additional feature that
may account for the supernormal anabolic effect. We have found that Fn-fs induce ex-
pression of IGF-I binding proteins (BPs) [65]. The induced BPs released from chon-
drocytes likely bind IGF-I and concentrate it around the cells. Thus, during Fn-f in-
duced proteolysis, the active IGF-I concentration around the cell may be greatly ele-
vated, leading to enhanced effects on PG synthesis.
Thus, these studies suggest that Fn-fs might have some role in basal metabolism or
light tissue damage where they might amplify anabolic processes. Initially they might
also make the tissue more refractory to further damage. However, if the damage pro-
gresses to severe damage, catabolic pathways may be additionally activated to assist in
tissue clearance. At present, we do not know if Col-fs and HA-fs cause the same effects.
Is There a Single Global Mechanism?
Our current studies are focusing on the signaling mechanisms of ECM fragments
beginning with receptor and ECM and continuing into activation of intracellular
kinases. Our proposed model for our Fn-fs that we think might apply to Col-fs and
HA-fs as well is based on an early observation that in fibroblasts, Fn enhances
clustering of integrins and activates tyrosine kinases [125]. Thus, Fn-fs might block or
disrupt integrin clustering and either block the Fn pathway kinases or activate different
kinases. Our unpublished work suggests that Fn-fs do disrupt alpha5 integrin clusters
and decreases their areas on the cell surface and that this is associated with enhanced
internalization of the receptor subunits and a movement of integrin interacting
components from cell membranes into the cytosolic compartments (unpublished). This
is consistent with the notion that HA-f may detether the CD44 receptor and also
enhance internalization [106]. Our preliminary data also suggest that Col-fs have
similar effects and decrease receptor clusters consisting of both Fn and collagen
binding integrins. Consistent with this observation, type I Col-fs have been shown to
promote disassembly of focal adhesion in smooth muscle cells [126]; thus integrin
disruption by type II Col-fs is certainly a possibility. Thus, there may be shared ability
among the ECM fragments to disrupt the native receptor clusters at the cell surface,
enhance internalization and disrupt the normal interactions with cytosolic accessory or
scaffolding proteins and this may initiate new signaling pathways. Thus, the matrix
itself might be thought of as a negative regulator of these ECM fragments pathways.
When the ECM is degraded, the activities of these fragment pathways may be liberated.
We earlier implicated the alpha5 integrin in Fn-f activities by chemical
crosslinking of Fn-f to chondrocytes and by antisense oligonucleotide inhibition of the
alpha subunit [127,128]. This is the subunit that comprises, with the beta1 subunit, the
classical Fn binding integrin. However, more recently we have also observed that Fn-fs
can be chemically crosslinked not only to the alpha5 subunit, but also to other subunits,
including the alpha1 and alpha2 that comprise collagen binding receptors and alpha3
and alphaV subunits that comprise Fn and thrombospondin binding receptors. Upon
further analysis by dual label confocal microscopy we have confirmed that not only do
Fn-fs bind in proximity to these integrins but so do Col-fs. This surprising observation
prompted us to investigate whether the fragments not only cross interacted with diverse
integrins but whether the integrins themselves were proximal to each other. Indeed, we
have found by confocal analyis that the alpha 1, 2, 3 or V subunits appear to colocalize
with the alpha5 subunit. We consider these observations very preliminary and are
attempting to devise alternate methods to verify. These observations have prompted us

to suggest a global mechanism in which Fn-fs and Col-fs bind to “integrisomes” or
integrin clusters through either direct interaction or indirect interactions mediated
through their native ligands or the ECM. These interactions would physically alter
these “integrisomes” to initiate altered signaling. There is also another possible
explanation. Because Fn-fs can bind to native Fn and certain Fn-fs can bind collagen
and specific Col-fs might bind both native collagen and Fn, the ECM may establish a
platform for both homotrophic and heterotrophic interactions that indirectly disrupt
“integrisomes” and alter signaling. Consistent with this proposal, Tuckwell et al. have
shown that denatured collagen can bind the alpha5 subunit by binding Fn which bridges
to the alpha5beta1 receptor [129]. But what of CD44? Would it communicate within the
“integrisomes”? Interestingly, it has been suggested that a C terminal Fn-f utilizes the
CD44 receptor [66], so perhaps there is a linkage between all three major ECM
fragment systems that occurs at the level of these receptor clusters.
In terms of a global mechanism for signaling downstream of receptors, some of the
first observations on effects of Fn-fs on signaling showed that Fn-fs activate MAP
kinases. Gemba et al. reported that an amino-terminal Fn-f activated MAP kinases in
human chondrocytes and that chemical inhibition of the three MAP kinases, ERK1/2,
JNK and p38 inhibited Fn-f mediated NO production [83]. Others have reported that
MAP kinases are activated by a central cell binding Fn-f and that inhibition of MAP
kinase activities decreases MMP-13 upregulation [63]. Others have confirmed the role
of MAP kinases in Fn-f upregulation of collagenase [130]. The role of Nf-kB has also
been shown in Fn-f upregulation of cytokines and chemokines [79] as well as a role of
PYK2 and PKC [131]. Our own unpublished work with bovine cartilage and bovine
chondrocytes shows the involvement of MAP kinases, Nf-kB, PYK2 and PKC in
MMP-3 and MMP-13 upregulation and cartilage damage (unpublished). However,
while NF-kB is often thought to be downstream of MAP kinases in a linear pathway,
our work suggests much more complexity. Tests of chemical inhibitors suggest that for
MMP upregulation there are separate pathways for PYK2, MAP kinases and Nf-kB.
Clearly, our work is showing that the kinase signaling pathways are complex, are not
necessarily linear, and may have several arms and that the common points or origin of
these arms may be far upstream.
Very little has been published on signaling pathways for Col-fs. Our preliminary
data suggest that Col-fs are slower at enhancing MMP levels than Fn-fs. This
difference is also reflected in a smaller degree of activation of Nf-kB and p-38.
Activation of JNK/SAPK is almost undetectable. Further, Col-fs do not appear to
greatly upregulate TNF-α or IL-1β even after 72 hours of treatment, yet Col-fs can
significantly deplete matrix PG in explants in 7 day cultures. Thus, by comparisons
between the Fn-f and Col-f systems, we might conclude that a noncytokine pathway is
sufficient for significant cartilage damage but that cytokines do enhance damaging
activity. Since these experiments are complex, much more work is needed to clarify the
role of cytokines in Col-f signaling. In terms of HA-fs, the most recent observations on
signaling activities of HA-fs, suggest that they utilize Nfkb and p38 MAP kinase [114]
and upregulate iNOS [115] which would generate NO as a possible mediator. Thus,
major points of similarity between the three systems are p38 MAP kinase and Nf-kB.
In summary of a global mechanism, based on published data, it appears that all
three ECM fragments utilize receptors to which their native precursors bind and that
their precursors are largely inactive. Our preliminary data suggest that Fn-fs and Col-fs
may utilize similar receptors and that these receptors are proximal to each other in the
membrane. Our preliminary data also suggest that Fn-fs and Col-fs disrupt integrin
clusters and enhance internalization. Observations with HA-fs also suggest a de-
dethering effect with enhanced internalization. We would propose that the enhanced
internalization of receptors disrupts interactions with cytosolic scaffolding proteins and
this exposes or creates new signaling pathways. Published data suggest that Fn-fs and
HA-f utilize Nf-kb and MAP kinases for various catabolic activities while our prelimi-
nary data suggest the same for Col-fs. Our preliminary data with Fn-fs suggest that
these pathways are complex and comparison of Fn-fs and Col-fs adds an additional
level of complexity in that cytokines may not be necessary for the early catabolic re-
sponse of Fn-fs and Col-fs but may play an important role over the longer term. These
similarities suggest to us that there may be a global mechanism which if attenuation is
prudent, is best done upstream closer to receptor.
Summary and Prospectus
This review has attempted to describe and compare three ECM fragment systems of
cartilage regulation. Their very existence points to the important role of the ECM itself
in regulating normal cartilage metabolism and to the concept that regulation by the
ECM does not occur through just one component but different components that can
interact with each other. Since ECM fragment pathways are derived from the most
abundant protein, type II collagen, as well as one of the least abundant proteins, Fn, this
suggests that there is a potential for other pathways derived from other relatively minor
components. Thus, it is likely that other ECM fragment systems will be discovered.
Further knowledge of their mechanisms is important for determining whether or not
there is a single global pathway with a single upstream point of intervention. However,
further knowledge of the role these systems play in regulation is crucial to our
understanding of whether intervention in diseases such as osteoarthritis would be
prudent. It is likely that these pathways should be attenuated in the disease process, but
complete attenutation, if possible, may not be prudent because of the simultaneous role
these pathways likely play in cartilage repair.
Acknowledgments
The authors thank past support from the Arthritis Foundation of the Greater Chicago
Chapter and the North Central Chapter, the National Institute of Arthritis and Muscu-
loskeletal Diseases Specialized Center of Research (SCOR) Grant, the Dr. Ralph and
Marian C. Falk Medical Trust Fund, the North Dakota EPSCoR, and the Eugene W.
Cornatzer Chair Trust.
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IOS Press, 2007
VI
Pathophysiological Relevance of PPAR to

Osteoarthritis: From the Control of
Inflammation to Cartilage Protection?
Arnaud BIANCHI, Mélanie KIRCHMEYER, Marie-Madeleine GALTEAU
and Jean-Yves JOUZEAU *
Laboratoire de Physiopathologie et Pharmacologie Articulaires (LPPA),
UMR 7561 CNRS-UHP Nancy 1, Avenue de la forêt de Haye, BP 184,
54505 Vandœuvre-lès-Nancy, France
Abstract. Peroxisome proliferators activated receptors (PPAR) are ligand-

inducible nuclear transacting factors comprising 3 subtypes, PPARα, PPARβ/δ
and PPARγ, which play a key role in lipids and glucose homeostasis. All PPAR
subtypes have been identified in joint cells and their activation resulted in a tran-
scriptional repression of pro-inflammatory cytokines (IL-1, TNFα), early inflam-
matory genes (NOS2, COX-2, mPGES-1) or matrix metalloproteases (MMP-1,
MMP-13), at least for the γ subtype. These anti-inflammatory and anti-catabolic
properties were confirmed in animal models of joint diseases although much less
data are available for experimental osteoarthritis (OA) than for polyarthritis. PPAR
agonists were also shown to stimulate IL-1 receptor antagonist (IL-1Ra) produc-
tion by cytokines-stimulated cells in a subtype-dependent manner. So, PPAR ago-
nists are able to reduce joint inflammation and to prevent cartilage destruction, al-
though many effects were obtained at a higher concentration than required to re-
store insulin sensitivity or to lower circulating lipids levels. Besides, PPAR ago-
nists were able to modulate the differentiation and/or activity of bone cells, but
data are lacking for their effect on OA-associated sclerosis of subchondral bone.
Although promising, the therapeutic insight of PPAR agonists in OA warrants ad-
ditional proofs that could be obtained indirectly from the follow-up of diabetic
and/or hyperlipidemic patients with OA treated daily with glitazones or fibrates.
Keywords. PPAR subtypes, gene transcription, cytokines, eicosanoids, metallo-

proteases, animal models, osteoarthritis
1. Peroxisome Proliferators Activated Receptors (PPAR)
1.1. Structure – Overall Expression and Functions
Peroxisome proliferators activated receptors, subtypes PPARα (N1RC1), PPARβ/δ

(FAAR, NUC1 or NR1C2) and PPARγ (NR1C3), are ligand-inducible nuclear transact-
*
Corresponding Author: Pr. Jean-Yves Jouzeau, LPPA, Phone: +33(3)83683950, Fax: +33(3)83683959,
E-mail: Jean-Yves.Jouzeau@pharma.uhp-nancy.fr.
78 A. Bianchi et al. / Pathophysiological Relevance of PPAR to Osteoarthritis
Figure 1. Schematic representation of the functional domains of PPAR. PPAR are composed of four distinct
functional regions: 1) the A/B domain located at the N-terminus, containing a ligand-independent activation
function (AF-1) responsible for PPAR phosphorylation; 2) the C domain or DNA binding domain (DBD)
promoting the binding of PPAR to the peroxisome proliferator response element (PPRE, a classical direct
repeat [DR] separated by one or two nucleotides) in the promoter region of target genes; 3) the D domain is a
hinge region responsible for the docking of cofactors, 4) the E/F domain or ligand-binding domain (LBD)
located at the C-terminus, responsible for ligand specificity and containing a ligand-dependent activation
function (AF-2) which promotes the recruitment of cofactors to assist the gene transcription process and is
necessary for the heterodimerization with RXR.
ing factors. They belong to the steroid receptors family including receptors for retinoids,
thyroid hormones or corticosteroids [1]. Their overall protein structure is composed by
six domains, termed generally from A to F, having key specific functions such as
ligand binding (LBD), interaction with DNA (DBD) or control of transactivation (AF
domains) (Fig. 1).
PPARα is expressed exquisitely in tissues contributing actively to fatty acids ca-
tabolism (mainly in liver, and less markedly in brown fat, kidney, heart or skeletal
muscle), where it regulates the expression of genes involved in fatty acid uptake and ω-
or β-oxidation [2]. PPARα is also expressed in endothelial and vascular smooth muscle
cells, as well as macrophages/foam cells, and contributes to the control of inflammation,
thereby opening insight to the treatment of atherosclerosis [3,4].
PPARβ/δ is the less well-characterized PPAR subtype despite its ubiquitous ex-
pression. It takes place in lipids metabolism by favouring the reverse transport of cho-
lesterol and oxidation of fatty acids [5], plays a major metabolic role in muscle and
adipose tissue [6] and has been linked to profound anti-obesity and anti-diabetic actions
in animal models [7]. Activation of PPARβ/δ has also been linked to cell proliferation
or apoptosis depending on the cell type and seems to play a key role in wound heal-
ing [8].
PPARγ is highly expressed in white and brown adipose tissue and less intensively
in cardiac and skeletal muscle [2]. It plays a pivotal role in adipocytes differentiation
and lipids storage [9] and its activation provides insulin sensitizing properties that have
entered the clinics [4,10]. PPARγ is also thought to be a negative regulator of inflam-
mation [11] since its activation is able to suppress pro-inflammatory cytokines produc-
tion [12,13] while being anti-inflammatory in several animal models [14,15]. Of par-
ticular note, many metabolic effects of PPARγ and β/δ agonists were supported by their
ability to correct the circulating imbalance between leptin and adiponectin [4,10], two
adipokines also found at abnormal levels in the synovial fluid of OA patients [16].
A. Bianchi et al. / Pathophysiological Relevance of PPAR to Osteoarthritis 79
1.2. Mechanism of Action
Activation of PPARs leads to the formation of heterodimers with retinoid-X recep-

tors [17] (RXRs). These PPAR-RXR dimers bind to DNA-specific sequences, called
peroxisome proliferators response elements (PPRE), to stimulate or dampen the tran-
scription of target genes. The classical PPRE consensus sequence is a direct repeat
(AGGTCA) separated by one or two nucleotides (DR1/2) (Fig. 1), which is unfortu-
nately not sufficient to predict the responsiveness to PPAR agonists since a perfect
consensus was reported to be possibly non functional [18] whereas a highly degener-
ated sequence was, on the contrary, demonstrated to be fully responsive [19]. In addi-
tion to their differential tissue distribution, PPAR subtypes vary in their selectivity and
sensitivity towards agonists and recruit distinct co-activator proteins follow-on in the
regulation of different sets of genes [1]. There are several mechanisms by which PPAR
activation can regulate the transcriptional machinery (summarized in Fig. 2).
Briefly, besides the classical PPRE-mediated effects and possible interference with
histone acetylation [20] (Fig. 3), PPAR can develop protein-protein interaction with
several transcriptions factors as NF-κB [21–23], AP-1 [24], NF-AT [25], STAT [26] or
egr-1 [27], thereby reducing the response to inflammatory cytokines or growth factors.
PPAR can also modulate the response to transcription factors by competing for the
recruitment of several co-activators [20] or by interfering directly with their DNA bind-
ing site, as demonstrated recently for AP-1 in the promoter region of MMP-1 gene [28].
Of particular note, additional PPAR-independent mechanisms have been reported for
PPAR agonists, especially the inhibition of NF-κB pathway by the natural PPARγ
ligand 15-Δ12,14-Prostaglandin J2 (15d-PGJ2) [29,30], most of which being attributable
to the high chemical reactivity of its cyclopentenone ring with substances containing
nucleophilic groups, such as cysteinyl thiol group of proteins [31].
1.3. PPAR Agonists
PPAR are considered as lipid sensors that can be activated either by endogenous com-
pounds, generally natural fatty acids and their metabolites (low-binding affinity
ligands), or by synthetic agonists (high-binding affinity ligands), including the anti-
lipidemic fibrates [32] and the anti-diabetic thiazolidinediones [33] (see Table 1).
1.3.1. Endogenous Compounds

Most endogenous PPAR agonists are eicosanoids derived from arachidonic acid
biotransformation by the lipoxygenases or cyclooxygenases pathways. Thus, the
5-lipoxygenase metabolite leukotriene B4 (LTB4) [34] is a potent agonist of PPARα
although 8S-HETE (8S-hydroxyeicosatetraenoic acid) has the highest affinity for this
subtype [32]. Furthermore, the cyclooxygenases metabolites PGA1 [35] and PGI2 [36]
are preferential ligands of PPARβ/δ. Finally, by-products from the 15-lipoxygenase
pathway, 9-HODE (9-hydroxy-octadecadienoic acid) and 13-HODE, and the
cyclooxygenase-derived dehydration product of prostaglandin D2, 15d-PGJ2, are selec-
tive agonists of PPARγ [37,38]. Interestingly, 15d-PGJ2 was demonstrated to be pro-
duced in the late resolution phase of experimental acute inflammation and is postulated
to be a negative regulator of inflammation [39]. Plenty of fatty acids metabolites,
Figure 2. Schematic representation of the transcriptional machinery control by activation of PPAR. A) After
ligand binding (variable translocation depending on the subtype), the PPAR-RXR heterodimer binds to a
peroxisome proliferator response element (PPRE) located in the promoter region to control (generally acti-
vate) the transcription of a target gene; B) After ligand binding, the PPAR-RXR heterodimer competes with a
transcription factor (TF) for the recruitment of common co-activators, therefore resulting in the suppression
of TF-dependent transcriptional activity; C) After ligand binding, the PPAR-RXR heterodimer squelches a
transcription factor (TF) by direct protein-protein interaction, therefore resulting in the suppression of
TF-dependent transcriptional activity by reduction of its translocation; D) After ligand binding, the
PPAR-RXR heterodimer binds to a peroxisome proliferator response element overlapping (PPRE composite)
the binding site of a transcription factor (TF) in the promoter region of a target gene, therefore suppressing
TF-dependent transcriptional activity by competitive DNA binding.
PPAR 9-cis retinoic

agonists acid
PPAR RXR PPAR RXR
N-CoR
SMRT N-CoR
SMRT
RE
PP
AR
CBP/p300
PP > SRC-1
R
RX
Gene Transcription Gene Repression
Figure 3. General model for transcriptional activation by PPAR. Binding of PPAR agonists and 9-cis retinoic
acid to a PPAR-RXR/corepressor complex, which is not bound to DNA, results in the dissociation of the
corepressors (Nuclear receptor CoRepressor [N-CoR], Silencing Mediator for Retinoid and Thyroid hormone
receptor [SMRT]) and activation of the PPAR-RXR heterodimer. The activated PPAR-RXR heterodimer
binds to a peroxisome proliferator response element (PPRE) located in the promoter region of a target gene
and recruits co-activators (preferentially CAMP response element Binding Protein [CBP] and the related
protein p300 [CBP/p300], or Steroid Receptor Coactivator 1 [SRC-1]) which have intrinsic histone acetyl-
transferase (HAT) activity. Finally, acetylation of core histones removes the electrostatic attraction between
negatively charged DNA and positively charged lysine, allowing the loosening of the nucleosomal structure
with a subsequent chromatin remodelling. The subsequent recruitment of large protein complexes, including
RNA polymerase II, leads to initiation of gene transcription. In some cases, as the control of COX-2 gene
expression in IL-1-stimulated synovial fibroblasts, the recruitment of CBP/p300 can be competitive with
other transcription factors, resulting in a decrease in HAT activity and secondary reduction of gene transcrip-
tion.
including those originating from transcellular metabolism, can activate PPAR with a
variable subtype selectivity [40]. However, the theory of an endogenous control of in-
flammation by PPAR activation suffers from actual limitations. Firstly, most endoge-
nous compounds have a low binding affinity to PPAR (Kd generally over the micromo-
lar range) and, as a consequence, high concentrations of ligands will be necessary to
activate the system. Secondly, data are lacking to demonstrate that these metabolites
can be produced endogenously in sufficient amounts to activate PPAR, especially for
15d-PGJ2 [41], even if intracellular concentration could overcome plasma levels for
Table 1. PPAR agonists used in in vitro studies with joint cells
PPAR PPAR PPAR

Compounds Cell type [References]
α β/δ γ
Synthetic high-binding affinity
ligands
Clofibrate (clofibric acid) +# – ± C [47,52]
Fenofibrate (fenofibric acid) +# – ± C [52]
GW9578 ++ # – – Ocl (differentiation/activity) [59]
C [47,48,52]
Pyrixinic acid (Wy14643) ++ # – ± SF [27,60]
Obl (maturation) [61]
L165041 – ++ # ± Ocl (differentiation/activity) [59]
GW501516 – ++ # – SF [50]
C [62,63]
SF [60]
Ciglitazone – – +#
Ocl (differentiation/activity) [59,64]
GI262570 – – ++ # C [65]
Pioglitazone – – +# C [63]
C [28,48,52,66–68]
Rosiglitazone – – ++ # SF [20,50,55,57]
Obl (differentiation) [69]
C [47,66,68]
Troglitazone – – +# SF [20,27,57,70]
Natural low-binding affinity ligands
C [47, 48, 55, 62, 63, 65–67]
12,14 # SF [27, 50, 55, 60, 70]
15-deoxy-Δ -Prostaglandin J2 – – +
Ocl (differentiation) [64]
Obl (differentiation) [69,71]
Prostacyclin – +# – Obl [71]
++: very easy binding and/or strong activation in transactivation assays; +: easy binding and/or activation in
transactivation assays; ±: weak binding and or low activation in transactivation assays; –: no binding detected
and/or no activation in transactivation assays; #: this compound has been determined to have selectivity for
this PPAR isoform; C: chondrocytes; SF: synovial fibroblasts; Obl: osteoblasts; Ocl: osteoclasts
some metabolites. Thirdly, most fatty acids metabolites lack any marked selectivity for
a given PPAR subtype and the endogenous control of inflammation will depend on
whether they are produced concomitantly or sequentially and/or can compete for PPAR
binding. Besides these endogenous compounds, dietary n-3 polyunsaturated fatty acids
(n-3 PUFAs) could have some therapeutical relevance since they are natural ligands for
PPARα and PPARγ [32], but can also modify the endogenous metabolites produced
from membrane phospholipids [42].
1.3.2. Synthetic Ligands

Fibrates, as clofibrate, fenofibrate or Wy14643, are agonists of PPARα which were
developed as hypolipidemic agents through optimization of their lipid-lowering activity
in rodents before the discovery of PPAR [32]. Whereas the active metabolites of clofi-
brate and fenofibrate are dual activators of PPARα and γ, with an approximately
10-fold selectivity for PPARα, Wy14643 and GW9578 are 50 to 500-fold selective for
PPARα [43]. GW501516 is a newly synthesized agonist [44] which is > 1000-fold
selective for PPARβ/δ over the other subtypes [45], whereas L165041 is only 10-fold
more selective for PPARα over PPARγ [43,45]. Agonists of PPARγ include antidia-
betic molecules of the thiazolidinediones or “glitazones” family which have been de-
veloped initially through empirical screening in rodent models of insulin resistance.
Glitazones have an increased selectivity for PPARγ from troglitazone and pioglitazone
to rosiglitazone [43,45,46], although non thiazolidinediones derivatives, such as
GI262570, have a > 1000-fold selectivity for PPARγ over the PPARα and β/δ sub-
types [43].
As for any compound with pharmacological activity, the in vitro concentrations
used must be viewed critically to interpret the efficacy of PPAR agonists on joint cells.
In several studies, the PPARα agonist Wy14643 was tested above 50 µM [27,47,48]
and the PPARγ agonists troglitazone [27,47] or rosiglitazone [47,49,50] above 10 µM,
whereas the PPARβ/δ agonist GW501516 was active from 0.1 nM [50] to 100 nM.
Most of these concentrations are high compared to the respective binding affinity or
activity of PPAR agonists (generally < 1 µM for Wy14643 on PPARα < 10 nM for
GW501516 on PPARβ/δ and < 100 nM for rosiglitazone on PPARγ) in cell-based
transactivation assays [51] or reporter cell lines expressing human PPAR subtypes [45].
In contrast, PPAR-dependent activities were reported at lower concentrations, namely 1
µM of rosiglitazone [28] or Wy14643 [52] in rabbit chondrocytes monolayers although
the selectivity of high concentrations of PPAR agonists was confirmed with a panel of
subtype-selective target genes in rat chondrocytes embedded in alginate beads [53].
This discrepancy could be explained, at least in part, by species differences in the re-
sponse of PPAR to a given agonist [32] and by differences in the methodology (gene
reporter technology) or experimental conditions (pericellular environment [54]) used.
However, one can also underline that most anti-inflammatory effects were observed
only with a high concentrations of PPAR agonists whereas the stimulation of IL-1Ra
production occurred from a low agonist’s concentration. This suggests that the sensitiv-
ity of genes to a given PPAR agonist could depend on the location of any PPRE in the
transcriptional machinery of their promoter or, alternatively, that the anti-inflammatory
effects may be rather supported by the titration of transcription factors which requires a
higher agonist concentration and/or additional mechanisms.
Whatever the PPAR agonist, the time at which it is added relatively to the inflam-
matory stimulus is critical to interpret its potency in cell culture systems. As PPAR
agonists are expected to act as transcriptional regulators, it is not surprising that nu-
merous reports on joint cells were obtained with a pre-treatment time ranging from
several minutes [27,55,56] to few hours [57]. However, co-stimulation was also re-
ported to be efficient against cytokines [28,48,50,52,55], whereas the inhibitory effect
of 15d-PGJ2 on IL-1-induced MMP-1 expression decreased gradually with time after
addition of the stimulus [55]. In some experiments, PPARγ agonists provided an identi-
cal inhibition of IL-1-induced responses in pre-treatment or co-incubation [48], but
15d-PGJ2 was reported to be less inhibitory on TGF-β-induced activation of COL1A2
promoter in co-stimulatory conditions [58]. Taken together, these data confirm that the
transcriptional activity of PPAR agonists takes advantage from any pre-incubation but
suggest that their potency remains obvious when they are added at the same time as
cytokine challenge.
1.4. Expression in Joint Tissues
In the last ten years, PPARα, PPARβ/δ and PPARγ were shown to be expressed consti-
tutively in synovial fibroblasts [50,57] as well as chondrocytes from several species
[28,47,55,68]. In most cases, activation of PPAR resulted in inhibition of cytokine-
induced expression of pro-inflammatory genes or matrix metalloproteases (see below).
Nonetheless, contradictory data were reported on the ability of PPARγ agonists to in-
duce chondrocytes apoptosis [62,63,72]. PPAR subtypes were also reported in bone
cells where activation of the γ subtype was linked to inhibition of both osteoclastogene-
sis [64] and differentiation of progenitors into osteoblasts [73], although subtype-
specific PPAR agonists had a variable effect on bone resorption [59]. Besides, messen-
ger RNAs for PPARγ were detected in rat articular fat-pad [74] which is a potent
source of cytokines [75] and adipokines [16] production within the joints. In acute or
chronic inflammatory conditions, activation of PPAR took also advantage from the
expression of its three subtypes in monocytes and macrophages [12,23,76] as well as B
and T lymphocytes [77,78]. Taken together, these data demonstrate that PPAR sub-
types are expressed both in resident and infiltrating joint cells, suggesting that they may
have a pleitropic anti-inflammatory effect although in a cell-specific manner.
It is worth noting that the expression pattern of PPAR subtypes could be modified
in inflammatory conditions since PPARγ was reported to decrease in joint cells in re-
sponse to bacterial endotoxins [57] or IL-1 [50]. Such inflammation-induced pattern is
thought to contribute, at least in part, to the PPARβ/δ-dependent effect of rosiglitazone
in IL-1-stimulated chondrocytes [50]. Recently, it was also reported that the mRNA
levels of all PPAR subtypes decreased in chondrocytes stimulated with TGF-β [53],
further underlining that changes in PPAR levels could be key regulators of the pharma-
cological responses to their agonists [79].
2. Modulation of Key Inflammatory Genes by PPAR Agonists
2.1. Cytokines
In many cell types, the PPARγ agonists 15d-PGJ2 and thiazolidinediones were shown
to inhibit the transcriptional induction of genes playing a pivotal role in joint patho-
physiology as TNFα [12] or IL-1 [13]. Thus, PPARγ agonists were able to repress
LPS-induced TNFα expression [57] and IL-1-induced IL-1β production [50] in syno-
vial fibroblasts. These data were consistent with the ability of 15d-PGJ 2 to suppress
pro-inflammatory cytokines production in THP-1 cells stimulated with PMA [80] and
synovial fibroblasts from OA or RA patients [81]. A reduction of circulating levels of
TNFα, IL-1β and IL-6 was also reported in arthritic mice treated systemically with
rosiglitazone [82]. This suppressive effect of PPARγ agonists on cytokines was accom-
panied by an inhibition of NF-κB pathway in synovial fibroblasts [57,81] and chondro-
cytes [68] or by a reduced expression of phosphorylated I-κB (pI-κB) in inflamed joint
tissues [15]. More recently, the PPARα agonist fenofibrate was also demonstrated to
reduce the degradation of I-κB in IL-1-stimulated synovial fibroblasts [83], further
suggesting that the modulation of pro-inflammatory cytokines by PPAR agonists may
occur, at least in part, by inhibition of the NF-κB pathway.
Besides, PPAR agonists were reported recently to stimulate IL-1 Ra production in

synovial fibroblasts [50], chondrocytes [52] and THP-1 cells [80]. Although there was
some discrepancy, due to differences in cell types or experimental conditions [50,80],
these data demonstrated that PPARγ agonists were able to correct the imbalance be-
tween IL-1β and IL-1Ra towards a less pathological state. Furthermore, the stimulating
effect of PPAR agonists on IL-1Ra production was supported by activation of either the
β/δ [50] or the α [52] subtype. Finally, the stimulation of IL-1Ra was thought to con-
tribute to the inhibitory effect of several PPARα agonists on IL-1-induced MMPs ex-
pression in chondrocytes, although the exact mechanism remains to be elucidated [52].
2.2. Inducible Arachidonic Acid Cascade
Prostaglandins (PG), mainly PGE2, are well known arachidonic acid-derived lipid me-
diators that are produced in excessive amounts within inflammatory joints [84]. The
effects of PGE2 on joint tissues vary with the differentiation status of chondrocytes, but
it is generally accepted that PGE2 can contribute to the formation of an altered cartilage
matrix [85–88], the maintenance of synovitis [84,89,90] and to an accelerated bone
turnover [91,92]. Synthesis of PGE2 is a multi-step process involving a preferential
enzymatic coupling between constitutive and inducible isoforms of phospholipases A2
(PLA2), cyclooxygenases (COX) and terminal PG synthases [93]. Besides COX-2,
membrane Prostaglandin E synthase-1 (mPGES-1) was shown to play a key limiting
role in the stimulated synthesis of PGE2 in synovial fibroblasts [94] and chondro-
cytes [95]. PPARγ agonists were reported to stimulate the basal expression of COX-2,
which has a PPRE consensus site in its promoter [96], but this did not result necessarily
in an enhanced production of PGE2 [67]. In contrast, PPARγ agonists were able to sup-
press cytokine-induced PGE2 production in human [68] and rat [67] chondrocytes, as
well as in human synovial fibroblasts [27], with a greater inhibitory potency on
mPGES-1 than on COX-2. Although there was some species differences in the contri-
bution of PPARγ to the effect of 15d-PGJ2 [67], this agonist was therefore considered
to act as a “dual agent” on arachidonic acid cascade in OA chondrocytes [55]. The
transcriptional regulation of human COX-2 and mPGES-1 by pro-inflammatory cyto-
kines involves overlapping, but distinct, signalling pathways [27,97] which were inhib-
ited by interference of PPARγ agonists with histone acetylase p300 activity [20] or the
transcription factor early growth response factor-1 (Egr-1) [27], respectively. A major
role of NF-κB was suggested in the control of mPGES-1 transcriptional activity in rat
chondrocytes [67], but an indirect regulation cannot be ruled out since NF-κB was also
shown to regulate the early expression of Egr-1 [98]. These data underline that PPARγ
agonists have a multi-step regulatory role on inducible arachidonic acid cascade and
provide a rationale for possible negative feedback regulatory loops in joint cells [70]
and during the resolution phase of acute inflammation [39].
It is important to recall that, beside their inhibitory property on prostaglandins syn-
thesis, some non selective COX inhibitors were able to activate PPARα and γ in trans-
activation assays [99], whereas inhibition of PPARβ/δ by sulindac sulfide might
contribute to its antiproliferative potency on colon cancer cells [100]. Although provid-
ing an additional mechanism to some genomic effects of NSAIDs, as their
COX-independent chemopreventive properties [101], the therapeutical relevance of
these data remains uncertain in OA for at least three reasons: i) PPAR modulation by
NSAIDs was observed at doses which were consistent with adipocytes differentiation
but far above those required for inhibition of COX isoenzymes in chondrocytes [102]
and synovial fibroblasts [103]; ii) loss of PPAR stimulation by COX-derived metabo-
lites [40] could be insufficiently compensated by the weak agonist potency of NSAIDs;
iii) increased levels of lipoxygenase-derived metabolites (see § 1.3.1), secondary to
COX-inhibition, could compete with NSAIDs for activating PPARs [104].
3. Modulation of Extracellular Matrix Remodelling by PPAR Agonists
3.1. Matrix Metalloproteases (MMPs)
Matrix metalloproteases (MMPs) are thought to play a major role in cartilage degrada-
tion in OA [105] even if the promising efficacy of their inhibitors in animal models
[106,107] has not been confirmed in OA patients [108] or raised safety concern [109].
Amongst MMPs, stromelysin-1 (MMP-3) and aggrecanases (ADAMTS-4 and -5) were
implicated in the degradation of proteoglycans, though at different cleavage sites in the
interglobular domain of aggrecan [110], whereas collagenase-1 (MMP-1) and -3
(MMP-13) contributed actively to the cleavage of specific collagens [111]. Gelatinases
(MMP-2 and -9) may play a secondary role in cartilage breakdown since they have less
specificity for a given extracellular matrix component while contributing to the re-
moval of matrix fragments [112]. PPARγ agonists were shown to reduce IL-1-induced
MMP-1 expression or activity in synovial fibroblasts [55] and chondrocytes [28] and,
in the later case, this was accompanied by a reduced degradation of proteoglycans. The
transcriptional down-regulation of MMP-1 gene involved the reduction of activator
protein (AP)-1 binding [55] which was ascribed to DNA binding competition on a
composite PPRE/AP-1 site in the MMP-1 promoter [28]. More recently, the PPARα
agonist clofibrate was also shown to suppress the inducing effect of IL-1β on MMP-1,
-3 and -13 expression in rabbit chondrocytes, but this effect was supported by an in-
crease in soluble IL-1Ra production [52]. In human chondrocytes, PPARγ agonists
were also reported to inhibit IL-1β-induced MMP-13 production at the transcriptional
level, by interfering with the activation of AP-1 and NF-κB [48], a finding consistent
with the dual decrease of IL-1β and MMP-13 levels and the reduced severity of carti-
lage lesions in OA animals treated with pioglitazone [113]. Finally, agonists of PPARγ
prevented the expression of the aggrecanase-generated epitope NITEGE in cytokine-
treated rat chondrocytes while reducing the expression of MMP-3 and -9 and subse-
quent occurrence of the MMP-generated epitope VDIPEN [65]. Although there is no
firm demonstration that PPAR are playing a role, these data can be brought together
with the ability of n-3 PUFAs to prevent glycosaminoglycans release as well as
ADAMTS-4, MMP-3 and -13 expression in human OA cartilage exposed to IL-1 [42].
Taken together, these data support that PPAR agonists, mainly of the γ subtype, have
an anticatabolic potency towards MMPs and may prevent cartilage degradation in OA.
3.2. Growth Factors
Transforming growth factor (TGF-β) is a multifunctional cytokine that plays an impor-

tant role in tissue repair [114] but has a complex pathophysiological role in OA. On
one hand, TGF-β stimulates the synthesis of cartilage-specific components [115] and
counteracts the suppressive effect of inflammatory cytokines on them [116], while it
controls MMPs activity by the synthesis of their natural tissue inhibitors [117]. On the
other hand, its intra-articular administration [118] or joint overexpression [119] is in-
flammatory with the synovial-layer-dependent formation of osteophytes [120], whereas
it induces the expression of aggrecanase-1 in joint cells [121,122] with the subsequent
occurrence of matrix-derived neo-epitopes. In many other tissues, TGF-β contributes to
organ fibrosis by favouring matrix overload but its stimulatory effect on collagens
[58,123,124] and fibronectin [125] synthesis is reduced by PPARγ agonists. Such in-
hibitory potency may contribute to the preventive effect of some PPAR agonists on
skin [58], lung [124], kidney [126] or liver [127] fibrosis. It was demonstrated, very
recently, that selective agonists of either PPAR subtypes were able to suppress the
stimulatory effect of TGF-β on PGs synthesis and deposition by interfering with aggre-
can gene expression in chondrocytes maintained in tridimensional culture [53]. Al-
though it remains unclear to what extent such anti-anabolic effect may balance with the
respective cytokine suppressive potency of TGF-β and of PPAR agonists in cartilage,
these data suggest that PPAR agonists could be deleterious in situation of cartilage re-
pair while being protective in situation of cartilage degradation.
Insulin-like growth factor (IGF)-1 is another cytokine sharing in common with
TGF-β the ability to stimulate the production of extracellular matrix components [128]
and to counteract their degradation [129], although it remains less mitogenic, at least on
mature chondrocytes [130]. In OA cartilage, the increased expression of IGF-1 com-
bined with the normal level and functionality of IGF-1 receptors has led to the proposal
that chondrocytes become hyporesponsive to IGF-1, because the bioavailability of this
growth factor is reduced secondary to an increased production of IGF binding proteins
(IGFBP) [131]. In non articular cell types, PPARγ agonists were shown to inhibit sev-
eral biological responses to IGF-1 [132] while stimulating the production of IGFBP-1
[133,134]. More recently, selective agonists of either PPAR subtypes were demon-
strated to stimulate the expression of IGFBP-1, -2 or -5 and to decrease those of
IGFBP-6 whereas IGFBP-3 and -4 remained unresponsive in liver and kidney cell
lines [135]. Unfortunately, IGFBP-1 and -6 are not secreted by articular cartilage or
chondrocytes [131] and IGFBP-3 is expressed predominantly in OA conditions [136]
with a maximal content in the more severe lesions [137]. So, PPAR agonists may have
a poor theoretical potency to relieve the IGF-1 hyporesponsiveness of OA cartilage
although one cannot exclude that they could interfere with the cell-specific proteases
that compromise locally the functional activity of growth factor binding proteins [138].
4. Effect of PPAR Agonists in Animal Models of Rheumatic Diseases
4.1. Arthritis Model
Agonists of PPARγ were shown to reduce the severity of experimental polyarthritis in

rat [83,139] and mice [15,82,140]. A concomitant reduction of synovitis was reported
with the natural agonist 15d-PGJ2 [139,140] or several glitazones given preventively or
therapeutically [15,82,139], and more recently with the PPARα agonist fenofibrate [83].
Efficacy was associated with an overall reduction of inflammatory genes [15,82,140]
and oxidant stress [82,140], although contradictory data were reported for the effect of
PPARγ agonists on apoptosis of synovial fibroblasts [15,139]. However, one must con-
sider that the anti-arthritic effect of PPAR agonists was obtained by parenteral route
[139,140] or with high oral doses [83,139], supporting that their anti-inflammatory
effect necessitated much higher doses than required to lower circulating lipids or in-
crease insulin sensitivity. Indeed, the dose ratio between anti-inflammatory and insulin
sensitizing properties was estimated to be over 100-fold for troglitazone in rat, although
without obvious liver toxicity [139].
4.2. Experimental Osteoarthritis (OA)
There is only one report evaluating the effect of the PPARγ agonist pioglitazone in the
menisectomy model of OA in guinea pig [113]. When given at 20 mg/kg/day from
1 day after surgery until necropsy, pioglitazone reduced the severity and extent of carti-
lage lesions in the knee joint but was ineffective on osteophytes formation. Prevention
of macroscopic and morphologic changes was accompanied by a reduction of the per-
centage of chondrocytes staining positively for MMP-13 or IL-1β in OA cartilage, sug-
gesting that interference with IL-1 signalling in articular chondrocytes may contribute
to the protective effect of pioglitazone. As for polyarthritis, cartilage sparing was ob-
tained for a higher dose than used in antidiabetic studies although the circulating levels
remained in the range of those reported in humans treated with the highest recom-
mended dose of pioglitazone [113]. As non-insulin dependent diabetes mellitus is also
a systemic risk factor for the development of OA [141], there is possibility that glita-
zones could reduce cartilage destruction by their dual cytokines suppressive and insulin
sensitizing properties in diabetic patients with OA.
5. Conclusion and Future Trends
Several in vitro and animal studies have demonstrated that PPAR subtypes are ex-
pressed and functional in joint cells and that their activation down regulates the tran-
scriptional machinery to reduce pro-inflammatory cytokines and metalloproteases pro-
duction. From that point of view, PPAR agonists can prevent joint inflammation and
cartilage destruction to various degrees. However, one must also consider that most
anti-inflammatory effects were reported at concentrations of agonists far above their
binding affinity for PPAR subtypes. In addition, PPARγ agonists were able to prevent
fibrosis of soft tissues by reducing the synthesis of extracellular matrix components and
to cause bone mass loss in some mouse models [142]. This suggests that joint inflam-
mation is less sensitive to PPAR activation than lipids and glucose homeostasis and
that PPAR agonists could have a complex interplay with the turn-over of cartilage and
bone matrices. So, it seems logical to speculate that the modulation of OA by PPAR
activation will come to fruition if we can demonstrate firstly that diabetic patients
treated with PPAR agonists are at lower risk of developing OA and/or develop less
severe cartilage lesions without significant bone changes.
Note Added to Proof
During the processing of this manuscript, an additional work demonstrated that piogli-
tazone (30 mg/kg/day) reduced the development of cartilage lesions in the anterior cru-
ciate ligament transection model of OA in dog (Boileau C. et al., Arthritis Rheum 56
(2007), 2288-98). This protective effect was accompanied by a reduced expression of

matrix degrading enzymes (MMP-1, ADAMTS-5) and less activation of inflammatory
signaling pathways as ERK1/2, p38MAPK or NF-κB in OA cartilage.
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Part II
Signalling Mechanisms
IOS Press, 2007
VII
MAP Kinases
Charles J. MALEMUD
Department of Medicine, Division of Rheumatic Diseases, and Department of Anatomy,
Case Western Reserve University, School of Medicine, Cleveland, Ohio,
44106-5076 (USA)
Abstract. Mitogen-activated protein (MAP) kinase activation by cytokines and

other soluble mediators in articular chondrocyte cultures was shown to reproduce
critical components relevant to cartilage development, synovial joint inflammation
as well as human and animal osteoarthritic pathology. MAP kinase activation has
been shown to be critical in cartilage formation Cytokines, such as interleukin-1β
and tumor necrosis factor-α, soluble mediators, such as nitric oxide, and growth
factors, namely fibroblast growth factor, connective tissue growth factor, vascular
endothelial growth factor and hepatocyte growth factor activate specific MAP
kinases resulting in nuclear factor-κB activation. NF-κB is a transcription factor
which regulates matrix metalloproteinase gene expression, induces chondrocyte
programmed cell death, up-regulates chondrocyte cytokine gene transcription as
well as suppressing extracellular matrix protein biosynthesis, events that are con-
sistent with synovial inflammation and the resultant destruction of articular carti-
lage in osteoarthritis.
Introduction
The mitogen-activated protein (MAP) kinase family is made up of the extracellular

signal-regulated protein kinases (ERKs), p38 kinase in its various isoforms (i.e. α, β1,
β2, γ, δ) and C-Jun-N-terminal kinase (JNK) [1,2]. Osteoarthritis (OA) is now recog-
nized as a disease ‘process’ that is, in part, characterized by “non-classical inflamma-
tion” of synovial joints and a significant imbalance between anabolic and catabolic
pathways which ultimately result in the destruction of articular cartilage [3,4].
It is appreciated now more than ever that MAP kinases play a prominent role in
both the pathogenesis and progression of OA through their capacity to activate tran-
scription factors such as nuclear factor κB (NF-κB) that result in suppressed extracellu-
lar matrix (ECM) protein biosynthesis and up-regulated matrix metalloproteinase
(MMP) gene expression. NF-κB activation also regulates pro-inflammatory cytokine
gene transcription [5]. Because MAP kinases appear to play an integral role in OA
pathogenesis by altering synovial joint allostasis, it has become critical to identify
which MAP kinases are specifically activated during the early stages of OA. This in-
formation would be crucial for understanding which specific MAP kinases regulate
various cellular events such as chondrocyte proliferation, cytokine production, pro-
grammed cell death (i.e. apoptosis), ECM protein biosynthesis as well as ECM protein
turnover and degradation; processes that are all central to OA [6,7]. In addition, identi-
100 C.J. Malemud / MAP Kinases
fying which MAP kinases are activated in the course of developing gross and micro-
anatomic changes that are consistent with OA pathology may provide the fundamental
underpinning for developing small molecule inhibitors (SMI) of MAP kinases for use
in the future medical therapy of OA [8].
This review will focus on the substantial amount of compelling evidence that MAP
kinases are integral to the initiation and progression of OA by critically examining the
relevant literature demonstrating that specific MAP kinase activation occurs in chon-
drocyte cultures in response to cytokines and other mediators known to be critical to
OA pathogenesis and progression. Further, this review will also comment on the few
studies of MAP kinase activation in experimentally-induced animal models of OA.
MAP Kinase Activation
MAP kinase activation is required in order for MAP kinases to phosphorylate target
protein substrates. MAP kinase activation is generally carried out by at least seven up-
stream protein kinases (PKs) belonging to the MAP kinase kinase (MEK/MKK) protein
family [2]. At least 4 MKK genes have been cloned from mammalian cells [9]. MKK
activity is regulated by additional upstream MKKs (i.e. MKKKs and MKKKKs) that
are either tyrosine- or serine-binding proteins which may also require low molecular
weight GTP-binding proteins as co-factors for MKKK activation. In this regard, recent
evidence indicated that it is an MKK-MAP kinase complex organized by scaffolding
proteins that allows for specific MKKK activation selectivity by GTPases, additional
PKs and receptors [10].
Pro-inflammatory cytokine gene transcription, best exemplified by interleukin-1β
(IL-1β) and tumor necrosis factor-α (TNF-α) gene expression is elevated in OA carti-
lage and OA synovium [4]. IL-1β and TNF-α gene transcription and their biological
activities are regulated by MKK activation and MAP kinases. Thus, in the cellular
model proposed by Eder [11] on how MAP kinase activation regulates IL-1β and
TNF-α biological activities, MKKKs were proposed to be recruited to either IL-1 or
TNF-α signaling pathways where MKKKs became bound to the TNF-α receptor acti-
vating factor (TRAF) thus promoting MKK phosphorylation and downstream MAP
kinase activation. The Eder model [11] made a case for a common pro-inflammatory
cytokine pathway (i.e. ‘pathway redundancy’) so that either IL-1 or TNF-α could result
in MKK activation via TRAF with specific pathway bifurcation occurring downstream
at the JNK or p38 kinase activation site as well as through NF-κB activation. Addi-
tional evidence for the role of MKK activation involving a TNF-α gene expression
autocrine loop came from studies that showed that arctigenin, an inhibitor of MKK1
activity, blocked TNF-α mRNA transcription and TNF-α protein production by
Raw264.7 cells stimulated with lipopolysaccharide which was also dependent on acti-
vator protein-1 (AP-1) activity [12].
ERKs
ERK Structure
ERKs were originally isolated from a rat brain cDNA library probed with DNA encod-
ing the kinase domain of the insulin receptor-related protein [13]. The ERKs identified
C.J. Malemud / MAP Kinases 101
in that study contained all the amino acid residues conserved within protein-tyrosine
kinases. Subsequently, the human ERK gene was localized to chromosome
1p36.1 [14]. This study also provided the first evidence that ERK proteins contained a
receptor-like membrane-spanning structure. Of the ERKs that have been extensively
studied, ERK1 (i.e. p44) and ERK2 (i.e. p46) are the most abundant ERKs [15,16], but
other ERK forms (i.e. ERK-3, -5, -7, -8) have also been isolated [17–19]. Indeed, addi-
tional evidence has indicated that specific ERK1 isoforms, namely, ERK1b, a 46kDa
protein [20] which like ERK1 or ERK2 was activated by MEK1. However, ERK1b
activation did not parallel that of ERK1. ERK1c is a 42kDa protein which is an alterna-
tively spliced form of ERK1b found to be widely distributed in tissues and cells.
ERK1c could be activated by MEKs, although ERK1c activation and its slower inacti-
vation by phosphatases distinguished ERK1c from ERK1 [21]. In addition, Eblen et al.
[22] showed that Rac (a member of the Ras-oncogene family) in association with p21-
activated kinase (PAK) enhanced ERK2 signaling. In COS cells, MEK1 was found in
association with ERK2. Further, the lack of inducible binding between ERK2 and
MEK2 was likely to be a result of that fact that MEK2 is not a suitable substrate for
PAK. These results suggested the view that there likely existed a significant degree of
specificity in the MEK/ERK activation system [22].
ERK Activation
ERKs are rapidly phosphorylated in response to various extracellular stimuli, but espe-
cially by growth factors such as epidermal growth factor, nerve growth factor and insu-
lin-like growth factor-1 (IGF-1) [23,24]. When ERK activation is transient, ERK phos-
phorylating activity rapidly declines and evidence shows that c-Fos, a critical ERK
substrate becomes unstable and is not suitably activated [25]. However, if ERK phos-
phorylation is sustained or prolonged, c-Fos is phosphorylated by ERK and the 90kDa
ribosomal S6 kinase resulting in c-Fos becoming fully active. This ERK activation
mechanism also appears to be dependent on the ERK targeting DEF domain in c-Fos.
Thus, the ERK/DEF domain appears to be dominant in regulating ERK/c-Fos docking
which modulates cell cycle progression and cellular transformation [25].
ERK Nuclear Translocation
ERK translocation to the nucleus appears to be highly dependent on specific residues in

the ERK activation loop since ERK2 mutations created in this domain, especially in
residues 176–181, were shown to be responsible for ERK2/MEK1 dissociation upon
mitogenic stimulation [26]. In contrast, residues 176-181 as well as residues required
for ERK2 dimerization did not appear to play a significant role in ERK2 trafficking
through nuclear pores [26].
ERK Inactivation
ERKs are inactivated by MAP kinase phosphatases (MKPs). In the case of MKP3,
ERK deactivation occurs within a complex of MKP3 and ERK and further, MKP3 may
also prevent phospho-ERK homodimerization and any additional ERK activation by
MEK [27]. There appears to be significant MKP specificity. For example, when MKP4
was expressed in COS-7 cells, it blocked activation with the selectivity profile of
ERK > p38 kinase = JNK [28]. Whereas MKP5 and MKP7 selectively deactivated
p38α and β kinase, but not p38 γ and p38 δ kinases, MKP7 bound to and inactivated
JNK/SAPK, but not ERK [29].
ERKs and Chondrogenesis
Studies of in vitro chondrogenesis under basal conditions showed that ERK activity
declined as chondrogenesis occurred [30]. However, Murakami et al. [31] showed that
FGF-mediated up-regulation of Sox9, an important transcription factor that regulates
Type II, IX and Type XI collagen as well as link protein and aggrecan gene expression
during chondrogenesis [32,33] was ERK-dependent. In that regard, Sox9 gene expres-
sion could be ablated by inhibiting ERK 1/2 with an MEK SMI during in vitro chon-
drogenesis.
By contrast, ERK activity was also shown to be rapid, but transient, when in vitro
chondrogenesis was stimulated by growth-differentiation factor-5 (GDF-5) [34]. In
addition, the ECM of cartilage-constructs made on polylactic acid-co glycolic acid
(PGLA) scaffolds using human mesenchymal stem cells contained aggrecan and Type
II collagen when transforming growth factor-β was added to the PGLA scaffold [35].
The emergence of the chondrogenic phenotype appeared, however, to only partially
involve ERK signaling pathways as MEK inhibition resulted in down-regulation of
Type II collagen, but not aggrecan. These results appear to partially sustain previous
studies by Kim et al. [36] and Legendre et al. [37] who showed that maintenance of the
chondrogenic phenotype was only partially dependent on ERK 1/2 activity. Additional
studies by Yagi et al. [38] using small interference mRNA (siRNA) bcl-2 (an anti-
apoptosis protein) in the presence of caspase inhibitors have added an additional level
of complexity to the previously cited studies relating ERK activity to chondrogenic
expression [30–37] because Yagi et al. [38] showed that blc-2 siRNA blocked Sox9
expression in rat chondrocytes via MEK/ERK 1/2, suggesting that bcl-2 could also be a
critical regulator of MEK/ERK 1/2-mediated chondrocyte differentiation in vitro.
A recent analysis showed that functional cartilage loading in young female Wistar
rats induced AP-1 and Runx2 transcription factor activity that were, in part, dependent
on ERK activity [39]. These results suggested that ERK was also critical for the differ-
entiation and maturation of cartilage tissue development in vivo. In this regard, Lai
et al. [40] showed that β1 integrin interaction with ECM proteins could stimulate both
p38 and ERK 1/2 in the CFK-2 chondrocytic cell line. In that study, parathyroid stimu-
lating hormone inhibited both p38 and ERK 1/2 activation, but Indian hedgehog (Ihh)
protein expression was down-regulated primarily through ERK 1/2. Ihh has been
shown to be an important component in chondrocyte terminal differentiation and endo-
chondral ossification [41].
ERK Activation and Chondrocyte Proliferation
ERK activation was shown to play a prominent role in chondrocyte responses to fibro-
blast growth factor (FGF). Thus, FGF inhibited adult chondrocyte proliferation and an
SMI of MEK 1/2 blocked rat chondrosarcoma proliferation as well as ERK 1/2 and p38
signaling which was also dependent on inactivating the retinoblastoma family proteins
p107 and p130 [42].
Events Critical to OA Appear to Be ERK-Dependent
At the cellular level, OA pathogenesis and progression resulting in articular cartilage

destruction appears to involve various elements of the inflammatory response, includ-
ing cytokine gene up-regulation, prostaglandin production, MMP gene up-regulation,
synthesis and activation, ECM protein gene dysregulation, suppressed ECM protein
synthesis, apoptosis, systemic disturbances in the growth hormone/IGF-1 pathway as
well as inefficient chondrocyte proliferation and cartilage repair [2–4,43,44].
ERK Activation and OA

With regard to the role of ERK activation and its potential role in OA pathogenesis,
Martel-Pelletier et al. [6] showed that stimulation of nitric oxide (NO) by the pro-
inflammatory cytokine, IL-17 (which can create a chondrocyte anabolic/catabolic im-
balance) was dependent on MEK-1/2 and MEK-3/6 activity in cultured human os-
teoarthritis chondrocytes. The MEK-1/2 SMI, PD98059 was able to block IL-17 in-
duced inducible nitric oxide synthase (iNOS) and production of NO.
ERKs and Cytokine GeneUp-Regulation

Cytokine gene up-regulation in OA is best exemplified by the increased levels of IL-1
and TNF-α found in OA synovial fluid. The increase in IL-1 and TNF-α in OA is likely
a result of activating both synoviocyte and chondrocyte IL-1 and TNF-α [4]. It is evi-
dent, however, that activation of a specific MAP kinase such as ERK 1/2 alone yielded
only modest increases in IL-1 and TNF-α gene up-regulation whereas activation of
p38, JNK, ERK 1/2 and PI3K resulted in full TNF promoter activity [45].
Anabolic and Catabolic Imbalance in OA Are Partially Dependent on ERK Activity

IL-1 plays a central role in chondrocyte dysfunction in OA [6,7]. Active IL-1 was
found in synovial membrane isolated from OA joints [46] indicating that activated
synoviocytes are a primary source for IL-1 in OA and also critical to IL-6 up-
regulation. More recently, however, Fan et al. [47] showed that IL-1β induction of IL-6
and leukemia inhibitory factor (LIF), both recognized as playing a role in chondrocyte
MMP activity and ECM protein degradation was partially dependent on ERK 1/2 in
human osteoarthritis chondrocyte cultures, but neither IL-1 nor LIF were strongly ex-
pressed in OA cartilage. Further, Raymond et al. [48] identified an IL-1β-responsive
element in the MMP-1 (i.e. collagenase-1) promoter containing a consensus CCAAT
enhancer-binding protein (C/EBP) site at threonine-235 which was ERK-dependent
since the ERK SMI, PD98059, reduced C/EBP phosphorylation. That study [48] de-
fined a novel role for C/EBP in IL-1β-induced MMP-1 gene transcription in which
dominant-negative (dn) ERK1 and ERK2 constructs also suppressed IL-1β-induced
MMP-1 promoter transactivation. Selective ERK and NF-κB inhibitors were also
shown to partially, but significantly, block MMP-1, MMP-13 (i.e. collagenase-3) and
Type II collagen gene transcription in human chondrocyte cultures after IL-1β treat-
ment [49] suggesting that ERKs play a significant role in not only regulating MMP
gene transcription but also in maintaining the chondrogenic phenotype as well.
Diacerein through its active metabolite Rhein has been tested for its chondropro-
tective properties. Rhein was shown to protect bovine chondrocytes from IL-1β-
induced MMP-1 gene up-regulation and block ECM protein suppression [50] by inhib-
iting the MEK/ERK pathway as well as by suppressing downstream NF-κB and AP-1
activity.
ERKs and Chondrocyte Apoptosis

Apoptosis is a significant event in cartilage degeneration because apoptosis results in a
reduction in cartilage vitality and chondrocyte proliferation, both of which are required
for efficient cartilage repair [51]. In that regard, recent studies showed that BAY11-
7085, a potent anti-inflammatory agent used to treat rat adjuvant arthritis induced hu-
man chondrocyte apoptosis [52] which was dependent on sustained ERK 1/2 activa-
tion. Thus, ERK 1/2 inhibition might provide chondroprotection and prevent chondro-
cyte apoptosis induced by IL-1β or TNF-α [8].
Another site for suppressing the apoptosis cascade resides in the induction of
proto-oncogenes by external stimuli. Thus, Islam et al. [53] showed that cyclic hydro-
static pressure induced p53 and c-myc gene expression and apoptosis in human os-
teoarthritis chondrocyte suspension cultures. Based on the finding that myc suppressed
ERK signaling in chick embryo fibroblasts it has been suggested that the myc-binding
site (BMD) of the protein bin-1 (which negatively regulates myc activity) could be
exploited to regulate apoptosis via its ability to stimulate ERK activity [54].
Human chondrocytes treated with IL-1β produced elevated NO and cyclooxy-
genase-2 (COX-2) levels [55]. Notoya et al. [56] previously showed that human chon-
drocyte apoptosis induced by NO was COX-2 dependent. In this regard, Nieminen
et al. [57] demonstrated that IL-1β transiently activated ERK 1/2 as well as p38 kinase
and JNK in immortalized human T/C28a chondrocytes resulting in elevated COX-2
and prostaglandin E2 (PGE2) levels which were suppressed by PD98059. By contrast,
SMI p38 kinase and JNK inhibitors, SB203580 and SP600125, respectively, had more
variable effects on COX-2 activity and PGE2 production.
ERKs and Cartilage Repair

Tissue-engineering is a promising approach for stimulating cartilage repair in OA. For
cartilage repair employing artificial cartilage implants, tissue-engineered cartilage-
constructs would most likely have to be implanted in cartilage where high weight-
bearing occurs and where significant cartilage degeneration in OA has already been
identified. For this reason, it is imperative that the integrity of the tissue-engineered
cartilage ECM proteins be optimized so that biomechanical properties of the implants
are maintained. Thus, loss of the chondrocyte phenotype by modulation of cartilage-
specific gene expression would be expected to significantly compromise potential carti-
lage repair by these implants. In this regard, Wenger et al. [58] recently demonstrated
that human osteoarthritis chondrocytes grown on a scaffold of HYAFF®-11 (Advanced
Biopolymers, Termo, ITALY) in mini-bioreactors synthesized significant amounts of
sulfated-proteoglycan and Type II collagen, but not Type I or Type X collagen. How-
ever, cyclic hydrostatic pressure (CHP) employed at 5MPa using a sinusoidal fre-
quency of 1Hz significantly increased apoptosis in these cartilage-constructs. Studies
reported by Schulz-Tanzil et al. [59] also showed that loss of chondrogenic expression
by human chondrocytes in culture was characterized by reduced Type II collagen bio-
synthesis, α3 integrin expression, src-homology collagen (Shc) and ERK activity that
eventually resulted in chondrocyte apoptosis. It will therefore be interesting to deter-
mine the extent to which key signaling proteins in the ERK and Ras-mitogen-activated
PK pathway [59] contribute to CHP-induced apoptosis in tissue-engineered human
cartilage constructs and whether sustained ERK activity or ERK inhibition alters the
chondrogenic phenotype.
Finally, IGF-1 appears to be a critical circulating growth factor required for stimu-
lating chondrocyte proliferation and for up-regulating proteoglycan synthesis [60]. In
this regard, Starkman et al. [61] showed that PD98059 and another, MEK SMI,
namely, U0126 blocked IGF-1-stimulated ERK activation, but failed to alter IGF-1
stimulated proteoglycan synthesis. Instead, IGF-1 stimulated the PI3K/Akt pathway
which when blocked by Akt inhibitors suppressed IGF-1 stimulated proteoglycan syn-
thesis. Further, IGF-1 failed to stimulate aggrecan, decorin or biglycan mRNA levels in
the presence of PD98059 indicating that IGF-1-mediated proteoglycan synthesis up-
regulation occurred primarily through its effect on modulating protein translation.
P38 Kinase
P38 Kinase Structure
The most extensively studied p38 kinase isoform structure is that of p38α because of its
reported involvement in inflammation. This includes the critical role p38α plays in
modulating COX-2 and iNOS gene expression as well as in controlling NO production
induced by cytokines [61]. Although the functional distribution of α and β2 p38 kinases
are well documented, much less is known about the tissue distribution of β1, γ or δ p38
kinase isoforms [62,63].
A significant amount of p38 kinase structural data emerged from the development
of p38 SMIs, such as the diarylimidazole, triarylimidazole and triarylpyrrole
SMIs [64,65]. These studies revealed that the molecular basis for the specificity of
these SMIs resided in the p38 threonine-106 residue located in the p38 ATP-binding
pocket. Further, the peptide-substrate binding site and ATP-binding site for p38 was
structurally distinct from ERK2 [66]. Functional studies also revealed several differ-
ences between p38 kinase isoforms. Thus, Li et al. [67] showed that in contrast to p38α
and β, p38γ did not phosphorylate activating transcription factor-2 or MAPK activated
protein kinase-2, but was able to phosphorylate myelin basic protein. Further, p38δ
activity was not blocked by pyridinyl imidazole derivative, SB202190, an SMI of
p38α, β and γ isoforms. Nonetheless p38δ could be activated by MKK3 and MKK6,
which are known activators of the several p38 kinase isoforms [68].
P38 Kinase Activation
In a fashion similar to other MAP kinases, p38 kinase activation requires up-
stream MEK/MKK activity with MKK3, MKK4 and MKK-6 implicated in this proc-
ess [69,70].
Role of p38 in Chondrocyte MMP Production
The current interest in p38 and its role in chondrocyte MMP gene transcription stems
from the fact that the pro-inflammatory cytokines that up-regulate chondrocyte MMPs
also activate p38 signaling pathways [71–73]. P38 kinase is also prominently involved
in MMP-9 (i.e. 92Kda gelatinase) and MMP-13 gene up-regulation stimulated by cyto-
kines, such as IL-1β [74], indicating that MMPs with potent activity towards aggrecan,
Type II collagen and denatured collagen are regulated by p38 phosphorylation.
Geng et al. [75] were among the first to demonstrate that IL-1β and TNF-α caused
selective activation of human chondrocyte p38, ERK and JNK. In that regard Geng
et al. [75] showed that ERK 1/2 activation brought about by several factors, including,
platelet-derived growth factor, IL-6 and IGF-1 contrasted sharply with the more re-
stricted activation of p38 and JNK brought about by p38 and JNK activation. Geng
et al. [75] concluded that MAP kinase activation was likely to be early events in cyto-
kine-mediated initiation of the catabolic/anabolic imbalance thought to cause the early
surface lesions in OA animal and human cartilage. In a subsequent study, IL-17, was
also found to be capable of stimulating the expression of several additional mediators
of cartilage ECM protein degradation and apoptosis, including COX-2, iNOS, IL-1β,
IL-6 and MMP-3 (i.e. stromelysin-1) in human chondrocyte cultures [76]. The produc-
tion of these pro-inflammatory mediators was, in part, dependent on p38 activation
since the p38 SMI, SB203580, suppressed IL-17-mediated changes in COX-2, iNOS,
IL-1β, IL-6 and MMP-3. More recently, additional activators of chondrocyte MMP
gene transcription have been reported that also involve p38 activation. Thus, Mengshol
et al. [77] reported that IL-1 induced MMP-13 in the SW-1353 chondrosarcoma cell
line resulted in p38 and JNK activation via Runx2 transcriptional activity, but the
MMP-13 promoter response was primarily regulated by p38 acting via Runx2 and
AP-1 activation. The results of the studies reported by Mengshol et al. [77] were re-
cently confirmed by Pei et al. [78] who showed that over-expression of Runx2 stimu-
lated MMP-13 gene transcription but had no effect on MMP-1 in either SW 1353 chon-
drosarcoma cells or human articular chondrocytes. In that study [78], MMP-13 gene
transcription was found to be dependent on Runx2 phosphorylation that resulted from
p38 kinase activation.
Additional signaling pathways have been explored to determine their significance
in chondrocyte MMP gene up-regulation by IL-1. Thus, by employing licofelone, an
inhibitor of cyclooxygenases and 5-lipoxygenase, Boileau et al. [79] demonstrated a
dose-dependent inhibition by licofelone on IL-1-induced MMP-13 gene transcription in
human osteoarthritis chondrocytes. This inhibition correlated with reduced p38 and
AP-1 activity which also involved the cyclic AMP response element binding protein.
Of note, licofelone failed to inhibit ERK 1/2 or JNK activation.
MMP-1 and MMP-13 gene transcription inhibition was also seen in IL-1β-
stimulated human osteoarthritis chondrocytes after treatment with pomegranate fruit
extract which correlated with p38 inhibition, but not ERK or JNK inhibition [80]. Of
interest, hyaluronan oligosaccharides (HA-oligos) stimulated chondrocyte MMP-13
gene transcription which was found to be p38 kinase and NF-κB-dependent [81].
Whether hyaluronan depolymerization or fragmentation actually occurs within the
synovial joint inflammatory milieu remains to be determined as is the potential rele-
vance of HA-oligos to MMP-13-mediated cartilage degradation. Finally, the novel
highly specific p38 SMI R-130823 decreased MMP-1 and MMP-13 gene transcription
as well as PGE2 production in IL-1β-stimulated human and bovine chondrocytes fur-
ther supporting the view that p38 kinase is the predominant activated MAPK regulating
chondrocyte IL-1β-stimulated MMP-1 and MMP-13 gene transcription [82].
Potential Role for p38 in Chondrocyte Senescence, ECM Synthesis and Apoptosis
Reduced chondrocyte viability and proliferation limits repair of surface defects in aging
and OA articular cartilage [3]. Kang et al. [83] employed the p38 SMI SB203580 or dn
mutations in MKK6 or p38 to assess the role of p38 in rabbit articular chondrocyte
proliferation and senescence. They showed that p38 inhibition by any of these three
techniques stimulated chondrocyte proliferation during the active growth phase and
also extended chondrocyte life span which was telomere-independent [83].
The suppression of chondrocyte ECM protein biosynthesis by IL-1 will also di-
rectly affect cartilage repair. Thus, Radons et al. [84] recently showed using PK SMIs
that p38 and/or PI3K/JNK were involved in IL-1-induced chondrocyte IL-6 secretion,
the latter an important co-factor in regulating anabolic/catabolic balance in cartilage.
Apoptosis is also likely to limit cartilage repair in OA and may be responsible for
inefficient chondrocyte ECM production in OA [51]. NO is one of the strongest induc-
ers of chondrocyte apoptosis and, indeed, chondrocyte apoptosis and cartilage degrada-
tion have been linked in OA [85]. Recent studies have also shown that leptin can act as
a pro-inflammatory mediator and working synergistically with IL-1 stimulates iNOS in
cultured chondrocytes. IL-1/leptin added to human chondrocytes or the ATDC-5 mur-
ine chondrosarcoma cell line increased iNOS activity and was dependent, in part, on
p38 activation, but Janus kinase-2, PI3K and MEK-1 were also implicated in iNOS
gene transcription in this study [86].
Because articular cartilage oxygen tension levels are likely to be low in the joint,
the modulation of the transcription factor, hypoxia-inducible factor-1α (HIF-1α) may
also play a critical role in altering chondrocyte allostasis. In a recent study, Coimbra
et al. [87] found HIF-1α mRNA in both normal human and OA cartilage. In addition,
TNF-α increased chondrocyte HIF-1α mRNA which was partially blocked by p38
SMIs and NF-κB inhibition [87]. As a result of this finding, it will be of interest to de-
termine the extent to which HIF-1α regulates chondrocyte apoptosis under normoxic
and hypoxic conditions, since previous studies showed that TNF-α and NO which in-
duce apoptosis in cultured chondrocytes [51] was also found to regulate HIF-1α pro-
duction not only by a non-hypoxic reactive oxygen species-sensitive mechanism [88]
but also as a component of the inflammatory response by its capacity to stimulate iNOS
mRNA in activated macrophages [89].
P38 and Chondrogenesis
Under basal in vitro conditions, chondrogenesis was accompanied by p38 activa-

tion [30], whereas when ACDC-5 cell chondrogenesis was stimulated by GDF-5, p38
activation was slow and sustained [34] indicating that p38 activation was required for
chondrocyte terminal differentiation. More recently, Tuli et al. [90] showed that trans-
forming growth factor-β-induced chondrogenesis from adult bone marrow or trabecular
bone-derived mesenchymal progenitor cells resulted from p38 and ERK1 activation
and to a significantly lesser extent from JNK activation. Activation of p38 and ERK1
was also related to events which modulated N-cadherin and regulation of Wnt-7A gene
expression as well as the Wnt/β-catenin/T-cell factor pathway which appears to be
critical for N-cadherin-mediated mesenchymal progenitor cell condensation, a critical
event in chondrogenesis in vivo [41].
JNK
JNK Structure
JNK, also known as stress-activated protein kinase (SAPK), is found in 3 forms,

namely, JNK1, JNK2 [91,92] and JNK3 [93] which share about 50–80% homology
with each other, and in particular JNK3 and p38 which share 51% identity in primary
sequence [93]. Although JNK1 and JNK2 is found distributed among many tissues,
JNK3 appears to have particular significance in nervous system tissue in that it is al-
most exclusively expressed in brain with low levels expressed in kidney and testes [8].
The mouse JNK/SAPKα gene spans a region of about 36 kilo-bases and contains
13 exons, which represent about 8% of the gene sequence [94]. Splice variants are re-
sponsible for several forms of JNK/SAPKα generated by alternative splicing of exons 7
and 8. When a JNK promoter construct contained the activator protein-2 element the
JNK promoter activity was increased from 28% to 77% [94].
The Physiologic Significance of JNK
The physiological significance of JNK is primarily based on its capacity to bind and
phosphorylate the DNA binding protein, c-Jun, which increases c-Jun transcriptional
activity [73,95]. The JNK2 form was shown to bind to C-Jun with about a 25-fold
greater affinity than JNK1 and had a lower Km for c-Jun than JNK1 [95]. Further, it
was JNK2 that was primarily phosphorylated by human osteoarthritis chondrocytes in
response to IL-1β [96]. C-Jun is a well recognized component of the AP-1 complex
which is critical in cytokine and MMP gene regulation [97]. In addition, JNK has also
been reported to bind to and phosphorylate activating transcription factor-2 (ATF-2),
Elk-1, nuclear factor of activated T-cells (NFAT) and p53 [63].
JNK Activation
JNKs are activated by Rac and Cdc42 via MKK4 and MKK7 by phosphorylating JNK
on threonine-183 and tyrosine-185 [98]. MKK4 is activated primarily by IL-1β and
TNF-α [91,92] whereas MKK7 is activated by other environmental stressors, including,
IL-3, ligation of CD-40, B-cell antigen receptors for Fc, Ras/GTPases, heat, UV irradia-
tion, anisomycin, hyperosmolarity and TNF-α in hemopoietic cells and HeLa cells [98].
The JNK-interacting protein (JIP) group of scaffolding proteins was found to selec-
tively activate MKK7 by causing aggregation of the MMK7/JNK complex [99] and the
rat and human islet brain-1 protein was found to be homologous to JIP-1 [100]. In addi-
tion, the mouse and human MKK7 protein are highly structurally conserved, the latter
being very specific for JNK as evidenced by the fact that MKK7 activated JNK1, but
not p38 kinase in co-expression studies [98,101].
Role of JNK in Chondrocyte Cytokine Responses
Chondrocyte MMP gene expression induced by TNF-α was shown to be regulated

principally by JNK and p38 [102, 103] which resulted in AP-1 activation and MMP
gene up-regulation [104]. Phenyl-N-tert-butylnitrone (a spin trap agent) and epigallo-
catechin-3-gallate (EGCG) were both able to inhibit human osteoarthritis chondrocyte
Il-1β-induced JNK and p38 as well as IL-1β-induced MMP-1 and MMP-13 gene tran-
scription [96,105,106] confirming a previous report [107] which showed that IL-1β
activated rabbit chondrocyte JNK, ERKs and p38 in an IL-1β concentration- and time-
dependent manner. However, in that study [107], TNF-α only activated chondrocyte
JNK. Our laboratory recently found that TNF-α activated human chondrocyte JNK1,2
and p38 as well as STAT3 but had no effect on JNK, p38 or STAT3 protein levels as
determined by Western blotting (unpublished studies). Further, IL-1β was also shown
to cause a transient elevation in phospho-JNK, phospho-ERK and phospho-p38 in im-

mortalized human T/C28a2 chondrocytes which led to increased COX-2 and PGE2 lev-
els [57], and SP600125 a highly specific JNK SMI [8] but not its negative control
compound, namely, N1-methyl-1, 9 pyrazolanthrone, down-regulated COX-2 and
PGE2 in a dose-and time-dependent manner suggesting a post-transcriptional regulatory
mechanism.
Fibronectin (Fnf) fragments but not fibronectin (Fn) have potent biological activ-
ity, including the capacity to cause chondrocyte MMP gene up-regulation [108]. In that
regard, Forsyth et al. [109] showed that after IL-1β-treatment, a 120-kDa Fnf that binds
to the α5β integrin, but not intact Fn, or α2β1 and α5β1 integrin neutralizing antibodies,
increased human chondrocyte c-Jun and p38 phosphorylation as well as NF-κB activity
which led to a further increase in pro-MMP-13 and activated MMP-13 levels. IL-1 re-
ceptor antagonist protein did not suppress JNK or p38 activation, but did partially in-
hibit MMP-13, the latter finding suggestive of the presence of an IL-1 autocrine feed-
back loop. In addition to IL-1β, TNF-α and Fnf-mediated activation of JNK, Fanning
et al. [110] showed that mechanical static compression loads administered ex vivo
stimulated bovine cartilage explant SAPK/ERK-kinase-1 (SEK1) of the JNK pathway
with maximum SEK1 activation occurring at 1 hr and with a greater amplitude than for
either ERK 1/2 or p38. By contrast, the cartilage explant response to IGF-1 differed
considerably in that IGF-1 induced early, but transient ERK 1/2 activation, with no
sustained ERK 1/2 activation.
Finally, although chondrocyte apoptosis can be induced by IL-1β, TNF-α, and NO
as well as by biomechanical stress, [43,51,53], selective activation of JNK was insuffi-
cient to induce CC-139 fibroblast apoptosis unless the PI3K pathway was also inhib-
ited [111].
JNK and Chondrogenesis
JNK was not required for either basal in vitro or FGF-or GDF-5-stimulated chondro-
genesis [30,31,34] which was in contrast to the apparent requirement for p38 and ERK
activation under these conditions [30,34] However, Wnt-3a caused c-Jun expression
and c-Jun phosphorylation by chicken chondrocyte JNK which resulted in AP-1 activa-
tion [112]. In that study [112], AP-1 activation led to Sox9 suppression and reduced
Type II collagen synthesis. Thus, it appeared that JNK activation may regulate the
modulation of the chondrogenic phenotype by activating Wnt-3a-stimulated beta-
catenin/T-cell-factor/lymphoid enhancer-factor which is critical for determining the
sequence of events under which terminal chondrocyte differentiation occurs in vivo
[41]. In addition, the signal for chondrocyte terminal differentiation may also require
PI3K activation via protein kinase C (PKC) in CCN2/connective tissue growth factor
(CTGF)-stimulated chondrogenesis although a PKC-independent pathway involving
JNK was also reported [113].
MAP Kinases, Inflammation and OA
In vitro studies of chondrocyte MAP kinase activity as well as transcription factor acti-
vation by IL-1β, TNF-α, COX-2, NO and advanced glycation end (AGE) products have
led to the postulation that potential novel therapeutic agents directed against MAP
kinases could be employed to suppress the ‘non-classical’ inflammation of OA as well

as cartilage destruction in OA [4,43,114–118].
In addition to the MAP kinase pathways altered by cytokines and growth factors
that have already been discussed, 2 additional pathways involving MAP kinases war-
rant comment. Thus, Qureshi et al. [119] showed that the ERK 1/2 SMI, PD98059 or
U0126 were capable of potentiating TGF-β-stimulated production of tissue inhibitor of
metalloproteinases-3 (TIMP-3) in IL-1 activated bovine and human primary chondro-
cyte cultures as well as in the SW-1353 chondrosarcoma cell line. In that study [119],
ERK 1/2 inhibition reduced Sp1 activity which is a major transcription factor for regu-
lating TIMP-3 gene expression. The significance of this finding is related to the fact
that TIMP-3 is a critical endogenous MMP inhibitor and is the salient inhibitor of
MMP-13 and ADAMTS-4 which are the major enzymes implicated in collagen II and
aggrecan degradation in OA [43].
Interest in the role of MAP kinases in the neutral sphinogmyelinase-induced hy-
drolysis of sphinogmyelin to ceramide pathway which can be activated by IL-1 and/or
TNF-α first surfaced when Reunanen et al. [120] showed that ceramide-dependent in-
duction of MMP-1 by fibroblasts was inhibited by the MEK1 SMI, PD98059 and by
the p38 SMI, SB203580. Activation of C2-ceramide-mediated MMP-1 promoter activ-
ity was effectively suppressed by over-expression of MAP kinase phosphatase-1. Fol-
low-up studies showed that ceramide also induced apoptosis in rheumatoid arthritis
synovial cells [121], but Gerritsen et al. [122] showed that TNF-α-mediated apoptosis
was not due to ceramide activity in human synovial fibroblasts. More recent interest in
the ceramide pathway relative to OA pathophysiology resulted from studies showing
that the ceramide pathway in rabbit cartilage explants was induced by both IL-1 and
TNF-α, that C2-ceramide stimulated MMP-1, -3 and -13 mRNA and that C2-ceramide
induced rabbit chondrocyte apoptosis when employed at concentrations ranging from
10–4M to 10–5M [123]. Ceramide was also shown to induce aggrecanase activity as evi-
denced by ceramide-induced appearance of the C-terminal aggrecan neoepitopes
NITEGE373 and DIPEN341 in the culture medium when rabbit cartilage explants were
treated with C2-ceramide [124], but in this study no increase in MMP protein levels
was detected.
Vascular endothelial growth factor (VEGF), an angiogenic peptide has been
strongly implicated in synovial neoangiogenesis in rheumatoid arthritis [125]. Re-
cently, VEGF was shown to be increased when osteoarthritis chondrocytes were main-
tained under hypoxic conditions which also induced HIF-1α [126]. Further, hypoxia-
induced VEGF was blocked by p38 SMIs but not by JNK SMIs. By contrast,
IL-1-induced VEGF production was blocked by a JNK but not a p38 SMI, suggesting
that specific MAP kinase targeting will have to be employed to diminish pro-
inflammatory mediators in OA that are dependent on hypoxia and/or IL-1, respectively.
These results are also particularly noteworthy because Reboul et al. [127] showed that
OA, but not normal cartilage, expressed hepatocyte growth factor (HGF). HGF stimu-
lated MMP-13 gene transcription by human osteoarthritis chondrocytes that was JNK-
dependent, and also by an unidentified MAP kinase, which was not p38. Thus, sup-
pressing MMP-13 up-regulation in OA cartilage must take into account that several
specific MAP kinase pathways may be simultaneously activated by specific cytokines
as well as by inducible growth factors. This viewpoint is particularly relevant in con-
sidering any future medical management of OA which must reconcile the bifurcation
points whereby after IL-1 stimulation, chondrocyte MAP kinase activation, MMP gene
regulation and NF-κB activation are strongly correlated with one another in vitro. This
is particularly relevant since MAP kinase and NF-κB inhibition resulted in MMP gene
down-regulation [128]. Of note, Barchowsky et al. [129] had previously demonstrated
that IL-1 was superior to TNF-α in inducing c-Jun and ERK synthesis as well as c-Jun,
ERK and NF-κB activation in primary rabbit synovial fibroblasts with all three of these
activation events being required to up-regulate MMP-1 transcription. In addition, You
et al. [130] showed that IL-17B, a member of the IL-17 family, was strongly expressed
by chondrocytes during mouse limb bud development. This finding suggested that
IL-17 family proteins could also be expressed by adult chondrocytes so that the IL-17
pathway must be considered pertinent to the present discussion especially if chondro-
cyte IL-17 or IL-17 over-expression induces chondrocyte anabolic/catabolic imbalance
via MAP kinase and NF-κB activation. Indeed, Koenders et al. [131] recently reported
that either IL-17A over-expression or local IL-17A gene transfer increased MMP-3, -9,
-13 and ADAMTS4 mRNA as well as cartilage damage in IL-1-deficient mice in a
streptococcal cell wall-induced arthritis model. Thus, medicinal chemical or genetic
strategies designed to suppress NF-κB activation induced by cytokines using either PK
SMIs or direct NF-κB inhibition may eventually be made applicable for in vivo use to
suppress inflammatory responses in OA and, in fact, this approach is currently being
considered for future OA therapeutic intervention [132].
Finally, any strategy based on inhibiting MAP kinase-dependent alterations in
chondrocyte metabolism in vitro must also first show some efficacy in OA animal
models. In that regard, chondrocyte apoptosis was found to occur early after anterior
cruciate ligament transection in the dog. Chondrocyte apoptosis correlated with iNOS
and COX-2 induction which was dependent on MEK-1/2 and p38 kinase activity.
However, NF-κB inhibition did not alter apoptosis as measured by caspase-3 activ-
ity [133]. In addition, a recent study by Takahashi et al. [134] showed that Celecoxib, a
selective COX-2 inhibitor used in the clinical management of OA suppressed chondro-
cyte PGE2 as expected, but Celecoxib also suppressed p38 and ERK 1/2 activity before
and after NO induction. More importantly, MEK 1/2 inhibition by the SMI PD198306
partially suppressed cartilage OA pathology which was accompanied by suppression of
ERK 1/2 and MMP-1 activity [135]. These results indicated that ERK 1/2 activation
correlated with OA pathology, but a recent report by Longobardi et al. [136] showed
that TGF-β1-mediated chondrocyte mitogenesis was mediated by ERK 1/2. The IGF-I
response was regulated, in part, by ERK 1/2 suggesting that experimental manipulation
of ERK 1/2 while dampening OA changes might also limit cartilage repair pathways.
In summary, the cell culture and in vivo studies reviewed herein provide the impe-
tus for future investigation in which MAP kinase SMIs with strong specificity for the
various p38 kinase isoforms, ERKs and JNKs could be individually employed in OA
animal models and then if shown to be efficacious and non-toxic in animals could then
be employed in randomized controlled human OA clinical trials [8].
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IOS Press, 2007
VIII
Transcriptional Control of Chondrocyte

Gene Expression
Mary B. GOLDRING a,* and Linda J. SANDELL b
a
Research Division, The Hospital for Special Surgery,
Weill College of Medicine of Cornell University, New York, New York, USA
b
Departments of Orthopaedic Surgery and Cell Biology and Physiology,
Washington University School of Medicine at Barnes-Jewish Hospital,
St. Louis, Missouri, USA
Abstract. During cartilage formation and maintenance, the expression of chondro-

cyte-specific genes, such as those encoding type II collagen (COL2A1), aggrecan,
and cartilage-derived retinoic acid sensitive protein (CD-RAP), is regulated by
both activators and repressors that interact with the promoter or enhancer regions
of these genes. Cascades of both positive and negative transcription factors have
been found to determine developmental events in the embryonic growth plate. The
high mobility group protein Sox9, which is required for COL2A1 transcription
along with l-Sox5 and Sox6, plays a key role in cartilage formation and mainte-
nance, while Sp1 and the coactivator, CBP/p300, are required for constitutive ac-
tivity. The bHLH, HOX, SMAD, ETS, and STAT families consist of both positive
and negative regulators that directly or indirectly influence COL2A1 and CD-RAP
during chondrogenesis and chondrocyte hypertrophy. In osteoarthritis, activation
of mature articular chondrocytes may result in recapitulation of these developmen-
tal events and phenotypic modulation by the associated transcription factors. Cyto-
kine-induced transcription factors, including NF-κB, C/EBP, ETS, and AP-1 fam-
ily members that activate catabolic and proinflammatory genes, may then suppress
chondrocyte phenotype and cartilage repair mechanisms by inhibiting expression
of cartilage-specific genes. This review will focus on the transcriptional regulation
of COL2A1 and CD-RAP genes by factors involved in cartilage formation and
homeostasis, as well as in inflammatory and catabolic events that adversely affect
cartilage integrity.
Keywords. Gene regulation, chondrogenesis, chondrocyte, transcription factors,

cytokines
Introduction
As the unique cellular components of adult articular cartilage, chondrocytes are respon-
sible for maintaining the structural and functional integrity of the cartilage extracellular
matrix in physiological conditions [1,2]. The articular cartilage matrix is a complex
*
Corresponding Author: Mary B. Goldring, PhD, Hospital for Special Surgery, Caspary Research Building,
Room 528, 535 East 70th Street, New York, NY 10021, USA, E-mail: goldringm@hss.edu.
M.B. Goldring and L.J. Sandell / Transcriptional Control of Chondrocyte Gene Expression 119
mix consisting primarily of type II collagen (COL2A1) and other cartilage-specific

collagens, type IX (COL9) and type XI (COL11). In addition, the large aggregating
proteoglycan aggrecan makes up approximately 50% of the matrix, as well as a large
number other collagens, small proteoglycans, and other non-collagenous proteins [1].
In the absence of joint inflammation or other pathology, the turnover and remodeling of
the matrix components is very low, the half-life of collagen having been estimated at
greater than 100 years [3,4]. The glycosaminoglycan constituents on the aggrecan core
protein are more readily replaced and the half-life of aggrecan has been estimated to be
in the range of 3 to 24 years [4]. In osteoarthritis (OA), there is a loss of the steady-
state equilibrium between synthetic (anabolic) and resorptive (catabolic) activities re-
sulting in increased metabolic activity and progressive destruction of the cartilage ma-
trix (reviewed by Sandell [5]). Local loss of proteoglycans and cleavage of type II col-
lagen occur initially at the cartilage surface resulting in an increase in water content and
loss of tensile strength in the cartilage matrix as the lesion progresses. It is generally
agreed that the cartilage damage is associated with increased production of proteinases,
including the metalloproteinases (MMPs), MMP-1, MMP-3, MMP-8, MMP-13, and
MMP-14 [6,7] and the aggrecanases, ADAMTS-4 and -5 [8,9]. Synovial fluids from
patients with OA contain both aggrecanase- and MMP-generated aggrecan frag-
ments [10]. MMP-13-specific type II collagen cleavage products and MMP-13 itself
have been detected in OA cartilage [11,12].
In addition to the increased production of matrix-degrading proteinases in the early
stage of OA, there is a transient increase in chondrocyte proliferation, as well as evi-
dence of a general increase in synthetic activity and an alteration of the pattern of ex-
tracellular matrix synthesis, which is often interpreted as a repair response. Genomic
and proteomic analyses of global gene expression have detected increased expression
of the type II collagen gene (COL2A1) in early OA cartilage [13,14], possibly associ-
ated with the increased levels of anabolic factors such as bone morphogenetic protein
(BMP) 2 and inhibin βA/activin [13,15]. These and other transforming growth factor
(TGF)-β family members may stimulate aggrecan synthesis at the same time as pro-
moting the formation of fibrocartilage and osteophytes, bony structures at the periphery
of the joint surface. Type III collagen and type VI collagen, which are present at low
levels in normal cartilage: the chondroprogenitor splice variant of the type II collagen
gene, type IIA: and type X collagen, a marker of the hypertrophic chondrocyte that is
normally absent in adult articular cartilage, have been detected during certain stages of
OA or at atypical sites within OA cartilage [16,17]. Other genes associated with growth
plate development, such as MMP-9 and Indian hedgehog (Ihh) are detected in the vi-
cinity of early OA lesions, although the expression of Sox9, the master regulator of
cartilage formation, is decreased and does not correlate with active type II collagen
gene expression [7,18]. These observations have lead to the concept that the chondro-
cyte responds to early activation by attempting to revert to a progenitor phenotype and
to recapitulate developmental events [16,19].
Multiple mechanisms are likely involved in the disturbance of chondrocyte remod-
eling activities in OA. It has been proposed that mechanical disruption of chondrocyte-
matrix associations may lead to alteration of metabolic responses in the chondrocyte
[20]. More rapid matrix turnover may occur in the immediate pericellular zones com-
pared to the interterritorial zones of cartilage [12,21,22]. This suggests roles for chon-
drocyte cell surface receptors such as integrins and DDR2 in the response to mechani-
cal stress that may result in the disruption of normal remodeling of matrix components
[23–26]. In addition to acquired or age-related alterations in chondrocyte function and
120 M.B. Goldring and L.J. Sandell / Transcriptional Control of Chondrocyte Gene Expression
the effects of excessive mechanical loading, inflammation and accompanying dysregu-

lated cytokine activities may also contribute to cartilage catabolism [27,28]. OA is not
considered a classical inflammatory arthropathy, due to the absence of neutrophils in
the synovial fluid and systemic manifestations of inflammation. However, synovitis is
common in advanced OA involving infiltration of mononuclear cells, and expression of
proinflammatory mediators is observed in early and late OA [29]. Evidence from nu-
merous studies in vitro and in vivo indicates that interleukin-1 (IL-1) and tumor necro-
sis factor (TNF)-α are the predominant proinflammatory cytokines involved in the in-
duction of cartilage-degrading proteinases. The balance of these cytokines in relation to
anabolic factors may have profound effects on the ability of the chondrocyte to repair
the degraded matrix. The following review will compare and contrast the transcrip-
tional regulation of genes involved in cartilage matrix synthesis (COL2A1 and CD-
RAP) by anabolic and catabolic factors.
1. Transcription Factors Involved in the Regulation of Cartilage-Specific Genes
During the past two decades, many of the transcription factors that control the expres-
sion of cartilage-specific genes have been discovered and characterized in studies in
vitro and in vivo. Both positive and negative transcription factors have been found to
determine developmental events during chondrogenesis, the process by which mesen-
chymal condensations form the cartilage anlagen, which eventually forms the cartilage
of the articular joint or undergoes hypertrophy and endochondral ossification to form
bone. These events are controlled by cascades of both activators and repressors that
interact with the promoter or enhancer regions of chondrocyte-specific genes, including
those encoding type II, type IX, and type XI collagen, aggrecan, and the cartilage-
derived retinoic acid sensitive protein (CD-RAP). The high mobility group (HMG)
protein Sox9 plays a key role in cartilage formation and maintenance by permitting
transcription of cartilage-specific genes such as type II and type IX collagens, aggrecan,
and CD-RAP [30–34]. Sox9 activates COL2A1 transcription by binding to the first
intron enhancer through its high mobility group (HMG) DNA-binding domain and acts
cooperatively with L-Sox5 and Sox6 to regulate chondrogenesis in vivo [35,36]. These
and other SOX genes are regulated in a dynamic fashion during chondrogenesis by
members of the BMP/TGF-β family [37]. Other extracellular mediators that control
chondrocyte differentiation include Indian hedgehog (Ihh) via PTHrP, Wnt proteins via
β-catenin, and fibroblast growth factors (FGFs) via specific receptors that promote or
suppress proliferation (see for review [38]). The anabolic effects of IGF-I, BMP-2, and
FGF-2 on differentiated chondrocytes appear to be mediated, at least in part, by
Sox9 [35,39–42]. The transcription factors involved in positive and negative regulation
of chondrocyte differentiation to be discussed in the following sections are listed in
Table 1.
1.1. Sox9, L-Sox5 and Sox6
During skeletal development, COL2A1 expression is regulated in a coordinated fashion

by growth and differentiation factors, which modulate a series of transcriptional events
requiring elements within both the promoter and first intron regions [36,43,44]. Sox9
plays an essential role during sequential steps of chondrocyte differentiation and the
Table 1. Transcription factors involved in positive and negative regulation of chondrocyte differentiation
Positive* Sequence Negative** Sequence

HMG: Runt:
Sox9, L-Sox5, Sox6 (A/T)(A/T)CAA(A/T)G Runx2 (Cbfa1) TG(C/T)GGT
Zinc finger: Zinc finger:
Sp1 GGGCG Sp3 GGGCG
cKrox GGGAGGGGG
AP-2α GCCNNNGGC
CRYBP1 GAGAAAAGCC
NT2 GAGGAGGGGAG
Zfp60
Osx
bHLH: bHLH:
USF1, USF2 CANNTG (E-box) δEF1 CACCTG
Scleraxis CANNTG Twist CANNTG
DEC1 CACNAG Snail, Slug CAGGTG
Homeodomain: Homeodomain:
Hoxa13, Hoxd13 TNATNN Hoxc8, Msx2 TAATNN
Dlx-2 CNGTAANTG Dlx5 TAATTA
Cart1 TAATNNNATTA
ETS: ETS:
C-1-1 GGAA Erg GGAA
SMADs: Retinoic acid receptors:
Smad1 CAGACA (RAR/RXR) AGGTCA
Stat1 ATTCCTGTAAG NFATp(c2) GAGG
bZip:
JunB, JunD TGAC/G
Fra1, Fra2, FosB
c-Maf
C/EBPβ, C/EBPδ TTGAGAAA (COL2)
TTGGGAAA (CD-RAP)
*Designates those factors that promote cartilage-specific gene expression (COL2A1, CD-RAP) and other
events during chondrogenesis or in adult chondrocytes; many are induced by BMP/TGFβ family members.
**Designates factors that repress cartilage-specific gene expression and may promote transition to chondro-
cyte hypertrophy prior to endochondral ossification.
Sox9-binding intron enhancer is required for COL2A1 expression during chondrogene-

sis in transgenic mice in vivo [35,36,45,46]. In mouse chimeras generated with Sox9-/-
embryonic stem cells, the mesenchymal progenitors lacking Sox9 are excluded from
cartilage tissue and are unable to transcribe the COL2A1 gene [46]. L-Sox5 and Sox6
are co-expressed with Sox9 in differentiated chondrocytes and cooperate with Sox9 to
fully activate the promoter and maintain of COL2A1 expression both in vitro and in
vivo [35,47–49]. Several studies suggest that the proximal promoter may operate at
specific times during development when negative regulation is required via promoter
regions distinct from those required for positive regulation [36,50]. Sox9 also regulates
the transcription of genes encoding type IX collagen (Col9a1) [31], type XI collagen
(Col11a1) [32], aggrecan (Agc) [34], and matrilin-1 [51]. The cooperative effects of
L-Sox5 and Sox 6, however, are not observed in some situations. For example, L-Sox5
and Sox6 are not required for Sox9-dependent lineage commitment and prechondrocyte
differentiation in embryos [52], and the Col9a1 enhancer can be activated by Sox9
dimers in the absence of L-Sox5 and Sox6 [53]. Our own work on factors controlling
transcription of the COL2A1 gene has revealed both positive and negative regulatory
domains, many of which are not dependent upon the status of Sox9 binding to the en-
hancer (Goldring & Sandell, manuscripts in preparation). Thus, the interactions of SOX
proteins depend on the promoter context and the differentiation state of the chondrocyte.
1.2. Sp1 and Zinc Finger Proteins
Binding sites for the ubiquitous transcription factor, Sp1, were among the first identi-
fied in type II collagen genes [54–56]. Sp1, a primary transcriptional regulator of gene
expression, is responsible for stimulating or maintaining constitutive COL2A1 pro-
moter activity and a related family member Sp3 represses Sp1-mediated transactivation
by binding to the same site [57–60]. The zinc finger protein, cKrox, which also inter-
acts with GC-rich sequences, activates COL2A1 transcription in differentiated chon-
drocytes but inhibits constitutive activity in subcultured (dedifferentiated) cells [61].
1.3. Positive and Negative Regulation by Different Transcription Factor Family

Members
Transcription factors that are members of the homeobox (HOX), basic helix-loop-helix
(bHLH), and ETS families may have positive or negative effects on cartilage-specific
gene transcription. For example, the inappropriate expression of the C-1-1 variant of
the ETS factor Erg can block chondrocyte hypertrophy and endochondral ossifica-
tion [62–64]. HOX genes, which are important for patterning during limb development,
enhance (Hoxa13 and Hoxd13) or suppress (Hoxa11 and Hoxd11) transcription from
GC box-dependent promoters that drive expression of, for example, the BMP-4 gene
by interacting with GC box-binding proteins such as Sp1 [65]. BMPs also regulate pro-
moter activities via HOX proteins of the Dlx family [66]. Dlx2, which is stimulated by
BMP-2, acts via the intron enhancer to increase COL2A1 expression [67], whereas
Dlx5 and 6 promote transition to hypertrophy and Dlx5 binds to the Col10a1 promoter
and increases its activity [68].
E-box motifs, which are consensus-binding sites for bHLH proteins, are present in
promoter and enhancer regions of type II collagen and CD-RAP genes from different
species [50,69]. The interaction of δEF1 with conserved E-box sites containing
CACCTG or Snail family members Sna and Slugh with CAGGTG represses constitu-
tive activity of the COL2A1 promoter [50,70]. The bHLH protein, scleraxis, can dimer-
ize with other E box-binding proteins and is expressed at early stages of chondrogene-
sis in regions surrounding Sox9 [71–74]. DEC1 promotes chondrocyte differentiation
at early and late stages in response to PTHrP and cAMP [75]. Differential expression of
Id1, 2, 3, and 4 may influence chondrogenesis and phenotypic expression in mature
chondrocytes and chondrosarcoma cells [76–78]. The nuclear factor of activated T cells
NFATp(c2) suppresses chondrogenesis and inhibits aggrecan and COL2A1 gene ex-
pression in adult chondrocytes [79], whereas NFAT4 induces chondrogenesis by stimu-
lating BMP expression [80].
Negative transcription factor activity on chondrocyte-specific genes is necessary
for two reasons: (1) when required to down-regulate genes expressed in the chondro-
cyte and (2) to repress transcription in “non-chondrocytes”. The expression of most
cartilage genes is very high during maturation of the growth plate and expansion of
articular and hyaline cartilages. However, expression is much lower in hypertrophic
Figure 1. Positive and negative transcriptional regulation during cartilage development.
chondrocytes and in mature cartilage tissue. In fact, the negative regulators, δEF1 and
AP-2 are detected by immunohistochemistry in mature tissues and hypertrophic carti-
lage while the positive factor, Sox-9, is greatly reduced [43]. Factors that are known
primarily to suppress chondrocyte differentiation while promoting hypertrophy include
the retinoic acid receptor [81,82] and the zinc finger transcription factors, Zfp60 [83],
NT2 [84], CRYBP1 [85], AP-2 [86–88], and Osterix (Osx) [89]. Activation of PPARγ,
which interacts with retinoid X receptor (RXR), inhibits thyroid hormone-induced
chondrocyte hypertrophy [90] and suppresses both chondrogenic and hypertrophic
markers, while promoting adipogenesis [91]. This process may be mediated by the
PPARγ co-activator 1α (PGC-1α), which acts as a coactivator for Sox9 during chon-
drogenesis and interacts directly with Sox9 to promote Sox9-dependent transcriptional
activity [92]. AP-1 family members that are leucine zipper (bZip) proteins and can
form heterodimers, including c-Fos, Fra1, Fra2, FosB, JunB and ATF-2, are important
for the expression of Col10a1 and Mmp13 during chondrocyte hypertrophy in the
mouse embryo [93–97]. Fra 1 and FosB increase Col10a1 promoter activity by binding
to an AP-1 site [98]. MMP-13 is a target of c-Maf, which can form heterodimers with
other bZip proteins and regulates the differentiation of hypertrophic chondrocytes [99].
Thus, chondrocyte-specific gene expression depends upon the balance of positive and
negative factors interacting with the same DNA elements or with each other [38,100].
A diagram of expression patterns of various transcription factors during endochondral
bone development is shown in Fig. 1.
1.4. Negative Regulators in Tissue-Specific Gene Expression
CD-RAP is a secreted protein expressed by chondrocytes with an expression pattern

even more restricted than type II procollagen [69,101] and considered the most reliable
marker for cartilage [102]. During chondrogenesis, CD-RAP is co-expressed with
COL2A1, activated from the beginning of chondrogenesis, and expressed throughout
cartilage development. Many regulatory elements characterized thus far in the pro-
moter of the CD-RAP gene play similar roles to those in the COL2A1 or COL11A1
genes. For example, AP-2 at a low concentration is an activator, whereas it represses
CD-RAP promoter activity at a high concentration [86]. Sox9 binds the CD-RAP pro-
moter at –410 to –404 bp and activates transcription [103]. Upstream stimulatory factor
(USF) and δEF1 with an E-box located at –487 to –482 bp and activate or repress CD-
RAP gene expression depending on the proportion of the USF to δEF1 in the nuclei (Li
and Sandell, unpublished data).
Studies in transgenic mice have revealed that a –2251 bp promoter directs tissue-
specific expression of E. coli ß-galactosidase gene (LacZ) reporter gene, consistent
with the endogenous CD-RAP gene expression pattern. A truncation to a –2068 bp
promoter does not reliably express the reporter gene [104]. These results suggest that
the 183 bp DNA fragment between –2251 and –2068 bp contains important elements
that are responsible for tissue-specific expression of CD-RAP. When the 183 bp frag-
ment is removed from the native –3345 bp promoter, which directs only cartilage-
specific expression in vivo, the reporter gene is widely expressed in transgenic mice in
muscle, bone, nerve ganglion, lungs and cartilage [105]. This fragment confers tissue
specificity by repressing the expression of CD-RAP and COL2A1 in non-cartilage tis-
sues and contains several HMG-like sites, which are targets for binding of L-Sox5,
Sox6 and Sox9. Overexpression of these SOX proteins can activate CD-RAP promoter
activity via the 183-bp fragment. However, this fragment also contains a negative regu-
latory site for the transcription factor, CCAAT/enhancer-binding protein (C/EBP).
C/EBPβ and C/EBPδ are upregulated in chondrocytes by treatment with IL-1β, an in-
hibitor of CD-RAP expression, and all C/EBP isoforms repress gene transcription
through the C/EBP site within the 183-bp element [106]. Therefore, C/EBP is another
negative regulator for cartilage-specific genes. In fact, when the C/EBP binding site is
removed from the CD-RAP promoter, the gene is expressed in the muscle cell line,
C2C12 [105]. These results indicate that the presence of C/EBP in muscle and in other
non-cartilage tissues may contribute to the lack of expression of CD-RAP and other
cartilage-characteristic genes. The down-regulation of cartilage gene expression by
C/EBPs under the influence of pro-inflammatory cytokines will be explored later in
Section 2.4.
1.5. Protein-Protein Interactions and the Coactivator, CBP/p300
An additional control mechanism involves the coactivator, CREB-binding protein

(CBP) or its paralogue, p300, which does not interact directly with promoter DNA se-
quences, but serves as a bridge between DNA-binding proteins and the RNA poly-
merase II transcriptional machinery. Through intrinsic histone acetylase (HAT) activity,
CBP/p300 can directly acetylate the lysine residues of certain transcription factors that
are generally activators of gene transcription, including cAMP-responsive binding pro-
tein (CREB), NFκB, c-Jun family members, C/EBPs, and SMADs, and thereby serves
to integrate activities of various factors resulting in transcriptional synergy [107]. With

regard to chondrocyte-specific gene expression, CBP/p300 increases transcriptional
activities of the cartilage homeoprotein-1 (Cart1) [108] and BMP-responsive
Smad1 [109]. CBP/p300 also potentiates transcription by acetylation-dependent loosen-
ing of the chromatin structure, and a recent study indicates that its interaction with
Sox9 is required for COL2A1 promoter activity [110].
Both CBP and p300 elicit strong positive transcription of the CD-RAP and
COL2A1 genes when expressed in chondrocytes. In fact, the expression levels exceed
those induced by Sox proteins alone [59,111]. The mechanism for the increase in gene
transcription involves both the positive regulator Sox9 and the negative regulator
C/EBP. The CBP or p300 acts as a co-regulator by binding to the DNA-binding tran-
scription factors and increasing transcription by different mechanisms depending on the
target gene and availability of other factors. After binding to CBP or p300, Sox9 binds
to DNA with higher affinity, thereby increasing gene transcription [110]. The binding
of p300 or CBP to C/EBP inhibits the binding of this transcription factor to the DNA
thereby sequestering and rendering inactive the negative regulator [111].
1.6. Transcription Factors in the Growth Plate
Negative regulators of COL2A1, including Cbfa1/Runx2, δEF-1, C/EBP, and AP-2, are
highly expressed in hypertrophic cartilage [43], suggesting that down-regulation of
chondrocyte-specific genes is necessary before mineralization can occur. For example,
Runx2, together with Runx3 in the embryonic growth plate, stimulates chondrocyte
terminal differentiation [112–114] and increases the expression of type X collagen and
MMP-13 in hypertrophic chondrocytes [115–117]. Although Sox9 is a dominant tran-
scriptional activator for chondrogenesis, it also acts as a repressor of Runx2 activity
during chondrocyte hypertrophy by directly interacting through the HMG domain with
the runt domain of Runx2 [118]. Runx 2 partners with SMADs to regulate the type X
collagen gene in response to BMPs [119]. However, there is no SMAD site on the
Runx2 promoter, and many of the positive and negative regulators of chondrocyte hy-
pertrophy modulate Runx2 transcriptional activity. For example, Dlx3 is an activator
when it binds directly to the promoter of a target gene, where interaction with Runx2
can reduce Runx2-mediated transcriptional activation [120]. The homeodomain protein
Nkx3.2, which is an early BMP-induced signal required at the onset of chondrogenesis,
is a direct transcriptional repressor of Runx2 promoter activity [121,122]. The bHLH
factor Twist inhibits chondrogenesis [123] but prevents premature osteoblast differen-
tiation by transiently inhibiting Runx2 function [124]. Twist-1 is expressed exclusively
in the perichondrium, where it favors chondrocyte hypertrophy in a Runx2-dependent
manner, but paradoxically, it blocks Runx2 activation of the Fgf18 promoter by Runx2
[125]. Cooperation of the Groucho homologue Grg5 or the leucine zipper protein ATF4
with Runx2 promotes chondrocyte hypertrophy [126] or osteoblast differentiation [127],
respectively. Histone deacetylase 4 (HDAC4), which is expressed later in prehypertro-
phic chondrocytes, prevents premature chondrocyte hypertrophy by interacting with
Runx2 and inhibiting its activity [128]. The canonical Wnt/β-catenin-induced TCF/Lef
transcription factors suppress chondrocyte differentiation in early chondroprogenitors
and promote chondrocyte hypertrophy at later stages and subsequent endochondral
ossification [129,130] by binding to the Runx2 promoter [131]. Runx2 interacting with
BMP-induced Smad1 is required for the induction of GADD45β, a survival factor in
hypertrophic chondrocytes that acts as a transcription factor during the induction of
MMP-13 and COL10A1 [132]. The inhibition of COL2A1 promoter activity and gene
expression by GADD45β [133] suggests indirect and unexpected mechanisms by
which BMPs and TGFβ may regulate chondrogenesis.
The expression patterns of BMP-2, 4-, 6 and 7 contribute to progressive chondro-
cyte differentiation at different stages of cartilage development, and the local regulation
of their overlapping activities is governed by extracellular BMP antagonists, including
noggin, follistatin, chordin and twisted gastrulation. Depending upon the differentiation
stage of the chondrocyte, BMP-2 can induce COL2A1 or COL10A1 via Sox9 or Runx2,
respectively, presumably in cooperation with Smad1, 5, or 8 [134]. In contrast, TGFβ,
through phosphorylation of Smad2 and 3, acts as an early mediator of chondrogenesis,
but inhibits chondrocyte hypertrophy and COL10A1 expression. TGFβ induces aggre-
can gene expression via cross-talk between Smad2 and the ERK1/2 and p38 MAPK
pathways during differentiation in the chondroprogenitor ATDC5 cell line [135]. Al-
though BMPs induce type II collagen and proteoglycan synthesis in differentiated ar-
ticular chondrocytes, direct binding of either TGFβ- or BMP-induced SMADs to gene
promoters has been difficult to prove. In fact, TGFβ inhibits COL2A1 promoter activ-
ity by decreasing the ratio of Sp1 to Sp3 [58], but increases type II collagen synthesis
by a translational or post-translational mechanism involving the TGFβ-activated kinase
1 (TAK1) [136]. Recent findings indicate that TGFβ-induced Smad3 enhances Sox9
transcriptional activity and COL2A1 expression by interacting with Sox9 and enhanc-
ing the association between Sox9 and CBP-p300 [137].
The T-box transcription factor, Brachyury, another factor that is induced by
BMP-2, is upregulated during early stages of FGFR3-mediated chondrogenesis in the
osteochondroprogenitor cell line, C3H10T1/2 [138]. The inhibition of chondrocyte
proliferation by FGFR3 prior to hypertrophy involves Stat1 [139] and IFNγ-induced
down-regulation of COL2A1 transcription requires Stat1 and its activation by the
kinases Jak1 and Jak2 [140]. Together, these and other findings suggest the complexity
of the signaling pathways and downstream transcription factors that are involved in
regulation of the gene expression in chondrocytes during development and postnatal
growth.
2. The Regulation of Chondrocyte Phenotype by Proinflammatory Cytokines
The proinflammatory cytokines, IL-1β and TNFα, are in involved in the destruction of
the articular cartilage in both rheumatoid arthritis and OA [141,142]. The chondrocyte
is the cellular target of cytokine action in cartilage, and IL-1β and TNFα and their re-
ceptors colocalize with MMP production in superficial regions of OA cartilage [11].
IL-1β suppresses the expression of a number of genes associated with the differentiated
chondrocyte phenotype, including COL2A1 and CD-RAP [106,143,144]. Early studies
in vitro showed that IL-1 and TNFα are capable of inhibiting the synthesis of type II
collagen by chondrocytes by suppressing gene transcription and the levels of associated
mRNAs [143–146]. IL-1 and TNFβ also stimulate the synthesis of prostaglandin
E2 (PGE2), which feedback-regulates COL2A1 transcription in a positive man-
ner [147,148].
2.1. The Regulation of Chondrocyte Phenotype by Cytokine-Induced Signaling

Pathways
IL-1 and TNFα share the capacity to activate a diverse array of intracellular signaling
pathways, although the cell surface receptors and associated adaptor molecules are dis-
tinct (see Chapter 1). In chondrocytes, the JNK and p38 MAPK signaling pathways
predominate in the regulation of IL-1 and TNFα-induced genes. The inhibition of
COL2A1 expression by IL-1 or TNFα in chondrocytes involves the p38 [149], JNK,
and NFκB pathways [150]. Injurious mechanical stress and cartilage matrix degrada-
tion products are capable of stimulating the same signaling pathways as those induced
by IL-1 and TNFα [151–158]. Since these pathways also induce or amplify the expres-
sion of these cytokine genes, it remains controversial whether inflammatory cytokines
are primary or secondary regulators of cartilage damage and defective repair mecha-
nisms in OA [159]. Based on studies in animal models [160,161] and analysis of carti-
lage samples or body fluids from OA patients [162–166], controversy exists about
whether type II collagen, or the type IIA chondroprogenitor variant, is elevated or sup-
pressed, appearing to depend upon the zone of cartilage analyzed and the stage of OA.
The early increase in anabolic activity in OA cartilage may be associated with in-
creased expression of BMP-2 induced by IL-1 and TNFα [15]. BMP-2 would, in turn,
activate of COL2A1 transcription and permit interaction of the COL2A1 promoter with
cytokine-induced factors. Although signaling via the p38 MAPK pathway can regulate
gene expression by post-transcriptional mechanisms [167], phosphorylation events that
are downstream of ligand binding to cytokine receptors may result in induction and
activation of transcription factors that bind to DNA elements of target genes, as dis-
cussed below.
2.2. The Regulation of Chondrocyte Phenotype by Cytokine-Induced Transcription

Factors
Cytokine-activated transcription factors of the NFκB/c-Rel, C/EBP, ETS, and AP-1

families, which mediate the induction of MMPs, cyclooxygenase (COX) 2, and nitric
oxide synthase (NOS) 2 by IL-1 and TNFα [168–172], may also be involved in sup-
pressing the transcription of COL2A1 and CD-RAP [59,106] (Table 2). IL-1 may also
induce chondrocytes to synthesize other cytokines such as IL-6, which together with
the soluble IL-6 receptor that is not present in sufficient amounts in cultured cells,
down-regulates COL2A1, aggrecan, and link protein via the JAK/STAT pathway in
association with suppression of Sox9 [173]. Studies in knockout or transgenic mice
suggest that IL-1 or TNFα are not involved in the formation of articular cartilage in the
embryo. However, the cartilage remodeling initiated in response to trauma or inflam-
mation may involve inactivation of the chondrogenic transcription factors, via direct or
indirect interactions with cytokine-induced transcription factors. It has been proposed
that inhibition of Sox9 expression by IL-1 determines the regulation of COL2A1 gene
transcription by these cytokines [41,174,175], although expression of Sox9 and
COL2A1 does not always correlate [59,140,176,177]. Recent evidence indicates that
Sox9 overexpression in chondrocytes may either increase or decrease COL2A1 tran-
scription depending upon its concentration and the differentiation state of the
cells [178]. During chondrocyte hypertrophy, however, the suppression of Sox9 ex-
pression and activity by transcription factors such as Runx2 has been proposed as a
Table 2. Cytokine-induced transcription factors that influence chondrocyte gene expression
Transcription factor* Cytokine Sequence

NFκB (p65/p55) IL-1, TNFα, IL-17, IL-18 GGGRNNYYCC
AP-1 family (AP-1, ATF-2) IL-1, TNFα, IL-17, IL-18 TGACTCA
C/EBPβ, C/EBPδ IL-1, TNFα, IL-17, IL-18 TTG(A/G)GCAAA
ETS (Ets-1, PEA-3, ESE-1) IL-1, TNFα, GGAA
Egr-1 IL-1, TNFα, GCGGGGGCG
STATs:
Stat1 IFN-γ TTTCATATTACTCT
Stat3 Oncostatin M TTCTGGGAATT
*These transcription factors are generally negative regulators of COL2A1 or CD-RAP expression in adult
chondrocytes.
mechanism essential for endochondral ossification [179,180]. Although Runx2 is re-

quired for IL-1 induction of MMP-13 gene transcription in articular chondrocytes [169],
its role in the suppression of chondrocyte phenotype by inflammatory cytokines has not
been defined definitively.
2.3. Early Cytokine-Induced Transcription Factors: NFκB, AP-1, and EGR-1
The understanding of transcriptional regulation by cytokines in chondrocytes and other

connective tissue cells is incomplete, even with regard to genes that have been well
studied, such as the MMPs. Early work in other systems suggested that NFκB and AP-
1 are primary response factors for the regulation of IL-1β-induced genes. Further work
showed that AP-1 (c-Jun/c-Fos), one of the first transcription factors studied, was in-
sufficient for IL-1β -induced MMP-1 gene expression by fibroblasts, [181]. Further-
more, it has not been possible to attribute the regulation of a significant number of cy-
tokine-responsive genes to direct interaction of NFκB or AP-1 with DNA elements,
including those in the COL2A1 promoter, which does not contain functional binding
sites for these transcription factors. NFκB binds to and activates the BMP-2 pro-
moter [182], but it also down-regulates Sox9 expression by posttranscriptional destabi-
lization of mRNA [183]. TGFβ expression is also induced by IL-1β through activation
of the bHLH factor AP-4, which binds to a site overlapping with an AP-1 site [184].
The Jun activation domain-binding protein 1 (Jab1), which is a coactivator of
c-Jun/AP-1, inhibits BMP signaling by binding to Smad5 [185]. These findings suggest
alternative mechanisms.
Recently, we found that IL-1β-induced and activated EGR-1, an immediate early
growth response factor, inhibits COL2A1 promoter activity by binding to the –
131/+125 bp core promoter and displacing Sp1 from at least one of the GGGCG boxes
that overlap with the EGR-1 binding site [59]. This mechanism may also account for
the increased ratio of Sp3 to Sp1 binding to the Sp1 sites observed in response to IL-1
[60]. Since overexpression of CBP reverses the inhibition, it is probable that EGR-1
acts by disruption of the interactions among Sp1, CBP, and TATA-binding pro-
teins [59]. This early response appears to be transient, suggesting that complete tran-
scriptional repression of COL2A1 promoter activity may be dependent upon the bind-
ing of other IL-1β-induced factors to upstream promoter sequences (Fig. 2).
Figure 2. Mechanism of suppression of COL2A1 transcription by IL-1β in cartilage.
2.4. Inhibition of Chondrocyte Phenotype by C/EBP and ETS Factors
Other candidate IL-1-induced transcription factors include C/EBPβ and C/EBPδ, which
downregulate the expression of both CD-RAP and COL2A1 [106], and ETS factors.
These factors are present at low or undetectable concentrations in the cytoplasm and
are part of a cascade of cytokine-induced genes that are normally induced at intermedi-
ate and later time points. The subsequent sustained expression suggests involvement in
maintenance of the transcriptional response. C/EBP and ETS factors function as activa-
tors in the context of cytokine-induced genes such as COX-2 [168,186] and MMPs
[169,181,187].
The ETS factors constitute a family of at least thirty members that play central
roles in regulating genes involved in development, differentiation and cell prolifera-
tion [188]. Several ETS factors, including ETS-1 and PEA3, cooperate with AP-1 in
regulating MMP gene expression [169,181,187]. ESE-1, also known as ESX, ELF-3,
ERT, and JEN, is a novel ETS factor that is restricted to epithelial tissues under physio-
logical conditions [189,190]. ESE-1 is expressed in non-epithelial tissues undergoing
inflammation such as rheumatoid and, to a limited extent, OA synovium and in chon-
drocytes, as well as glioma cells, smooth muscle cells, synovial fibroblasts, osteoblasts,
and monocyte-macrophages, after treatment with IL-1β, TNFα, or lipopolysaccharide
(LPS) [172]. This induction relies on the translocation of the NFκB family members,
p50 and p65, to the nucleus and transactivation of the ESE-1 promoter via a high affin-
ity NFκB binding site [172,191]. Following induction, ESE-1 can directly activate tran-
scription of NOS2 [191] and COX2 [186] by binding to two or more functional ETS
sites in the respective promoters. Together these studies indicate that increased expres-
sion of these IL-1β-induced genes is mediated indirectly by NFκB via induction of
ESE-1, which then serves as a primary transcription factor that binds to and regulates
promoter activity of the target gene.
ESE-1 acts as a direct repressor of COL2A1 promoter activity by binding to at
least two tandem ETS sites upstream of –131 bp and accounts, in part, for the sustained
suppression by IL-1β (Fig. 2). Previous studies have shown that IL-1β-induced NFκB
inhibits COL2A1 gene expression by suppressing Sox9 promoter activity [174] or by
destabilizing Sox9 mRNA [183]. Consistent with those findings, adenoviral overex-
pression of IκB in chondrocytes blocks the suppression of COL2A1 mRNA by IL-1β
[192]. However, the constitutive levels of Sox9, L-Sox5 and Sox6 mRNA are not sup-
pressed by IL-1β, similar to findings for IFN-γ [140], and overexpression of the three
SOX proteins, which bind to the intronic enhancer [35], does not reverse the inhibition
of COL2A1 activity by IL-1β [193]. Thus, once the promoter is activated, the overex-
pression of Sox9 with L-Sox5 and Sox6 may further enhance constitutive expression of
COL2A1 but the inhibition by IL-1β-induced factors cannot be overcome. A recent
study also showed that NFκB is not required for modulation of Sox9-dependent
COL2A1 promoter activity by Bcl-2 [194].
Whereas ESE-1 is a potent inducer of events associated with inflammation and tis-
sue destruction [172,186,191,195,196], C/EBP proteins act as negative regulators of
chondrogenesis during skeletal development, where IL-1β is not known to play a role
[105,111]. Both IL-1β and TNFα increase the expression and protein concentration of
the C/EBPs, but the mechanism of action is somewhat different from that of ESE-1.
IL-1β stimulates C/EBPβ and δ synthesis in a dose- and time-dependent manner over
48 hours [105]. Significant increases in C/EBPβ and δ and repression of CD-RAP and
COL2A1 are observed by 24 hours, suggesting that the C/EBPs are later regulators
than ESE-1. Chromatin immunoprecipitation of endogenous DNA shows that the
IL-1-responsive binding site in the CD-RAP promoter is within the 183 bp element at
–2251/–2068 bp discussed previously [111], in addition to a second element in the 169
bp fragment at –1062 bp. Interestingly, TNFα also induces C/EBP expression and
down regulates CD-RAP and COL2A1; however, the -1062 bp site is the only target
binding site of the TNFα-induced C/EBP [197] (Fig. 3). Thus, both pro-inflammatory
cytokines can act together to further decrease expression of the cartilage-characteristic
proteins, but the mechanism of action is somewhat different and, therefore, additive.
2.5. Protein-Protein Interactions Involved in the Suppression of Chondrocyte

Phenotype by Cytokine-Induced Transcription Factors
Cytokine-induced C/EBPs and ETS factors act as repressors partly by blocking protein-
protein interactions among Sp1, Sox9, CBP, and the basal transcriptional machinery.
The early cytokine-activated events are usually associated with positive responses, but
in differentiated chondrocytes at a post-developmental stage of low matrix turnover, the
COL2A1 promoter cannot respond to negative regulation by cytokines unless it is acti-
vated. In cytokine-induced genes, the assembly of higher order nucleoprotein com-
plexes orchestrated by high mobility group (HMG)-I(Y) factors may be important for
integrating the responses to the induced signaling pathways [198,199]. Similarly, Sox9
and related HMG factors are architectural proteins that act to maintain the nucleosomes
in an open configuration, thereby exposing the endogenous, chromatin-integrated
COL2A1 promoter to constitutive factors that interact directly with the promoter [200].
Thus, the transiently transfected promoter, which serves as a convenient model for dis-
Figure 3. Mechanism of suppression of CD-RAP transcription by TNFα in cartilage.
secting constitutive and regulatory elements, would not be under the same constraints
in vitro as in vivo. For example, the COL2A1 promoter constructs express strongly in
chondrocytes even in the absence of the Sox9-binding intronic enhancer, suggesting
that the promoter is maximally active when expressed ectopically. Constructs spanning
the promoter through the first intron express at 10 to 20% of the activities of constructs
containing the promoter alone. However, overexpression of combinations of Sox9 with
L-Sox5 or Sox6 increases the activity of the complete construct without reversing the
inhibition by IL-1β-induced factors [193].
Similar to other ETS factors such as ETS-1, which binds to two cysteine-histidine
rich regions of CBP [201], ESE-1 can interact with CBP/p300 [202]. CBP/p300 acts as
a positive regulator of chondrocyte-specific gene expression by interacting with the
P/Q/S-rich region in the carboxy-terminus of Sox9 [110] and by binding to and seques-
tering negative factors such as C/EBP [111]. Although CBP overexpression interferes
with IL-1β-induced activation of Egr-1 and upregulates constitutive COL2A1 promoter
activity [59], it does not reverse the strong suppression by ESE-1. CBP/p300-dependent
activity requires HDAC, which may modulate CBP/300 activity directly or influence
its interactions with Sox9 [203]. Similarly, the CBP/p300-Ets-1 complex, which exhib-
its functional HDAC activity, promotes chromatin remodeling and modification of
transcription factors and adaptor proteins [204], In endothelial cells, IL-1β upregulates
p300 expression [202], possibly accounting for the incomplete inhibition of COL2A1
promoter activity by IL-1β n chondrocytes.
In addition to affecting chromatin-DNA structure, HMG proteins also act to pro-
mote transcription by recruiting transcriptional regulators such as NFκB, ATF-2/c-Jun,
and interferon regulatory factor-1 that bind directly to DNA. ESE-1 has two DNA
binding domains, a classical ETS domain that would bind the ETS sites in the COL2A1
promoter, and an A/T hook domain that is found also in HMG proteins and recognizes
the A/T-rich region of double-stranded DNA [189]. GST-ESE-1 pull-down assays us-
ing truncated Sox9 fragments indicate that ESE-1 also interacts with the HMG box
toward the N-terminus of Sox9, which is distinct from the CBP/p300-interacting site in
the C-terminus [110]. These results suggest that ESE-1 does not interfere with Sox9
binding to CBP, but since ESE-1 also binds CBP/p300, it could serve to sequester both
proteins and prevent their participation in promoter activation (Fig. 2). Experiments
showing that ESE-1 still suppresses COL2A1 promoter activity by ESE-1 in the pres-
ence of excess CBP and Sox proteins indicate that it cannot serve as a bridge to en-
hance complex formation. Other ETS factors, which could serve as positive regulators
in the maintenance of constitutive expression, may compete for ESE-1 binding sites.
The different roles that ETS factors play in chondrogenesis, as well as in regulating the
expression of MMPs and other collagen genes, suggest that the promoter context and
the relative concentrations of different factors determine the extent of repression or
activation of gene transcription by ETS factors [64,205,206].
3. Conclusion
In this review, we have focused on transcriptional regulation of the COL2A1 and

CD-RAP genes during cartilage development and in adult articular cartilage. The fac-
tors that are involved in the induction and maintenance of chondrocyte phenotype may
be similar in both processes, but the balance of transcriptional activators and suppres-
sors is disrupted when the chondrocyte is activated during OA and attempts to respond
to the changes in the microenvironment. The involvement of the IL-1-induced tran-
scription factors, such as NF-κB, C/EBPβ and δ, ESE-1, and EGR-1, is consistent with
early cytokine-activated events that are usually associated with gene activation but pro-
duce a negative response in the context of the COL2A1 or CD-RAP promoter. Al-
though outside the scope of this review, these same transcription factors could also
contribute to the expression of non-cartilaginous genes such as types I and III collagens,
which are increased by IL-1β in chondrocytes [143], particularly in the superficial zone,
and further contribute to the pathogenesis and progression of OA. The differential acti-
vation of upstream signaling events that result in induction of these transcription fac-
tors could explain the synergy and redundancy in cytokine responses. While proin-
flammatory cytokines stimulate potent negative transcriptional regulators of COL2A1
promoter activity, they may also stimulate the production of PGE2 and BMP-2, both of
which stimulate COL2A1 expression and may blunt the effects of the negative regula-
tors. Therefore, in the context of OA cartilage, the initial events that activate chondro-
cyte synthetic activity probably result in activation of the normally inactive COL2A1
promoter, which would then be susceptible to transcriptional repression. Thus, we con-
clude that multiple alternative mechanisms exist for the regulation of chondrocyte-
specific genes in adult articular cartilage once the synthetic activities of chondrocytes
are activated globally. Since transcription factors induced by proinflammatory cyto-
kines also upregulate genes associated with catabolic and inflammatory responses, in-
cluding COX2, MMP13, and NOS2, and similar signaling pathways may be induced by
adverse mechanical stress, the dissection of the molecular mechanisms involved may
lead to the development of targeted therapies for blocking destruction of the cartilage
matrix and promoting its repair.
Acknowledgements
Dr. Goldring’s research is supported by NIH grant R01-AG022021 and the Arthritis
Foundation. Dr. Sandell’s research is supported by NIH grants R01-AR36994 and
R01-AR045550.
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IOS Press, 2007
IX
Gene Expression Profiling of Human

Articular Chondrocytes and Osteoarthritis
Sergio A. JIMENEZ * and Sonsoles PIERA-VELAZQUEZ
Thomas Jefferson University, Jefferson Institute of Molecular Medicine,
Philadelphia, PA 19107
Abstract. The recent development of high throughput genomic profiling technolo-

gies such as cDNA microarrays combined with advanced computational ap-
proaches have provided basic and clinical investigators with the ability to identify
and characterize high-resolution expression profiles of numerous disease states and
to dissect molecular networks that underlie specific disease phenotypes. In the
field of osteoarthritis (OA) and cartilage research, the application of microarray
technology holds the promise that it may allow the identification of molecular sig-
natures specific for OA in articular cartilage chondrocytes which could provide
clues to the elucidation of the pathogenetic mechanisms involved or responsible
for the disease. Some of these molecular signatures may also be of great value in
patient management and clinical care by providing potential biomarkers of utility
as diagnostic or prognostic tools and as markers of the effectiveness of disease
modifying therapies for OA. The aim of this chapter is to provide an overview of
the relatively few investigations that applied microarrays to the study of human ar-
ticular cartilage and OA.
Keywords. Functional genomics, gene expression profiling, cDNA microarrays,

osteoarthritis, chondrocytes, articular cartilage, cytokines, growth factors
Introduction
The recent development of high throughput genomic profiling technologies such as

cDNA microarrays [1–3], combined with advanced computational approaches [4–7],
have provided basic and clinical investigators with the ability to identify and character-
ize high-resolution expression profiles of numerous disease states and to dissect mo-
lecular networks that underlie specific disease phenotypes. Within a few years follow-
ing their introduction, microarrays are now routinely used in almost every line of bio-
medical research with the most impressive examples of the successful utilization of this
technology in cancer research [8–13]. In addition to tremendous advances in clinically-
oriented studies, the introduction and widespread application of microarray technology
in basic research has allowed the identification and characterization of many molecular
*
Corresponding Author: Sergio A. Jimenez, MD, Thomas Jefferson University, Jefferson Institute of Mo-
lecular Medicine, 233 S. 10th Street, Room 509 BLSB, Philadelphia, PA 19107-5541, Phone: 215-503-5042,
Fax: 215-923-4649, E-mail: sergio.jimenez@jefferson.edu.
144 S.A. Jimenez and S. Piera-Velazquez / Gene Expression Profiling
pathways and of novel targets for therapeutic intervention. In the field of osteoarthritis
(OA) and cartilage research, the application of microarray technology holds the prom-
ise that it may allow the identification of molecular signatures specific for OA in articu-
lar cartilage chondrocytes which could provide clues to the elucidation of the pathoge-
netic mechanisms involved or responsible for the disease. Some of these molecular
signatures may also be of great value in patient management and clinical care by pro-
viding potential biomarkers of utility as diagnostic or prognostic tools and as markers
of the effectiveness of disease modifying therapies for OA. The aim of this chapter is to
provide an overview of the relatively few investigations that applied microarrays to the
study of human articular cartilage and OA, ranging from basic studies in characteriza-
tion of the chondrocyte phenotype and chondrogenesis to the analysis of the effects of
cytokines and growth factors on various aspects of chondrocyte biology and finally to
the assessment of human OA tissues.
1. Gene Expression Analysis of Chondrocyte Differentiation and Chondrocyte

Phenotype
Articular cartilage chondrocytes are highly differentiated cells responsible for the main-
tenance of the integrity of the tissue extracellular matrix (ECM). To produce and main-
tain a properly functional articular cartilage matrix the chondrocyte normally displays a
specific pattern of gene expression. This pattern changes dramatically in response to
structural or mechanical alterations in their surrounding matrix and in response to vari-
ous growth factors and cytokines [14,15]. The chondrocyte response may lead, in cer-
tain situations, to longstanding changes in the phenotype of the cell, and therefore to an
inability to properly repair or maintain the cartilage ECM. This is exemplified by the
phenotypic changes in the patterns of production or in the temporal or spatial distribu-
tion of the synthesis of interstitial collagens, fibroblast-type proteoglycans, and produc-
tion of ECM proteins that occur during the development of OA, de-differentiation in
culture, or in response to cartilage injury [16–19]. Thus, the maintenance of the chon-
drocyte-specific phenotype plays a crucial role in the preservation of the normal struc-
ture and biomechanical properties of articular cartilage and very likely also in the
pathogenesis of tissue destruction in OA.
Culture of chondrocytes in monolayers for prolonged periods or upon repeated
passages leads to the loss of their spherical shape and to the acquisition of an elongated
fibroblast-like morphology accompanied by profound biochemical changes including
the arrest of the synthesis of the cartilage-specific collagens (types II, IX and XI) and
proteoglycans (aggrecan), initiation of synthesis of the interstitial collagens (types I, III
and V), and an increase in the synthesis of fibroblast-type proteoglycans (versican) at
the expense of aggrecan [20,21]. The chondrocyte phenotype can be re-expressed in
these cells by culturing them in suspension, agarose, alginate beads, or on a hydrogel
substrate [22–24]. These changes in the biosynthetic profile of de-differentiated chon-
drocytes resemble some of the phenotypic changes seen in OA chondrocytes [17–19].
Numerous studies have investigated the mechanisms that underlie the phenotypic
instability of chondrocytes employing microarray gene expression analysis of chondro-
cytes cultured under conditions that allow them to either preserve their differentiated
phenotype or under conditions that lead to their de-differentiation. In a pioneering pa-
per, Stokes et al. described studies which examined the gene expression profile of dif-
ferentiated versus de-differentiated chondrocytes in vitro comparing chondroctyes cul-
S.A. Jimenez and S. Piera-Velazquez / Gene Expression Profiling 145
Table 1. Relevant genes that exhibit a 2-fold or greater difference in expression between differentiated and
de-differentiated human fetal chondrocytes (HFCs)
Higher Expression in Differentiated HFCs Higher expression in De-differentiated HFCs
Gene Fold Gene Fold
Extracellular matrix proteins Extracellular matrix proteins

Matrilin 3 27.6 Hexabrachion 4.7
COL11A2 11.3 Chitinase 3-like protein 1 4.4
Dermatan sulphate proteoglycan-3 6.5 Fibrillin 1 2.8
Fibromodulin 5.9 Fibulin 1 2.5
Col11α1 4.9 Collagen Iα1 2.1
Col9α2 4.9
Aggrecan 4.4
Chondroitin sulfate proteoglycan-3 2.5
Col9α3 2.5
Cartilage linking protein-1 2.2
COMP 2.1
Matrilin 1 2.1
Transcription factors Transcription factors
Hypoxia-inducible factor 1α 3.4 TWIST 2.6
Sox-9 3.5 Freac-4 2.1
RING zinc finger protein RZF 2.2 RXR-β 2.0
Hox-B6 2.1
Zinc finger protein 35 2.0
MADS/MEF2-family transcription factor 2.0
Growth factors/cytokines/extracellular mediators Growth factors/cytokines/extracellular mediators

Frizzled-related protein 10.2 Insulin-like growth factor-binding protein 4 3.6
precursor
IGF-II 6.3 Insulin-like growth factor binding protein 3 2.6
precursor
Melanoma growth reg protein (CD-RAP) 3.9
BMP-6 2.0
Adhesion proteins Adhesion proteins
Del-1 integrin binding protein 3.5 Cadherin 11 2.8
Epithelial V-like antigen (EVA) 3.4
tured on polyHEMA, which allows the preservation of the chondrocyte-specific pheno-

type, with chondrocytes cultured as monolayers on tissue culture plastic to induce the
loss of their phenotype [25]. In these studies, the microarray hybridization was per-
formed employing the UniGEM Human V Microarray (Genome Systems, Inc.) which
contains approximately 5000 known genes and 3000 ESTs. Table 1 lists the genes
which showed a 2-fold or greater difference in expression grouped in the following
four categories: (1) extracellular matrix proteins, (2) transcription/gene regulatory fac-
tors, (3) growth factors/cytokines/extracellular signaling molecules, and (4) cell adhe-
sion proteins.
A large number of chondrocyte-specific ECM protein genes were down-regulated
whereas numerous genes encoding ECM proteins associated with the fibroblastic phe-
notype including COL1A1 and tenascin were upregulated in de-differentiated chondro-
cytes. Among the gene regulatory factors whose mRNA levels were differentially regu-
lated were the chondrogenic factor SOX9, the O2-regulated hypoxia-inducible factor
1α (HIF-1α), the basic-helix-loop-helix transcription factor Twist, the winged-helix
transcription factor Freac-4 and the retinoic acid receptor RXR-β. In the category of
Figure 1. Northern analysis of mRNA isolated from differentiated and de-differentiated human chondroctyes
hybridized with cDNA probes for human COL2A1 and TWIST (A); IGF-II, IGFBP-4, RXR-β and BMP-6
(B); and HIF-1α (C). Hybridization with GAPDH was performed to control for differences in sample load-
ing. pH: polyHEMA (differentiated); Pl: tissue culture plastic (de-differentiated). Adapted from ref. [25].
(Reproduced with permission from the publisher).
growth factors/cytokines/extracellular signaling molecules, differentiated chondrocytes

expressed higher levels of transcripts for the frizzled-related protein, FRZB, and for
insulin-like growth factor-II (IGF-II), cartilage derived-retinoic acid induced protein
(CD-RAP) and bone morphogenetic protein-6 (BMP-6).
Interestingly, higher levels of the transcripts for two insulin-like growth factor
binding-proteins, IGFBP-3 and 4 were observed in the de-differentiated chondrocytes.
In the category of cell adhesion proteins there was an increase in the levels of tran-
scripts for the bone-associated adhesion protein, cadherin-11, in the de-differentiated
chondrocyte cultures. Northern-blot analyses of transcripts from selected genes that
showed substantial differential expression were performed to validate the microarray
results. Figure 1 shows the results of Northern hybridization analyses for expression of
COL2A1, TWIST, IGF-2, IGFBP-4, BMP-6, RXR-β, and HIF-1α transcripts in differ-
entiated vs. de-differentiated chondrocytes.
In summary, these extensive microarray studies documented the dramatic change
in phenotype of chondrocytes during differentiation and de-differentiation in vitro as
evidenced by the down-regulation of numerous genes associated with the ECM of carti-
lage and the up-regulation of ECM genes associated with an undifferentiated mesen-
chymal cell phenotype. Transcription and regulatory factors that showed differential
expression between the two cell states such as SOX9, TWIST, and HIF-1α were of
substantial interest as it is likely that they might play a role in controlling the pheno-
typic difference. The validity and applicability of these results to human OA was
documented by the demonstration by PCR that many of the differentially expressed
transcripts could be detected in fresh normal and OA articular cartilage or in freshly
isolated chondrocytes prepared from these tissues (Fig. 2). For example, it was notable
that TWIST expression was substantially increased in articular cartilage samples from
patients with OA, providing strong confirmation of the functional relevance of the mi-
croarray results obtained during de-differentiation of human chondrocytes in culture.
Figure 2. RT-PCR analysis of the expression of selected genes in intact adult human normal and OA carti-
lage and in chondrocytes isolated from these tissues. Total cellular RNA was isolated directly from cartilage
samples of OA patients or from freshly isolated OA chondrocytes. (A) RT-PCR analysis for the expression of
HIF-1α, IGFBP-3, IGFBP-4, IGF-2, TWIST, Del-1 and β-Actin in RNA directly isolated from the cartilage
of four different patients with OA. (B) RT-PCR analysis for the expression of cadherin-11 as in (A). (C) RT-
PCR analysis for expression of TWIST in RNA from freshly isolated chondrocytes from adult human normal
(N) and OA cartilage (OA). From ref. [25]. (Reproduced with permission from the publisher).
In a similar study, Finger et al. [26] performed DNA microarray analysis to inves-
tigate the molecular phenotype of a human chondrocyte cell line derived from juvenile
costal chondrocytes by immortalization with origin-defective simian virus 40 large T
antigen (Line C-20/A4) cultured as de-differentiated chondrocytes (monolayer) or as
differentiated cells (cultured on alginate beads). In these studies, the Clontech Human
Cancer Array 1.2 was employed and the results were validated employing quantitative
PCR. These investigators found that in monolayer cultures, numerous genes involved
in cell proliferation were strongly upregulated. Of the cell cycle-regulated genes, only
two, the CDK regulatory subunit and histone H4, were downregulated when the cells
were cultured in alginate beads, findings consistent with the ability of these cells to
proliferate in suspension culture. In contrast, the expression of several genes that are
involved in pericellular matrix formation, including MMP-14, COL6A1, fibronectin,
biglycan and decorin, was upregulated when the C-20/A4 cells were transferred to sus-
pension culture in alginate. These results indicated that although immortalized chon-
drocyte cell lines were not identical to primary chondrocytes in their patterns of gene
expression, they may nevertheless serve as valuable models for examining chondrocyte
function and pathophysiology when the scarcity, lack of availability and loss of the
chondrocyte-specific phenotype with serial passages limit the use of primary chondro-
cytes.
Gene expression profiles during the in vitro redifferentiation process of human ar-
ticular chondrocytes isolated from clinical samples from patients undergoing an autolo-
gous chondrocyte transplantation therapy were studied by Tellheden et al. [27].
Monolayer expanded human articular chondrocytes from four donors were cultured in a
pellet system and the redifferentiation was investigated by microarray analysis. The
culture expanded chondrocytes redifferentiated in the pellet model and the gene expres-
sion pattern changes included an increase in expression of genes encoding type II col-
lagen and other cartilage-specific matrix proteins, a strong downregulation of extracel-
lular signal-regulated protein kinase (ERK-1) and an upregulation of p38 kinase and
SOX9 genes, suggesting that redifferentiation closely mimicked some aspects of the
signaling processes involved in early chondrogenesis. Thus, these data showed that
adult human articular chondrocytes expanded from the cells remaining following
autologous chondrocyte transplantation in monolayer cultures retain the ability to redif-
ferentiate under certain permissive culture conditions and form cartilage like matrix in
vitro. The microarray data further suggested that this process involves the participation
of genes known to be expressed in early chondrogenesis.
In a more recent study of gene expression during chondrocyte differentiation and
de-differentiation, Goessler et al. [28] investigated the expression of distinct markers
during the de-differentiation of human chondrocytes in cell culture using microarrays
focusing on transforming growth factor β (TGFβ) pathways. In chondrocytes undergo-
ing de-differentiation, the gene for TGFβ1 was consistently expressed, while the gene
for TGFβ2 was not expressed under any conditions. The genes for TGFβ3, TGFβ4 and
TGFβi were activated with ongoing de-differentiation. TGFβ-receptor 3 was constantly
expressed, while the genes for TGFβ-receptors 1 and 2 were not expressed at any time.
The genes for LTBP1 and LTBP2 were activated with ongoing de-differentiation,
whereas the gene for LTBP3 was constantly expressed. Immunohistochemical staining
was employed for validation of the microarray results. The results suggested that
TGFβ3, TGFβ4, TGFβi, LTBP1 and LTBP2 participate in the process of de-
differentiation, while TGFβ1 and TGFβ2 might not be involved in this process. Of the
TGFβ-receptors, only the TGFβ receptor 3 appeared to be involved in de-
differentiation.
In other studies, the same group of investigators [29-31] examined the changes in
the expression patterns of various collagens and various regulatory proteins during on-
going culture and de-differentiation of nasal septum human chondrocytes maintained in
primary cell culture for 1, 6, and 21 days employing a microarray which allowed to
examine the expression of more than 9,000 individual human genes. After 6 and 21
days, collagen IX and X genes were downregulated, whereas collagen XI genes were
activated. Collagens I and II genes were downregulated initially but were reactivated
after 21 days. The results with analysis of TGFβ1 gene expression were grossly similar
to those reported in their previous study [28]. The genes encoding integrins β1, β5, and
α5 were upregulated from day 1 to day 21; integrin β3 was downregulated. Although
all data were not shown, the authors concluded that these studies suggested that genes
for collagens III, IV, VII, IX and XI might be new markers for the de-differentiation of
chondrocytes. Collagen II gene expression might reflect more closely the synthetic
activity of the cells rather than their de-differentiation and that integrins β1, β5, and α5
might be involved in signal transmission involved in the de-differentiation process.
An interesting study performed a comprehensive analysis of gene-expression pro-
files in human articular hyaline cartilage presumably representing fully differentiated
chondrocytes and meniscus fibrocartilage presumably representing fibrous de-
differentiation of chondrocytes by means of a cDNA microarray consisting of 23,040
human genes [32]. The profiles of the two types of cartilage were compared with each
other and with those of 29 other normal human tissues. Remarkable similarities were
found between the patterns of gene expression of the two cartilaginous tissues. It
should be mentioned, however, that the normal articular cartilage employed for these
studies was obtained from the patello-femoral joint of patients undergoing total knee
replacement for OA and although the tissues had a “normal” macroscopic appearance,
there were no histologic studies to confirm their normality, thus, it is likely that the
gene expression profile of these tissues may have reflected some early OA changes or
early changes of de-differentiation which rendered them more similar to the meniscus
fibrocartilage. Given the remarkable lack of differences in the gene expression profiles
of hyaline cartilage and meniscus fibrocartilage, the authors then compared the patterns
of gene expression of the two types of cartilage to those of 29 other non-cartilaginous
normal tissues. They identified 24 genes that were specifically expressed in both carti-
laginous tissues. Among these, the most important genes expressed specifically in carti-
laginous tissues (both hyaline and fibrocartilage) compared to non-cartilage tissues
were WNT7A (a member of the WNT family of secreted signaling molecules), COMP,
GLG1 (a membrane bound sialoglycoprotein involved in chondrogenesis), and GPS2
(a polypeptide which regulates RAS and MAPK pathways). A high level of expression
of SOX9 was found in both cartilages, however, SOX9 was also substantially ex-
pressed in 9 of the other non-cartilaginous normal tissues. The cartilage profiles were
also compared with the profiles of gene expression in human mesenchymal stem cells.
Twenty-two genes that were differentially expressed in cells representing the two carti-
laginous lineages (11 specific to each type) were identified. These could serve as mark-
ers for predicting the direction of chondrocyte differentiation.
2. Gene Expression Analysis of Chondrogenesis
Chondrogenesis, the process by which uncommitted pluripotent mesenchymal stem

cells evolve into the specific cells that populate all cartilaginous tissues in the body, is
one of the most crucial events during the embryonic development of all vertebrate spe-
cies. Chondrogenesis leads to the formation of permanent cartilaginous tissues that
undergo endochondral bone formation in the skeleton as well as the cartilage of the
growth plate and other organs such as the upper respiratory tract and the structural
components of the inner ear. Chondrogenesis is also responsible for the formation of all
articular surfaces and, therefore, even minor alterations in this highly organized and
extremely complex process are likely to lead to the development of serious disorders
ranging from chondrodysplasias to OA. Although remarkable progress has been ac-
complished in recent years in the elucidation and understanding of the multiple steps
and pathways involved during chondrogenesis and of the crucial molecular participants
in this process, there still remains a large body of information that needs to be revealed.
The recent application of microarray studies has provided substantial insights and has
opened novel pathways of investigation into this crucial process.
In one of the earliest studies, Sekiya et al. [33] followed the chondrogenic process
induced by recombinant human BMP-2, -4, and -6 in human mesenchymal stem cells
(MSC) isolated from the bone marrow from normal adult donors. The cells were cul-
tured in a pellet system for 21 days in chondrogenic medium containing TGFβ-3 and
dexamethasone with or without BMP-2, -4, or -6. Microarray analyses were performed
employing the HG-U95A array which contains 12,526 genes and the results were con-
firmed by PCR assays. The results showed that critical regulatory genes for chondro-
genesis and numerous chondrocyte-specific genes were expressed in a specific time
sequence in response to BMP-2. The most important changes included a greater than
100-fold increase in the expression of COL2A1, COL9A3, COL10A1, COL11A1 and
A2, COMP, dermatopontin, matrilin-3, aggrecan, fibromodulin and PTHrP-R. Other

genes with increased expression, although to a lesser extent, included SOX9, Forkhead,
SOX5 and Indian Hedgehog transcription factors and WNT5, IGFB5 and retinol bind-
ing protein (RBP). A notable decrease in FRIZZLED 2, cadherins 4 and 13, and in-
tegrin α3 genes was also observed. The results from this study displayed numerous
similarities to those described by Stokes et al. [26] reviewed in the previous section,
thus, confirming the validity of both observations.
In another related study, Goessler et al. [34] investigated the expression of in-
tegrins using microarray analysis during chondrogenic differentiation of human MSC
in comparison with de-differentiating human chondrocytes harvested during septo-
plasty emphasizing changes in adhesion proteins and their receptors. During chondro-
genic differentiation of MSC, the genes for fibronectin-receptor (integrin α5β1), fi-
bronectin and the GPIIb/IIIa-receptor were downregulated. The genes encoding com-
ponents of the vitronectin-receptor (integrin αvβ3) and CD47 were constantly ex-
pressed and the integrin-linked kinase (ILK) gene was downregulated. In contrast, ILK,
CD47, and ICAP genes were activated with ongoing de-differentiation of adult chon-
drocytes. The authors concluded that a candidate for signal-transmission involved in
de-differentiation is the fibronectin receptor (integrin α5β1) in conjunction with its
ligand, fibronectin. The GPIIb/IIIa-receptor might assist the process of de-
differentiation. Other receptors, e.g., for vitronectin and osteopontin (integrin αvβ3) or
their ligands, do not seem to be involved in the signaling events required for de-
differentiation.
Osawa et al. [35] performed similar studies to those of Sekiya [33] and
Goessler [34] employing instead of human MSC the mouse embryonal carcinoma cell
line ATDC5, a cell line which provides an excellent model system for a detailed analy-
sis of the chondrogenesis process in vitro. To understand better the molecular mecha-
nisms of endochondral bone formation, the gene expression profiles during the differ-
entiation course of ATDC5 cells were examined using an in-house microarray harbor-
ing 2,913 full-length-enriched cDNAs. The cDNA microarray was constructed from a
full-length-enriched cDNA library prepared with mixtures of mRNA obtained from the
ATDC5 cells and from mouse cartilage and bone and separately from E14 mouse fetus.
The results were validated by real-time RT-PCR and some were verified by Northern
hybridization analyses and immunohistology of developing murine growth plates. Fol-
lowing chondrogenic induction, 507 genes were up- or down-regulated by at least
1.5-fold. Genes for growth factor and cytokine pathways were significantly increased
in expression during late stages of chondrocyte differentiation and included decorin,
osteoglycin and asporin genes, which have been shown to bind to TGF-β and BMPs.
The authors emphasized the results suggesting that small leucine-rich proteoglycans
and asporin may play an important role in the regulation of chondrogenesis and that the
coordinated interaction between a number of intercellular signaling molecules is likely
to take place in the late chondrogenic stage.
3. Gene Expression Analysis of Cytokine and Growth Factor Effects on

Chondrocytes and Cartilage
The crucial role that cytokines play in the pathogenesis of rheumatoid arthritis, OA,
and other rheumatic diseases has been given extraordinary scientific attention since the
pioneering and pivotal studies of Saklatvala and Dingle [36] demonstrating the pres-
ence of a protein from synovium capable of exerting a profound catabolic effect on

articular cartilage. The protein termed initially catabolin was subsequently identified as
interleukin-1 (IL-1) [37]. It is generally accepted that the discovery of this pleotropic
protein is among the most important scientific discoveries of the last three decades and
has generated intense research activity particularly within the field of OA and carti-
lage [38–42]. It is, therefore, not surprising that numerous genomic profiling and mi-
croarray studies have been performed in order to increase the understanding of the
complex effects cytokines and related growth factors exert on chondrocytes and on
articular cartilage structure and function.
Vincenti and Brinckerhoff [43] conducted one of the earliest studies applying mi-
croarray analysis to identify putative immediate early genes involved in IL-1β effects
on articular cartilage employing the SW1353 chondrosarcoma cell line stimulated with
IL-1β. The Clontech Atlas Human Cancer 1.2 K array which contains 1,176 unique
genes was employed. Although the number of genes in this array is very small and the
genes contained had been selected by the manufacturer for their relevance to cancer,
this analysis identified alterations in the expression of genes encoding multiple tran-
scription factors, cytokines, growth factors and their receptors, adhesion molecules,
proteases, and signaling intermediates that may contribute to inflammation and carti-
lage destruction in arthritis. Among the numerous transcription factor genes upregu-
lated following IL-1 treatment, those belonging to the NF-κB and AP-1 family were the
most prominently increased. In contrast, IL-1 caused significant reduction in expression
of HOX-4A, retinoblastoma-like protein 2 and SMAD5 genes along with a substantial
decrease in type II collagen transcripts. Among genes for cytokines and growth factors,
LIF and IL-6 were increased whereas BMP4 was reduced. A potent stimulation of
genes for several MMP family members including collagenase-3, matrilysin and metal-
loelastase was also observed although that for collagenase-1 was not changed. Of sub-
stantial interest was the reduction in FRIZZLED 2 expression which has been also ob-
served in other studies during chondrocyte de-differentiation. Thus, this analysis has
identified numerous IL-β-responsive genes that warrant further investigation as media-
tors of disease in OA.
In a similar study, Gebauer et al. [44] investigated the response to IL-1β of the
same human chondrosarcoma cell line SW1353 in comparison with primary human
chondrocytes (PHC) by gene expression analysis assayed using a custom-made oli-
gonucleotide microarray representing 312 chondrocyte-relevant genes. The expression
levels of selected genes were confirmed by real-time PCR. Although gene expression
profiling showed only limited similarities between SW1353 cells and PHCs at the tran-
scriptional level, both types of cells showed similar changes with respect to catabolic
effects following IL-1β treatment. In similarity to the results obtained by Vincenti and
Brinckerhoff [43], MMP-3 and MMP-13 genes were strongly induced by IL-1β in both
systems. The gene encoding IL-6 was also found to be up-regulated by IL-1β in both
cellular models. However, the MMP-1 gene was found to be increased whereas expres-
sion of genes for intercellular mediators such as LIF and BMP-2 was not induced by
IL-1β in SW1353 cells. This study also identified NFκB as an important transcriptional
regulator of IL-1β-induced genes in both SW1353 cells and PHCs. One important con-
clusion from these studies was that SW1353 cells are a cell line with only very limited
similarities to PHC except for the study of induction of protease expression in response
to IL-1β.
In another related study, Aigner et al. [45] applied cDNA-array technology em-
ploying the Clontech Atlas 1.2 K array to study gene expression patterns of primary
human normal adult articular chondrocytes isolated from one single donor cultured
under anabolic (serum) or catabolic (IL-1β) conditions. Serum and IL-1β significantly
altered gene expression levels of 102 and 79 genes, respectively. The anabolic effects
of serum supplementation were confirmed by a marked stimulation of expression of
various collagen genes (COL2A1, COL11A1, COL6A1, A2, and A3, and COL16A1)
(types II, XI, VI, and XVI) as well as some proteoglycan genes including biglycan. The
catabolic effects of IL-1β treatment were reflected in substantial downregulation of
genes for COL2A1, aggrecan, decorin and biglycan expression and a marked increase
in expression of several MMP genes (MMP-1, MMP-3, MMP-13 and MMP-14). In
similarity to results from other studies discussed above, IL-1β upregulated the expres-
sion of numerous other genes including LIF, IL-6, Rho8 and members of the NF-κB
protein cascade. Also in agreement with other studies, a reduction in expression of
FRIZZLED 2, vimentin and osteonectin genes was observed upon IL-1β treatment.
Comparative gene expression analysis with previously published data from whole nor-
mal and OA cartilage showed significant differences compared to the changes detected
in OA cartilage indicating that the IL-1β stimulation did not appear to be a good model
for the gene expression alterations in OA chondrocytes.
In another study, the changes in global chondrocyte gene expression in response to
fibronectin fragments (FN-f), were investigated for the expression of various cytokine
genes by cDNA microarrays and were confirmed employing a cytokine protein ar-
ray [46]. Two microarrays were employed; one was a 268 cytokine receptor genes con-
taining array from Clontech and the other the Affimetrix U133A gene chip microarray.
Compared with untreated control cultures, stimulation by FN-f resulted in a > 2-fold
increase in IL-6, IL-8, MCP-1, and growth-related oncogene β (GRO-β) gene expres-
sion. Constitutive and FN-f-inducible expression of GRO-alpha and GRO-gamma were
also noted by RT-PCR and confirmed by immunoblotting. Inhibitor studies revealed
that FN-f-induced stimulation of chondrocyte chemokine gene expression was depend-
ent on NF-κB activity, but independent of IL-1. The studies showed the ability of FN-f
to stimulate chondrocyte expression of multiple proinflammatory cytokine and
chemokine genes suggesting that damage to the cartilage matrix with production of
FN-f can induce a potent proinflammatory state responsible for further progressive ma-
trix destruction.
To examine the additive or synergistic effects of interleukin-1 (IL-1) in combina-
tion with the IL-6/LIF related protein, oncostatin M (OSM), that may be involved in
mechanisms of cartilage repair and degradation, Barskby et al. [47], employed gene
microarray and real-time PCR experiments using RNA from human chondrosarcoma
SW1353 chondrocytes and primary human articular chondrocytes. The Affimetrix Hu-
man U133A and B microarrays were employed and the results were validated by real-
time PCR. The combination of IL-1 and OSM markedly up-regulated either coopera-
tively or synergistically the expression of numerous genes, including those encoding
MMP-1, -3, -10, -12, -13, and -14, cytokines, chemokines, extracellular matrix compo-
nents, and proteins and mediators involved in signal transduction. Real-time PCR con-
firmed a synergistic induction of genes for several MMPs, activin A, pentraxin 3
(PTX-3), and IL-8. The results demonstrated that the potent proinflammatory cytokine
combination of IL-1 plus OSM synergistically and coordinately up-regulated many
genes including those encoding several MMP involved in cartilage degradation. The
results also showed that the cytokine effects are not solely catabolic since some genes
with anabolic functions were also upregulated. Thus, this gene-profiling study empha-
sizes the complex processes that mediate articular cartilage degradation mediated by
members of the interleukin family of cytokines through the coordinated expression of
multiple genes.
4. Gene Expression Analysis of OA
Although there is a large body of information regarding numerous genes whose expres-
sion is either increased or decreased during the development of OA and the participa-
tion of their encoded products in the pathogenesis of the disease has been extensively
studied, the vast majority of these studies have employed biochemical and molecular
analyses of a few genes with putative participation in this process. The introduction of
microarray technology, on the other hand, allows the evaluation of the transcriptional
expression of very large numbers of genes or even of the entire genome transcriptional
activity at a specified point in time or under the influence of a particular stimulus or
putative pathogenetic factors/mechanisms. Although there are some important limita-
tions with this approach, this powerful technology has already become successfully
applied to the study of human OA and important and valuable information has already
been obtained from the few published studies.
Most of the work employing microarrays to study human OA was performed by
Aigner and collaborators [48–55]. In early studies, these authors utilized samples from
single or few OA patients and employed the Clontech Atlas Human Cancer 1.2 array
which is a limited microarray containing only 1,176 genes of relevance to cancer re-
search [48–50]. Despite these limitations, these early studies provided valuable infor-
mation regarding changes in the expression of numerous genes in OA and revealed
activation as well as phenotypic instability of articular chondroctyes. However, given
the limited number of patient samples studied and the very small number of genes able
to be examined employing this array, a broader gene expression profile of OA chon-
drocytes was performed [51]. This recent study compared normal articular cartilage (18
specimens, from subjects ages 45–88 years), cartilage with early OA (20 specimens,
from subjects ages 43–91 years obtained at autopsy), and OA cartilage obtained at the
time of total knee replacement (21 samples with mild OA and 19 samples with moder-
ate or severe OA from patients ages 61–84 years). This large gene expression profiling
study was performed with 78 normal and disease human articular cartilage samples
using a custom-made complementary DNA array covering > 4,000 genes. Several com-
parisons were made according to the absence or presence of OA lesions and to their
degree of severity based on Mankin’s scale. Many differentially expressed genes were
identified, including the expected up-regulation of genes encoding matrix macromole-
cules and anabolic and catabolic mediators. Some of the most relevant genes are listed
on Table 2.
The genes for types I, II and III collagens were strongly up-regulated in moder-
ate/severe OA cartilage, which is consistent with the findings of numerous previous
studies likely reflecting the general metabolic activation of OA chondrocytes rather
than de-differentiation. The genes encoding collagen types VI, IX and XI were also
found to be significantly up-regulated, but to a much lesser degree. In contrast, genes
Table 2. Genes up- or down-regulated in cartilage lesions with moderate/severe late-stage OA compared
with normal samples [51]
Gene Fold change
Collagen, type I, 1 (COL1A1) 9.55

Selenoprotein H (SELH) 9.27
Tenascin C (hexabrachion) (TNC) 8.06
Collagen, type III, 1 (COL3A1) 7.37
Collagen, type II, 1 (COL2A1) 6.07
Collagen, type V, 1 (COL5A1) 5.81
Interleukin-1 receptor, type II (IL1R2) 5.34
Fibronectin 1 (FN1) 4.69
Protease, serine, 11 (IGF binding) (PRSS11) 4.17
Collagen, type I, 2 (COL1A2) 3.41
Secreted protein, acidic, cysteine-rich (osteonectin) 3.34
Cartilage acidic protein 1 (CRTAC1) 3.01
Collagen, type VI, 2 (COL6A2)C2 2.96
Cartilage intermediate-layer protein (CILP) 2.72
Collagen, type XI, 1 (COL11A1) 2.66
Collagen, type VI, 1 (COL6A1) 2.58
Fibromodulin (FMOD) 2.56
Caspase 10, apoptosis-related cysteine 2.53
Chitinase 3-like 2 (CHI3L2) 2.11
Tissue inhibitor of metalloproteinases 1 (TIMP1) 2.05
Glutathione peroxidase 3 (plasma) (GPX3) 0.12

CCAAT/enhancer binding protein (C/EBP), delta 0.15
Thioredoxin-interacting protein (TXNIP) 0.29
Stromelysin 1 (MMP3) 0.29
Stearoyl-CoA desaturase 4 (SCD4) 0.31
Serine hydroxymethyltransferase 2 (mitochondrial) 0.32
Metallothionein 1E (functional) (MT1E) 0.33
Dual-specificity phosphatase 1 (DUSP1) 0.33
Cytokine-like protein C17 (C17) 0.35
BTG family, member 2 (BTG2) 0.36
Metallothionein 1G (MT1G) 0.38
Insulin-like growth factor binding protein 6 (IGFBP6) 0.43
Metallothionein 1X (MT1X) 0.43
Transducer of ERBB2, 1 (TOB1) 0.45
Glutamate-ammonia ligase (glutamine synthase) (GLUL) 0.48
Nicotinamide N-methyltransferase (NNMT) 0.48
Superoxide dismutase 2, mitochondrial (SOD2) 0.48
Figure 3. Dendrogram and heat map of the top 50 genes selected from the analysis of normal versus late-
stage OA cartilage. A nearly perfect separation between the normal samples and the late-stage OA samples is
seen. Adapted from ref. [51]. (Reproduced with permission of the publisher).
for noncollagenous matrix proteins were generally less up-regulated in OA chondro-

cytes, except for those encoding fibromodulin, cartilage intermediate-layer protein,
fibronectin, tenascin, and osteonectin/secreted protein, acidic and rich in cysteine. Ex-
pression of the SOX9 gene which encodes one of the most important cartilage
transcription factors involved in chondrogenesis and in the maintenance of chondrocyte
phenotypic stability was significantly down-regulated in OA cartilage. One important
finding was the down-regulation of the genes for superoxide dismutases 2 and 3 and
glutathione peroxidase 3. This observation suggests that continuous oxidative stress to
chondrocytes and the cartilage matrix may be a pathogenetic mechanism in OA. An-
other important observation was that only 15 genes were significantly up- or down-
regulated between normal and early degenerative cartilage lesions. In contrast, the
genes that were differentially expressed between normal and severe OA cartilage were
significantly higher. An ontology analysis revealed a broad spectrum of differentially
regulated genes and emphasized the up-regulation of numerous genes involved in ex-
tracellular matrix formation, the downregulation of genes involved in oxidative damage
defense, and changes in expression of numerous genes for cytokines or genes involved
in cytokine signaling. One interesting finding was that many genes related to the IL-1
pathway were not up-regulated, but instead down-regulated in OA chondrocytes. This
included IL-1β itself as well as IL-6, IL-8, and LIF. Figure 3 shows a biased cluster
analysis of the 50 most highly differentially expressed genes in the analysis of normal
compared with advanced or severe OA cartilage showing a nearly perfect separation of
the normal samples from the OA samples.
Thus, these findings provide a large reference data set on global gene alterations in
OA cartilage and suggest major mechanisms underlying central biologic alterations that
occur during OA such as the role of oxidative stress.
Another study of global gene expression patterns in OA chondrocytes was per-
formed by Tardif et al. [56] who compared gene expression patterns in normal and os-
teoarthritic (OA) human chondrocytes using the Atlas Human 1.2 microarray. Of the
novel genes identified, the authors focused on follistatin, a BMP antagonist, and three
other BMP antagonists, gremlin, chordin, and noggin, in normal and OA chondrocytes
and synovial fibroblasts. The genes for all BMP antagonists except noggin were ex-
pressed in chondrocytes and synovial fibroblasts. Follistatin and gremlin genes were
significantly up-regulated in OA chondrocytes whereas chordin was weakly expressed
in normal and OA cells and the gene for noggin was not expressed at all. Production of
follistatin protein paralleled the gene expression pattern. Follistatin and gremlin were
expressed preferentially by the chondrocytes at the superficial layers of cartilage. IL-1β
had no effect on follistatin but reduced gremlin expression. Conversely, BMP-2 and
BMP-4 significantly stimulated expression of gremlin but down-regulated that of fol-
listatin. Thus, the results of this study show the possible involvement of follistatin and
gremlin in OA pathophysiology.
In a similar study, Sato et al. [57] analyzed the differences in gene expression pro-
files of chondrocytes in intact and damaged regions of cartilage from the same knee
joint of patients with knee OA. Gene expression profiles in regions of intact and dam-
aged cartilage (classified according to the Mankin scale) obtained from patients with
knee OA were examined. Five pairs of intact and damaged regions of OA cartilage
were evaluated by oligonucleotide array analysis using a double in vitro transcription
amplification technique and the Affimetrix U133A and B high density arrays. The mi-
croarray data were confirmed by real-time quantitative PCR. About 1,500 transcripts,
which corresponded to 8% of the expressed transcripts, showed > or = 2-fold differ-
ences in expression between the cartilage tissue pairs. The expression of some genes
related to the wound-healing process, including cell proliferation and interstitial colla-
gen synthesis, was higher in damaged cartilage compared to cartilage from intact re-
gions, similar to the findings for genes that inhibit matrix degradation. Comparisons of
the gene expression profile differences with real-time quantitative PCR data supported
the validity of the data. Differences between intact and damaged regions of OA carti-
lage exhibited a similar pattern among the 5 patients examined, indicating the presence
of common mechanisms that contribute to cartilage destruction.
Concluding Remarks
The extraordinary potential of large-scale gene expression profiling employing mi-

croarray technology for arthritis research as discussed by Grant et al. [58] has just be-
gun to be realized in the fields of articular cartilage and OA investigations and an
enormous body of valuable information has already been obtained in the relatively lim-
ited number of studies which employed this approach. Despite the initial skepticism
and serious methodological concerns about the validity and applicability of large-scale
genomic profiling [59,60], and the subsequent appreciation of substantial limitations of
this novel technology as pointed out in numerous publications [e.g., 61], it has become
apparent that rigorous evaluation of the results and prudent, conservative and innova-
tive interpretation of the massive amounts of data obtained can, indeed, provide valu-
able biological insights into the pathogenesis and disease mechanisms of a variety of
complex disorders [62], including OA. The search for specific genes involved in the
pathogenesis of OA using cDNA array technology has allowed the identification of
several molecules potentially relevant to the disease process, among them SOX9,
FRIZZLED and follistatin. Thus, a new era of OA research has arrived with the suc-
cessful application of large-scale microarrays followed by high throughput validation
of the results employing real-time PCR. These studies will undoubtedly advance
greatly our current understanding of OA pathogenesis and will provide molecular sig-
natures of OA chondrocytes which will be useful as molecular biomarkers for the diag-
nosis of OA, identification of its clinical subsets, and evaluation of disease-modifying
therapies in the not too distant future.
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Part III
Effectors and Different Pathways
IOS Press, 2007
Prostaglandin E2 and Osteoarthritis:

The Role of Cyclooxygenases,
Prostaglandin E Synthases
and 15-Prostaglandin Dehydrogenases
Odile GABAY a,1, Marjolaine GOSSET a,1 and Francis BERENBAUM a,b,*
a
UMR 7079 CNRS, Physiology and Physiopathology Laboratory,
University Paris 6, 7 quai St-Bernard, Paris, 75252 Cedex 5, France
b
Department of Rheumatology, UFR Pierre et Marie Curie, Saint-Antoine hospital,
75012 Paris, France
Introduction
Osteoarthritis (OA) is characterized mainly by degenerative changes in joint cartilage

resulting in loss of cartilage, alterations of subchondral bone, and a local inflamma-
tion [1]. During inflammatory process, membrane phospholipids lead to eicosanoid,
products of the acid arachidonic cascade. Among them, Prostaglandin E2 (PGE2) plays
an important role.
Role of Prostaglandin E2 in Cartilage Homeostasis and in OA
The prostaglandins (PG) are a group of fatty acid compounds that have many effects
throughout the body, including activity in inflammation, smooth muscle contraction,
regulating body temperature, and effects on certain hormones. PGE2 is synthesized by
many cell types and tissues. It has long been considered as the principal prostaglandin
in arthritic diseases as well as in age-related diseases such as OA [2]. Notably, Bonner
et al. have shown that the concentration of arachidonic acid, the precursor of the pros-
taglandins, increased markedly with age [3].
PGE2 and Cartilage Degradation
PGE2 is highly expressed in arthritis [4]. To understand the mechanism by which pros-
taglandins could modulate the arthritis-induced cartilage degradation, McCoy et al.
1
The authors participate equaly to the redaction of the manuscript.
* Corresponding Author: Pr. F. Berenbaum, UMR 7079 Paris VI-CNRS, Physiology and Physiopathology
Laboratory, University Paris 6, 7 quai St-Bernard, Paris, 75252 Cedex 5, France. Phone: +33 144-27-22-83,
Fax: +33 144-27-51-40, E-mail: francis.berenbaum@sat.aphp.fr.
164 O. Gabay et al. / Prostaglandin E2 and Osteoarthritis
Figure 1. Role of Prostaglandin (PG)E2 in OA process. Two hypothesis of action are proposed for PGE2 in
OA: the decrease of synthesis of glycosaminoglycans and the increase of Matrix Metalloproteases (MMPs)
synthesis. PGE2 acts mainly by the binding on the EP2 and EP4 presents on cells membranes of chondro-
cytes, synoviocytes and osteoblasts.
have studied mice lacking each of the four known PGE2 receptor, named EP1-4 recep-
tors, after generation of collagen-induced arthritis. The deletion of EP1, EP2, EP3 re-
ceptors did not affect the development of arthritis but EP4 receptor deficient mice re-
sulted in an absence of cartilage degradation in collagen-induced arthritis. Thus, PGE 2
contributes to cartilage degradation in part by binding to the EP4 receptor [5].
The exact mechanisms of PGE2 actions leading to cartilage degradation are
not well understood. However, two main hypothesis are proposed: the first one is a
decreased synthesis of glycosaminoglycans [6]. The second one is an increase of
MMP synthesis [7]. Pelletier et al. have shown that the synthesis of MMP-1 is eico-
sanoid–dependent in human OA synovial membrane explants [8]. Moreover, the
IL-1β–induced MMP-2 activation and expression were found to be dependent on PGE2
in human chondrocytes [9]. Finally, MMP and TIMP expressions are regulated by
PGE2 in equine chondrocytes [10]. However, PGE2 suppressed MMP-1 expression
through C/EBP/NF-κB/MEKK1 suppression in synovial fibroblasts [11] (Fig. 1).
PGE2 and Apoptosis
Chondrocyte death may contribute to the progression of OA: OA cartilage has a higher
number of apoptotic chondrocytes than does normal cartilage in animal models [12]
and in humans [13]. Nitric Oxide (NO) and PGE2 play a crucial role in chondrocyte
death [14]. NO, generated from sodium nitroprusside has been shown to induce apop-
tosis in human articular chondrocytes, and IL-1β induced NO inhibits chondrocytes
proliferation via PGE2 [15,16]. Moreover, Notoya et al. have shown that NO induces
O. Gabay et al. / Prostaglandin E2 and Osteoarthritis 165
COX-2 expression through ERK1/2 and p38 kinase pathways resulting in an increase
of PGE2 release in human OA chondrocytes [14]. Therefore, PGE2 may sensitize chon-
drocytes to the cell death induced by NO resulting from an autocrine-paracrine mecha-
nism. Finally, Miwa et al. have shown that PGE2 induces apoptosis through c-AMP-
dependant pathway in articular chondrocytes [17].
PGE2 and Chondrocyte Differentiation
Regulation of chondrogenesis and chondrocyte maturation by prostaglandins has been

of interest these last years. Prostaglandins regulate chondrocyte phenotype, but their
role in chondrocyte differentiation is not yet clear [18]. Jakob et al. have shown that
PGE2 reduced collagen type I expression and doesn’t improved chondrogenesis [19].
Moreover, Li et al. have demonstrated that PGE2 inhibits chondrocyte differentiation
through Protein Kinase A and C (PKA and PKC) signalling [20]. However, another
study has shown opposite effects of PGE2 depending on its concentration: low levels of
PGE2 increase proteoglycans synthesis whereas high doses decrease it [21].
In addition, prostaglandins receptors EP1 to 4 seems to play an important role dur-
ing the maturation of the chondrocyte but the role of PGE2 seems to be linked to this
state of maturation. A full assessment of all subtypes of receptors in chondrocytes need
to be better defined [22].
PGE2 Synthesis and Degradation in OA Articular Cells
The synthesis of PGE2 is the terminal step of a sequence of enzymatic reactions, in-
cluding the release of arachidonic acid (AA) from membrane phospholipids by phos-
pholipase A2 (PLA2) and conversion of this substrate to prostaglandin H2 (PGH2) by
cyclooxygenase (COX)-1 and COX-2. PGH2 is subsequently metabolized by PGE syn-
thase (PGES) to form PGE2. The COX-1 isoform is expressed constituvely by many
cell types, whereas COX-2 requires specific induction by inflammatory mediators such
as lipopolysaccharides (LPS) and cytokines [23]. The prostaglandin E synthase catalyse
the conversion of PGH2 to PGE2. Three isoforms of PGES have been cloned [24–26]
including cytosolic PGES and two microsomal forms: glutathione-specific mPGES-1
and glutathione non-specific mPGES-2. cPGES is constituvely expressed and unre-
sponsive to inflammatory stimuli whereas mPGES-1 is inducible in an inflammatory
context [24,25,27]. The coordinate regulation and functional coupling of mPGES-1 and
COX-2 have been reported [27] (Fig. 2).
After synthesis, PGE2 needs to be released from cells in order to exert their ex-
tracellular effect. First, passive diffusion permits a slow exit of PGE 2 from the
cells [28]. Second, the transporters MRP2 and 4 (multidrug resistance proteins type 2
and 4) seem to be implicated in PGE2 efflux [29,30]. The specific prostaglandin trans-
porter is implicated in the influx of PGE2 into the cells but not in the efflux [31].
PGE2 usually act on cells as hormones in a paracrine or autocrine way by interac-
tion with specific receptors on cells membranes. PGE2 has 4 distinct receptors, namely
E-prostanoid (EP) 1 to EP4, encoded by distinct genes and with an expression depend-
ing on the cell type. These receptors belong to the G protein-coupled cell-surface re-
ceptor family. Recently, functional EP1-4 receptors were described at the nuclear
membrane of many cells [32] but their role in articular cells remain unknown. If PGE 2
does not bind with its specific receptors, this eicosanoid is rapidly converted to an inac-
Figure 2. The acid arachidonic cascade leading to PGE2 synthesis and its degradation. The conversion of
arachidonic acid to prostaglandin (PG)E2 occurs by sequencial enzymatic reactions involving isoformes of
cyclooxygénase (COX) and PGE synthase (PGES). COX-1 and cytosolic PGES are constitutive isoforms
whereas COX-2 and mPGES-1 isoforms are regulated by pro-inflammatory stimuli. After release from cells,
PGE2 interact with specific EP receptors. Its degradation is catalyzed by the cytosolic 15-PG dehydrogenase
(15-PGDH) into the inactive metabolite 15-ketoPGE2.
tive metabolite (13-14-dihydro 15-keto PGE2) by the prostaglandin 15-dehydrogenase

(15-PGDH) pathway.
A. The COX Enzyme: Structure, Regulation and Role
Cyclooxygenase, also known as prostaglandin endoperoxide H synthase, catalyze the

conversion of arachidonic acid to PGH2, the common precursor of all prostaglandins
synthase. Three isoforms of COX, namely COX-1, COX-2 and COX-3, have been de-
scribed (Table 1).
Whereas COX-1 is constituvely expressed in various cell types to maintain
homeostasis, COX-2 is the inducible COX isoform, implicated in PG synthesis in an
inflammatory context. COX-2 is implicated in many pathophysiological processes,
such as inflammation, pain, Alzheimer’s disease, cancer, angiogenesis and arthri-
tis [33]. COX-3 is a recently described variant of COX-1 as a result of the first intron
conservation. COX-3 is also called COX-1 V1 [34]. The focus of this article is to
present the expression and the regulation of each COX isoform in articular tissue.
Table 1. Cyclooxygenase (COX) and PGE synthase (PGES) isoforms
Enzymes/ Molecular Cellular Expression Tissue Functions in Structure

Chromosome weight/ location in joint distribution joint
mRNA size in joint
COX-1 72 kDa Nuclear constitutive ubiquitous unknown EGF-like,
9q32-q33,3 3 kb membrane- membrane
endoplasmic and catalytic
reticulum with a heme
binding site
domains
COX-2 72 kDa Nuclear inducible Stimulus- Inflammation, Idem than
1q25,2-25,3 4–4,5 kb membrane- induced in pain COX-1
endoplasmic tissues
reticulum
COX-3 65 kDa Endoplasmic constitutive Variant of unknown Idem than
(variant of 5,2 kb reticulum- COX-1 COX-1
COX-1) Nuclear &COX-2
9q32-q33,3 membrane
mPGES-1 15–16 kDa Nuclear inducible Ubiquitous Inflammation, MAPEG
9q34,4 14,8 kb membrane pain family
mPGES-2 33 kDa Golgi- constitutive skeletal mus- unknown Thoredoxin
9q33-q34 2 kb cytoplasm cle homology
domain
cPGES 26 kDa Cytoplasm – constitutive ubiquitous unknown Hsp90 co-
12q13,13 1,9 kb nuclear chaperone
membrane p23
Common Features and Differences Between COX-1 and COX-2
COX-1 and COX-2 are encoded by 2 different genes, respectively on the chromosome
9q32-q33.3 [35] and on the chromosome 1q25.2-q25.3 [36]. These enzymes are
72 KDa proteins with 60% homology [37]. They present common crystal structures and
are composed of three domains: (1) a N-terminal epidermal growth factor (EGF)
domain, (2) a helical membrane binding domain and (3) the large catalytic C terminal
domain [38]. This catalytic domain allows COX to converse AA to PGH2 by two
sequential enzymatic reactions, (a) cyclooxygenation of AA in PGG2 and (b) reduction
of PGG2 to PGH2. The kinetic properties of the two enzymes are quite similar [39,40].
However, COX-1 and COX-2 differentially use the AA substrate pool. Whereas COX-
1 acts on AA at high concentration (≥ 10µM), reflecting commonly an exogeneous
source of AA, COX-2 is biologically active at weak AA concentration (≤ 2.5µM)
resulting from an endogeneously release [41]. Moreoever, COX-1 and COX-2 show
differences in subcellular localizations. As COX-1 localizes equally in endoplasmic
reticulum and nuclear membrane of endothelial cells, COX-2 is preferentially found at
the nuclear envelop [42]. Recently, COX-2 have been shown to be localized in the
perinuclear region of articular cells. Kojima and colleagues reported that IL-1β induces
the localization of COX-2 in the perinuclear region of synoviocytes and chondro-
cytes [43,44] Moreover, we suggest that, as IL-1β does, mechanical stress induces the
localization of the COX-2 enzyme in the perinuclear region of chondrocytes [45].
Figure 3. Stimuli, signaling pathways and promoter response elements implicated in COX-2 and mPGES-1
expression. COX-2 and mPGES-1 expressions are increased in articular cells by various stimuli including the
pro-inflammatory cytokines interleukin (IL)-1β and tumor necrosis factor (TNF)-α, the reactive oxygen
species Nitric Oxyde (NO) or the mechanical stress. Moreover, hypoxia has been described as an inhibitor of
COX activity. All these stimuli activates two main intra-cellular signaling pathways, the mitogen activated
protein kinase (MAPK) and the NF-κB pathway. These signal lead to gene transcription activation of both
COX-2 and mPGES-1 genes, implicating promoter response elements.
Regulation of COX Expression
COX-1
Many differences exist between COX-1 and COX-2 in their transcriptional regulation.
COX-1 is an housekeeping gene. It lacks a CAAT or a TATA box and presents two
Sp1 cis-regulatory elements implicating in constitutive expression of COX-1 [46]. In
contrast, COX-2 gene presents a variety of response elements which explains, in part,
its inducibility by multiple inflammatory mediators, cytokines or growth factors.
Stimuli Involved in COX-2 Regulation (Fig. 3)

The pro-inflammatory cytokines IL-1β, TNFα, IL-6, the Leukemia Inhibitory Factor
(LIF) and LPS are potent activators of COX-2 expression leading to PGE2 increase in
all articular cells, ie chondrocytes, synoviocytes and subchondral osteoblasts
[23,47–53]. These effects are reversed after a dexamethasone or an NSAID treatment
[23,47,51,54].
Accumulating evidences suggest that the nitrated oxygen species and especially
nitric oxide (NO) modulate the cyclooxygenase activity in articular tissue. NO plays a
role in physiology but also in the OA inflammatory process [2]. This mediator is
spontaneously released by OA chondrocytes and synoviocytes [55]. Manfield and

colleagues reported that the inhibition of the NOSynthase (NOS) activity inhibits
the COX activity and therefore the PGE2 release in both bovine chondrocytes and
human OA cartilage [49]. However, cartilage explants treated with the NOS inhibitor
L-NMMA significantly increased COX-2 expression and subsequent PGE2
synthesis [54]. Therefore, divergent effects of NOS on COX exist, depending on the
cell-type and the COX isoform affected [55].
Mechanical stress (MS) is a key regulator of cartilage matrix turn-over but could
be deleterious when excessive loading is applied like in obesity. MS is definitely
identified as the main risk factor for OA [56,57]. Many authors have described the role
of MS on COX-2 expression. Compressive stress applied on cartilage explants or shear
stress applied on primary cultured chondrocytes triggers COX-2 mRNA and protein
expression [45,58,59]. This effect could be mediated by an increased NO release [59].
As mature articular cartilage is an avascular tissue, the oxygen supply to resident
chondrocytes could be a limiting factor for the cyclooxygenase activity. Mathy and
colleagues described that an hypoxic environment blocks COX-2 activity in bovine
chondrocytes [60] although COX-2 gene is already up-regulated in hypoxic condition
by IL-1. In fact, decrease in O2 tension triggers the expression of many factors like the
Hypoxia-inducible factor-1 (HIF-1), which is actually known as an activator of COX-2
expression [61]. The molecular mechanisms through which hypoxia modulates COX-2
activity and expression remain to be determined.
Finally, the role of estrogens in the development of OA has been suggested. First,
estrogens receptors are expressed on cartilage cells [62]. Second, Morisset and
colleagues reported that, in bovine chondrocytes, 17-β estradiol is as potent as
dexamethasone in preventing basal, but not TNFα− and IL-1β -induced COX-2 mRNA
expression [51]. Therefore, 17-β estradiol may play a role in cartilage homeostasis.
Signaling Pathways Involved in COX-2 Regulation (Fig. 3)

The signaling pathways involved in COX-2 expression are tissue-specific and depend
on the stimulus. Among them, MAPK are well described pathways in many cell types,
and especially in chondrocytes and synovial cells. p38 MAPK and JNK/SAPK are
implicated in ΤΝFα and IL-1β-induced COX-2 expression in human articular
chondrocytes [63]. p38 MAPK inhibitor (SB-202190, SB-203580, R-130823)
prevented also IL-1β−induced COX-2 expression in human synovials fibroblasts, as in
human chondrocytes cell line and in bovine chondrocytes [64–66]. Recently, a study
confirm the role of p38 MAPK but describes also, the role of Erk1/2 and Jnk in IL-1β
induced COX-2 expression [67]. Moreover, Faour and colleagues observed that IL-17-
induced COX-2 expression involve a restricted MAPK profile, the
MKK3/6/SAPK2/p38 cascade in human chondrocytes and synoviocytes [68]. Finally,
we demonstrate the implication of MAPK in mechanical stress-induced COX-2
expression. The p38 MAPK inhibitor SB203580 , the JNK kinase inhibitor SP600125 and the
specific ERK 1/2 inhibitor PD98059 significantly inhibit the COX-2 mRNA expression in
murine cartilage explants submitted to dynamic loading (personal communication).
Regulation of COX-2 Expression at the Promoter Level (Fig. 3)

Promoter region of the COX-2 gene contains a TATA box and various putative
transcriptional regulatory elements, such as nuclear factor-kB (NF-kB), the CAAT
enhancer binding protein (C/EBP), the cyclic adenosine monophosphate response

element (CRE) and peroxisome proliferator-activated receptors-responsive elements
(PPREs) [69].
Two consensus NF-kB binding sites are descibed on the COX-2 promoter. NF-kB
is a classical pathway triggered in an inflammatory context [70]. This pathway is in-
volved in the regulation of COX-2 induced by IL-1β in RA synoviocytes [71,72] and
induced by TNFα and IL-1β in human chondrocytes cell line [73,74]. The role of
NF-kB signalling was confirmed by an antisens strategy [73,75]. In contrast, Thomas
and colleagues found a role of NF-kB in the regulation of the human COX-2 promoter,
dependent on the binding of C/EBP in cultured rabbit chondrocytes [76].
The CCAAT-enhancer-binding protein (C/EBP) is critical for the stimulation
of COX-2 gene. In human synovial fibroblasts, C/EBP is involved in
TNFα−induced COX-2 expression [77]. Particularly, the role of C/EBPδ and C/EBPβ
factors were highlighted in articular chondrocytes transfected with COX-2 pro-
moter containing mutations in C/EBP cis regulatory elements, and stimulated with
IL-1 β [76].
Concerning the ATF/CRE element, there is actually little evidence for its role in
the regulation of COX-2 expression in articular tissues. Faour and colleagues described
the role of IL-17 on the increased COX-2 gene expression in both human chondrocytes
and synovial fibroblasts through the ATF-CRE enhancer site of the promoter. In fact,
mutation of this site is sufficient to abrogate induction of COX-2 promoter activ-
ity [68].
Peroxisome proliferator-activated receptors (PPARs) are ligand-activated tran-
scriptions factors, belonging to the nuclear receptor superfamily. After heterodimerisa-
tion with retinoid X receptor RXR, they bind to PPAR-responsive elements (PPREs) in
the promoter region of gene, like for COX-2. The 15-Deoxy-Δ12. 14–PGJ2 (15d- PGJ2),
the end-product metabolite of PGD2 is a potent activator of PPARγ. 15d- PGJ2 acts as a
dual agent on the regulation of COX-2 in human osteoarthritic chondrocytes. When
cells are stimulated with IL-1β, addition of 15d- PGJ2 partially reduced COX-2 expres-
sion whereas 15d- PGJ2 triggers COX-2 expression without PGE2 production in cells in
basal condition [78]. In RA synoviocytes, 15d-PGJ2 suppressed IL-1β-induced PGE2
synthesis through the inhibition of cyclooxygenase (COX-2) expression [79]. It seems
that 15d-PGJ2 exerts a negative feedback on the AA cascade in both synoviocytes and
chondrocytes in an inflammatory context (IL-1).
Regulation of COX-2 Expression at a Post-Transcriptional Level

COX-1 and COX-2 also show major differences in mRNA splicing, stability and trans-
lational efficiency. Post-transcriptional regulations on the 3’-untranslated region (UTR)
of COX-2 mRNA resulting in its stabilization, seem to be involved in the regulation of
COX-2 mRNA degradation. Such regulation has been reported by several authors in
chondrocytes submitted to IL-1α [51] and in both synoviocytes and chondrocytes
stimulated by IL-17 [68].
Expression and Role of COX in Articular Tissues
Vane first described in 1971 the existence of an enzymatic activity inhibited by aspirin
and indomethacin and implicated in the synthesis of prostaglandins. This enzyme was
called “cyclooxygenase” ans was thought to be unique [80]. In 1991, three groups
described the cyclooxygenase type 2 isoform (COX-2) inducible in an inflammatory

context [36,81,82]. Then, many authors studied the role of this enzyme in joint
inflammation using several models, like adjuvant arthritis, carrageenan-induced paw
inflammation or type II collagen-induced arthritis. Therefore an increased expression of
COX was described in articular tissues of rats treated with intraperitoneal injection of
streptococcal cell wall (SCW) or intradermal injection of Freund’s adjuvant [83]. In
1996, Anderson and colleagues were the first group to report that COX-2 plays a
proeminent role in adjuvant arthritis. After adjuvant injection in paws, an increased
expression of COX-2 mRNA and protein and a local PGE2 overrelease were found. The
use of a selective COX-2 inhibitor, SC58125, rapidly reduced the level of PGE2 in paw
tissues [84]. This result was confirmed by Kang and colleagues in 1996 [85]. Recently,
COX-2 antisense oligodeoxynucleotide injected after development of adjuvant-induced
arthritis in rats significantly suppressed induction of arthritis in a dose-dependent
manner [86].
The role of each COX isoform on type II collagen-induced arthritis has been
recently assessed. Selective inhibitors of COX-1 (FR122047, SC-560) did not inhibit
paw edema and PGE2 release in arthritic model whereas selective COX-2 inhibitor
(FR140423) did [87]. Therefore, COX-2 overexpression may be responsible to the
increase of PGE2 production implicated in edema, swelling and cellular infiltration in
joints.
In human OA cartilage and synovial samples, COX-2 mRNA and protein
expression were observed [54,85,88–90]. Moreover, OA cartilage samples sponta-
neously release more PGE2 (50-fold) than normal cartilage and 18-fold higher than
normal cartilage stimulated with cytokines or endotoxins [54]. Interestingly, IL-1β
triggers a strong increase of COX-2 activity in human OA synovial membrane but a
weak increase in articular cartilage. Authors suggest that the induction of COX-2 by
synovial cells in response to IL-1β is linked to proteoglycan degradation in OA [88].
The use of specific COX-2 inhibitors confirm the role of this enzyme in overrelease of
PGE2 in OA and may result in some beneficial effects in this disease.
COX-3
COX-3 is a recently described derivative of COX-1 as a result of the first intron

conservation. COX-3 is also called COX-1 V1. At this time, COX-3 mRNA is
described in both canine and human cortex and aorta, in the rodent heart, kidney and
neuronal tissues [34] and in mouse costal cartilage [45]. But conclusive evidence
regarding the existence of a human COX-3 protein is lacking.
Conservation of the first intron of COX-1 probably leads to the modification of the
active site conformation. COX-3 is more sensitive to acetaminophen than COX-1 and
COX-2 suggesting that COX-3 could be the target of this drug [91]. No regulation by
inflammatory mediators or mechanical loading [45] has been described yet. Only a
COX-3 mRNA expression in a human colon cancer cell line under osmotic stress has
been recently described [92]. As COX-1 and COX-3 are derived from the same gene,
these enzymes share the same promoter. No regulatory sites of COX-1 promoter by
mechanical stress or pro-inflammatory cytokines have been described. This is consis-
tent with the fact that COX-1 is constitutively and ubiquitously expressed.
B. The Prostaglandin E Synthase (PGES): Structure, Regulation and Roles
PGE synthase (PGES) is the last step of the enzymatic reactions of the arachidonic acid
cascade leading to PGE2 production. Three isoforms of this enzyme have been cloned.
Two of them are membrane-bound enzymes, called membrane-associated-PGES
(mPGES) type 1 (mPGES-1) [93] and mPGES-2 [26]. mPGES-1 is inducible and func-
tionally linked with COX-2. The cytosolic PGES (cPGES) is a protein constitutively
expressed in a large variety of cells and tissues, linked to COX-1 to promote early
PGE2 production during the inflammatory process [25]. PGES are expressed in articu-
lar tissues, cartilage, synovium and subchondral bone [94].
1) mPGES-1
Structure and Properties

The human mPGES-1 gene maps to chromosome 9q34.3. It spans 15 kb and is divided
into three exons. The primary structure of mPGES-1 from different animal species
shows a high degree of sequence homology (≈ 80%) [95]. This enzyme is a gluthatione
(GSH)-requiring perinuclear protein, member of the membrane-associated proteins
involved in eicosanoid and glutathione metabolism (MAPEG). Two amino acids are
conserved in the MAPEG superfamily: Arg110, essential for the enzymatic function,
and Tyr117. The mutation of Arg110 abrogates mPGES-1 catalytic function, implying
an essential role for this residue [96]. All MAPEG proteins have similar molecular
masses of 14–18 kDa and mPGES-1 is a 16 kDa protein. Finally, mPGES-1 is a 10
angström projection structure and constitutes a trimer in the crystal, after electron crys-
tallography [96].
Expression, Function and Regulation of mPGES-1 in Articular Tissues (Fig. 3)

mPGES-1 is localized to the superficial layers of human OA cartilage, where OA dam-
ages first appear and in which IL-1 β is also present [97]. Accumulating evidences im-
plicate mPGES-1 in the pathogenesis of OA. PGE2 exerts various physiological func-
tions through the EP receptors (EP1, EP2, EP3 and EP4). EP2 and EP4 are detected in
synovial fibroblasts from arthritis patients. Selective agonists for the EP2 and EP4 re-
ceptors increase mPGES-1 expression, in addition to PGE2, suggesting that PGE2
strongly enhance the expression of mPGES-1 in rheumatoid synovial fibroblasts and
OA synovial fibroblasts. The same mechanism is suggested in chondrocytes [98].
Moreover, non steroidal anti-inflammatory drugs, such as selective COX-2 inhibitors,
decrease the expression of mPGES-1 in IL-1β stimulated rheumatoid arthritis (RA)
synovial fibroblasts [99].
Recently, mPGES-1 inhibitors development have provided evidence of the in-
volvement of this enzyme in the inflammatory process [100].
In OA chondrocytes, osteoblasts and synoviocytes, mPGES-1 is up-regulated by
pro-inflammatory cytokines [27,101]. In chondrocytes, IL-1β but not IL-6 and IL-4 are
able to increase mPGES-1 [43]. IL-1β stimulates ERK and p38, but not JNK, MAP
kinases in chondrocytes leading to PGE2 and mPGES-1 release [102].
Moreover, the cyclopentenone 15d-PGJ2, known to have anti-inflammatory prop-
erties, decrease PGE2 synthesis in a dose-dependant manner. mPGES-1 expression is
completely abolished with a high dose of 15d-PGJ2 in rat chondrocytes stimulated by
IL-1β through inhibition of the NF-κB pathway, and in human OA chondro-

cytes [97,103].
Interestingly, recent findings show that mPGES-1 is also a mechanosensitive gene
in cartilage [104].
The putative promoter of the human mPGES-1 gene does not contain transcrip-
tional elements presents in the COX-2 promoter. It reveals the presence of two
GC-boxes, Barbie boxes, an aryl hydrocarbon regulatory element (ARE), lacks a
TATA box, and contains binding sites for C/EBPα and β, AP-1, two progesterone re-
ceptors and three GRE elements [105,106]. It was recently shown that the zinc-finger
containing transcription factors Egr-1 binds specifically to GC rich elements in the
mPGES-1 promoter region and facilitate the mPGES-1 gene transcription [105]. Fur-
ther investigations are needed to better define gene regulatory mechanisms that modu-
late the expression of mPGES-1.
mPGES-1 Deficient Mice

Involvement of mPGES-1 in pathophysiological events had been clarified by studies
with knockout mice mPGES-1 deficient mice are viable and fertile and develop nor-
mally [107].
In a collagen induced arthritis model, mPGES-1 deficient mice developed milder
arthritis than wild type, with reduced pain and inflammation [108]. Similar phenotype
have been observed in mice lacking cPLA2, COX-2 or EP4, revealing a metabolic flow
of the cPLA2/COX-2/mPGES-1/EP4 pathway leading to the development of inflamma-
tory arthritis.
2) mPGES-2

The gene for human mPGES-2 maps to chromosome 9q33-q34, in the vicinity of
COX-1 and mPGES-1 genes. It spans 7 kb and consist of 7 exons [109].
Watanabe et al. purified and identified a protein of 33 kDa that possessed a GSH-
independent PGES activity from bovine heart tissue [110]. They had previously re-
ported the existence of two separate mPGES enzymes in rat tissues [111].
mPGES-2 does not show a close similarity to mPGES-1, the overall structure of
this enzyme being rather distinct from mPGES-1. The catalysis of PGH2 to PGE2 by
mPGES-2 does not require the presence of Gluthation, as it does for mPGES-1. The
crystallisation of mPGES-2 show a dimer attached to lipid membrane by anchoring the
N-terminal section [112]. The amino acid sequence of mPGES-2 was highly conserved
among monkey, bovine and human cDNA. Since the evaluation of the amino acid se-
quence of mPGES-2 did not reveal any homology with any GHS-transferase, it was
concluded that mPGES-2 did not belong to the MAPEG family [112]. After analysis of
the 377 amino-acid sequence of mPGES-2, it appears that a consensus region,
Cys110-x-x-Cys113 carries the enzymatic activity of mPGES-2 [113]. mPGES-2 is first
synthetized in the Golgi membrane; then, its terminal hydrophobic domain is removed
after a proteolytic process and this enzyme is released into the endoplasmic reticulum.
Expression, Function and Regulation of mPGES-2 in Articular Tissues

mPGES-2 is constitutively expressed in chondrocytes and RA synovial fibroblasts and
is not affected by IL-1β stimulation, nor by mechanical stress [45,44].
Although mPGES-2 can be coupled to both COX-1 and COX-2 to produce PGE2
in response to acute and chronic inflammation, it seems that a modest preference for a
coupling with COX-2 has been demonstrated [114].
Thus, mPGES-2 may have a role in the production of PGE2 for homeostasis. Nev-
ertheless, a potential role in inflammatory diseases remains to be demonstrated.
Little is known about the regulation of this enzyme. Recently, the promoter of
mPGES-2 has been cloned. It contains multiple Sp1sites and a GC box, without TATA
box motif [109].
mPGES-2 is activated by various thiol reagents and is also stimulated by the addi-
tion of gluthatione and 2-mercaptoethanol [26].
3) cPGES

cPGES is highly conserved among animal species (≈ 95%) and its gene consists in
8 exons. cPGES, a 23 kDa cytosolic protein identical to p23, is a co-chaperone that
binds the ATP-dependent conformation of Heat shock protein-90 (Hsp-90). The
N-terminus cPGES has a Tyrosine residue, Tyr 9. Mutation of this residue abrogates the
enzymatic activity of cPGES. It is a GSH-requiring enzyme, constitutively expressed in
a wide variety of cells [25].
Expression, Function and Regulation of cPGES

cPGES expression is largely constitutive and ubiquitous, and is not affected by in-
flammatory stimuli. cPGES activation in cells requires its binding to Hsp90 [115]. This
enzyme is functionally coupled to COX-1: it is able to convert COX-1- but not COX-2-
derived PGH2 to PGE2 in cells [25]. Although PGE2 production by COX-1 is generally
considered to be constitutive, more studies are ongoing and suggest that cPGES may
undergo a translocation from the cytosol to the nucleus membrane to form an assem-
blage with COX-1 in order to up-regulate PGE2 production rapidly after cell stimula-
tion [116].
cPGES may physiologically contribute to PGE2 production for maintenance of
homeostasis.
In activated cells, phosphorylation of cPGES by casein kinase 2 (CK2) occurs in
parallel with an increased cPGES activity and PGE2 production. CK2 regulates cPGES
by a phosphorylation process. In vitro, phosphorylation of cPGES by CK2 increase the
affinity of cPGES for PGH2. CK2 inhibitors decrease cPGES phosphorylation and
PGE2 synthesis. This process is facilitated by interaction with Hsp90: these 3 molecules
form a complex [25]. Addition of Hsp90 inhibitors resulted in the dissociation of this
cPGES/Hsp90 complex and a decrease of PGE2 production [115].
Immediate increase in PGE2 production, as a consequence of cPGES activity, was
seen in vivo when rat fibroblasts were stimulated by bradykinin, dexamethasone and
p38 MAPKinase inhibitor indirectly suppress cPGES activation [117].
cPGES Deficient Mice

Knockout mice lacking cPGES/p23 are peri-natally lethal. Heterozygotes are viable,
fertile, and appear normal, despite a decrease in cPGES/p23 protein level [118].
C. The 15-PGDH Enzyme: Structure, Regulation and Role
15-hydroxyprostaglandin dehydrogenases (15-PGDH) are the key enzymes implicated

in the biological inactivation of prostaglandins. 15-PGDH enzymes catalyse the
oxidation of the 15(S)-hydroxyl group of prostaglandins to form inactive 15 keto-
prostaglandins [119]. Two types of 15-PGDH have been indentified. The 15-PGDH
type 1 is also called NAD+ dependent 15-PGDH and exhibit an important specificity
for prostaglandins [120]. 15-PGDH type 2, which used NAD+ or NADP+ as cofactors,
interacts with more substrates [121] and presents much higher Km values for
prostaglandins than 15-PGDH type 1. Therefore 15-PGDH type 1 is considered as the
major enzyme involved in the catabolism of prostaglandins and notably of PGE2 which
is one of its favourite substrate.
The NAD+-dependent 15-PGDH was purified in 1972 in human placenta [122].
This enzyme is ubiquitously expressed in mammalian tissues with highest activities in
lung, kidney and placenta, but is also present in mouse costal cartilage [45,123]. The
human 15-PGDH gene is localized to 4q34-q35. The cDNA of the enzyme has been
cloned and encodes a 266 amino acids protein [124]. The structure of this cytosolic
enzyme is a dimeric one composed of two identical subunits with a molecular weight
of 29 KDa [125]. Nevertheless it has been proposed that the monomeric enzyme might
be active [126]. The N-terminal region of 15-PGDH type 1 contains the binding site for
NAD+ and therefore is essential for its enzymatic activity. After its binding to NAD+,
15-PGDH interacts with its substrate at the C-terminal region. Then, the catalytic
reaction releases the PG and eventually NADH [127].
The regulation of 15-PGDH expression has been abundantly studied in cancer
[128–130]. Moreover, 15-PGDH mRNA and protein expressions were altered in
inflammed mucosa from patients with inflammatory bowel disease [131]. Moreover,
IL-1β and TNFα reduced 15-PGDH mRNA expression in human colonocytes and
trophoblasts cells from chorioamniotitis [132]. These results highly suggest a key role
of 15-PGDH in some pathophysiological processes. Actually, the down-regulation of
proteins involved in PGE2 inactivation is a largely unrecognized mechanism of
inflammation and should be studied in other inflammatory diseases such as OA.
Interestingly, a mechanical stress applied on mouse cartilage explants at physiological
ranges stimulate 15-PGDH mRNA expression in cartilage but its response is delayed
compared to the COX-2 and mPGES-1 induced overexpression [45].
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IOS Press, 2007
XI
NO and Other Radicals in the

Pathogenesis of Osteoarthritis
Martin LOTZ, MD
Division of Arthritis Research, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, CA 92037
mlotz@scripps.edu
Keywords. iNOS, apoptosis, superoxide, peroxynitrite
Introduction
A large body of information supports a role of free radicals in the pathogenesis of ar-
thritis and studies on experimental models of arthritis suggest that inhibitors of their
production or radical scavengers are of potential therapeutic value. The most important
free radical species in biological systems are derivatives of molecular oxygen, sulphy-
dryl or nitrogen compounds, polyunsaturated fatty acids and quinones and quinone-like
compounds. Previously this field was predominantly concerned with the oxygen-
derived free radicals superoxide and hydroxyl radical. The production of superoxide by
intact cells was first demonstrated by Babior in 1973 [1] who showed that leukocytes
incubated with latex particles produced an activity that reduced cytochrome c and had
the characteristics of superoxide. Since the demonstration that nitric oxide (NO) can be
produced by mammalian cells in 1987, interest in this area of research has rapidly ex-
panded. First, because of the diverse physiologic and pathogenetic effects of NO and
second, because of the interactions of NO and superoxide to form peroxynitrite, a
highly reactive species which may account for much of the free-radical induced toxic-
ity.
Regulation of Nitric Oxide Production
The first demonstration of nitric oxide (NO) release from mammalian cells was in the
vascular endothelium where it was established as the endothelium-derived factor that
causes smooth muscle relaxation. Since then NO has been shown to be produced in
many tissues and to regulate diverse cell functions. The production of NO by leuko-
cytes is associated with non-specific defense against certain microorganisms; it acts as
a neurotransmitter in the CNS and in non-adrenergic-non-cholinergic peripheral neu-
rons [2]. The role of NO in the pathogenesis of inflammatory diseases varies with the
M. Lotz / NO and Other Radicals in the Pathogenesis of Osteoarthritis 183
NOS
L-Arginine + O2 Citrulline + NO
NADPH NADP+
NO + O2 NO 2– / NO3–
NO + O2– –
O-O-N-O
–
O-O-N-O + H+ H-O-O-N-O
H-O-O-N-O OH + NO 2 / NO3–
The generation of NO is catalyzed by NO synthases (NO) which require L-arginine, NADPH and molecular
oxygen as substrates. L-arginine is oxidized at a terminal nitrogen on the guanidino group, resulting in the
formation of citrulline.
NO is unstable and highly reactive. Nitrite (NO2–) and nitrate (NO3–) are stable and measurable end products.
In the presence of nitric oxide and superoxide (O2–) the formation of peroxynitrite occurs (– O-O-N-O).
Peroxynitrite can protonate to produce peroxynitrous acid (H-O-O-N-O). Peroxynitrous acid decays rapidly
to form the hydroxyl radical (OH) and nitrogen dioxide (NO2) or nitrate (NO3–).
Figure 1. NO synthesis and interactions.
type of inflammatory stimulus and the organ involved. A significant function of NO in

cartilage and bone is suggested by the high levels of NO production in these tissues.
NO and equal amounts of citrulline are enzymatically formed from L-arginine by
nitric oxide synthases (NOS) which require NADPH, tetrahydrobiopterin, and molecu-
lar oxygen as cofactors (Fig. 1). Two classes of enzymes are known, the constitutive
NO synthases (cNOS) and inducible nitric oxide synthase (iNOS). Tissue-specific sub-
types, have been described for cNOS, notably the neuronal and the endothelial cell
cNOS [2]. Neuronal and endothelial cell cNOS are encoded by distinct genes but can
also be expressed in other cell types. The activities of cNOS are regulated by the intra-
cellular free calcium concentration and the Ca2+ binding protein calmodulin. At resting
Ca2+, both isozymes are inactive; they become fully active after an increase in intracel-
lular levels of Ca2+. Besides the conversion of L-arginine, cNOS generates H2O2 and
reduces cytochrome p450, activities that are Ca2+/calmodulin-dependent. Other redox
activities, the reduction of nitroblue tetrazolium to diformazan (NADPH-diaphorase
activity) or of quinoid-dihydrobiopterin to tetrahydrobiopterin, by cNOS appear to be
Ca2+/calmodulin-independent.
A second class of NOS is represented by the inducible NOS which was originally
isolated from mouse macrophages. Only a single gene for iNOS has been identified.
cDNA sequences cloned from human hepatocytes, articular chondrocytes and bone
cells are identical [2,19]. The inducible enzyme from murine macrophages displays
184 M. Lotz / NO and Other Radicals in the Pathogenesis of Osteoarthritis
only 50% sequence identity to the neuronal enzyme. Like neuronal cNOS, macrophage
iNOS has recognition sites for flavin-adenine-dinucleotide (FAD), flavin-
mononucleotide (FMN), and NADPH and also has a consensus calmodulin binding
site. In contrast to cNOS, iNOS binds calmodulin tightly without a requirement for
elevated Ca2+. This may explain why iNOS is independent of Ca2+ and elevated
calmodulin and appears to be activated simply by being synthesized. Inducible NO
synthase activity appears slowly after exposure of cells to cytokines such as IL-1, TNF
or IFNγ and bacterial products and its expression is sustained. The inducible NOS can
produce much larger amounts of NO than the constitutive forms and it is thought that
the release of NO by iNOS accounts for the proinflammatory effects of NO.
NO Production by Chondrocytes
iNOS expression has been demonstrated in various cell types. Within the joint, chon-
drocytes appear to be the major cell source of NO. In chondrocytes the expression of
iNOS is readily inducible by a broad spectrum of stimuli. Articular chondrocytes pro-
duce increased levels of NO in response to low concentrations of IL-1 [3]. This con-
trasts with most other cell types where multiple stimuli are required for iNOS induc-
tion. Even within the mesenchymal cell lineages the production of NO by chondrocytes
is unique. Undifferentiated mesenchymal stem cells do not express iNOS but after
chondrocytic differentiation, cells expressed iNOS and produced NO following stimu-
lation with IL-1. Mesenchymal stem cells having undergone adipogenic and osteogenic
differentiation did not produce NO after IL-1 stimulation [4]. Moreover, this induction
of iNOS expression and in human OA cartilage derives from a glucocorticoid-
insensitive mechanism [5]. In addition to IL-1 various other cytokines, extracellular
matrix degradation products [6,7], BCP, CPPD and MSU crystals [8–10] stimulate
iNOS expression in chondrocytes.
Mechanical loading plays a fundamental role in the physiological and pathological
processes of articular cartilage and NO production can be induced or inhibited, depend-
ing on the type of mechanical stimulation. Application of shear stress upregulated nitric
oxide and was associated with increases in chondrocyte apoptosis [11]. Shear stress
suppresses collagen II and aggrecan mRNA expression and this is dependent on
NO [11]. Mechanical injury also caused a significant loss of viable chondrocytes.
Death of cells could be largely prevented by addition of N(G)-monomethyl-L-arginine
to inhibit nitric oxide NO [12]. In contrast, dynamic compression to chondrocytes cul-
tured in agarose, downregulates the release of NO and enhances cell proliferation and
proteoglycan synthesis [13]. Cyclic tensile strain in chondrocytes inhibits IL-1-induced
iNOS expression [14].
Cartilage is avascular and functions under hypoxic conditions. The formation of
nitrotyrosine and peroxynitrite are dependent on oxygen tension [15]. A hypoxic envi-
ronment fully blocks COX-2 activity but favors iNOS gene expression in cultured
chondrocytes [16].
In vivo expression of iNOS in arthritis-affected cartilage has been demonstrated in
human tissue and in joints from animals with experimental OA [17,18]. The presence
of nitrotyrosine was associated with aging and with the development of OA in cartilage
samples from both monkeys and humans [19].
Molecular NO Effects
NO reacts with other radicals, with carbohydrates, proteins, lipids and nucleic acids.
NO can bind iron and thus regulate the activity of a large number of enzymes. Well
characterized is NO binding to the heme iron in guanylate cyclase which is at the active
site of the enzyme. This results in a conformational change and activation of the en-
zyme. Nitric oxide stimulates the mono-ADP-ribosylation of the glycolytic enzyme
glyceraldehyde-3-phosphate dehydrogenase. Associated with ADP-ribosylation is a
loss of enzymatic activity. This may be relevant as a cytotoxic effect of NO comple-
mentary to its inhibitory actions on iron-sulfur enzymes like aconitase and electron
transport proteins of the respiratory chain [20]. N-terminal groups of some proteins can
be modified by nitric oxide, perhaps by deamination [21]. Nitric oxide can also cause
genomic alterations. In vitro, NO deaminated deoxynucleosides, deoxynucleotides, and
intact DNA and caused DNA strand breakage [22]. Similar DNA damage can also oc-
cur in vivo and observed DNA sequence changes were consistent with a cytosine-
deamination mechanism [23]. NO reacts in the presence of specific protein thiols to
form S-nitrosoprotein derivatives that have endothelium-derived relaxing factor-like
properties. Human plasma contains approximately 7 microM S-nitrosothiols, of which
96% are S-nitrosoproteins, 82% of which is accounted for by S-nitroso-serum albu-
min [24]. By contrast, plasma levels of free nitric oxide are only in the 3-nM range.
Peroxynitrite
Many cell types produce both NO and superoxide. The generation of these two radicals
can lead to the formation of peroxynitrite [25] (Fig. 1). The formation of this relatively
long lived, strong oxidant from the reaction of nitric oxide and superoxide may con-
tribute to inflammatory cell-mediated tissue injury [26]. Because superoxide and nitric
oxide can react with each other to form peroxynitrite, they modulate each other’s half
life and the quality of their biologic effects. Superoxide can limit the effects of NO by
directing it to peroxynitrite and some NO effects such as vasodilation are prolonged in
the presence of superoxide scavengers [27]. Conversely, NO can be regarded as a scav-
enger of superoxide anion and this suggested that NO may provide a chemical barrier
to cytotoxic free radicals. Some effects of these radicals are clearly dependent on the
formation of peroxynitrite. The inhibition of aconitases is only observed in the presence
of peroxynitritie but not by NO in the absence of superoxide. Peroxynitrite is capable
of oxidizing a variety of molecules, including sulfides, thiols, deoxyribose, lipids,
ascorbate, α1-protease inhibitor. In the case of α1-protease inhibitor peroxynitrite oxi-
dizes the methionine residue to sulfoxide and inactivates the protein. Peroxynitrite also
inactivates the tissue inhibitor of metalloproteinase-1 (TIMP-1) activity towards gelati-
nase-A. High concentrations of peroxynitrite caused protein fragmentation while lower
concentrations inactivated TIMP-1 without altering the molecular weight [28]. Per-
oxynitrite initiates lipid peroxidation and this mechanism contributes to O2- and
NO-mediated cytotoxicity [29]. Reactive peroxynitrite anion may also exert cytotoxic
effects in part by oxidizing tissue sulfhydryls [30].
Peroxynitrite can decompose to products that nitrate aromatic amino acids. Such
nitro-aromatics may be ‘markers’ of NO-dependent oxidative damage. Nitrotyrosine
residues were first demonstrated in atherosclerotic plaques and subsequently in syno-
vial fluids. Serum and synovial fluid from patients with the rheumatoid arthritis contain
3-nitrotyrosine. By contrast, body fluids from normal subjects and patients with os-
teoarthritis contain no detectable 3-nitrotyrosine. The demonstration of nitrotyrosine
formation represents evidence for the production of peroxynitrite in vivo since tyrosine
is nitrated by peroxynitrite but not by nitric oxide [31].
NO Effects on Cell Growth and Survival
NO donors inhibit cell proliferation and endogenous NO production is at least in part

responsible for the growth inhibition induced by cytokines or other agents. Conversely,
factors that stimulate cell proliferation often inhibit NO formation [32,29]. Blanco et al.
first reported that high concentrations of the NO donor sodium nitroprusside (SNP)
induced apoptosis-like cell death in cultured human chondrocytes [33]. However, IL-1,
an inducer of NO production in chondrocytes did not induce chondrocyte apop-
tosis [34]. But in combination with an oxygen radical scavenger hypoploidy and DNA
fragmentation were observed. It was proposed that the balance between intracellular
NO and ROS may determine the type of chondrocyte death, with a low concentration
of ROS promoting apoptosis in the presence of NO and a high concentration of ROS
promoting necrosis. Del Carlo reported that NO itself is not cytotoxic for human chon-
drocytes but may be when combined with superoxide. Under certain conditions of oxi-
dative stress NO can even be protective against cell death [35].
In cultured human chondrocytes IL-1β induces binding of annexin V but cell death
or a causal relationship between NO generation and annexin V binding was not demon-
strated [34]. The NO donor SNP increased caspase-3 activity about 2.5 fold in human
OA chondrocytes. A caspase-3 specific inhibitor peptide caused a partial inhibition of
nucleosomal DNA fragmentation as analyzed by ELISA suggesting that cell death and
cleavage of chromosomal DNA induced by exogenous NO in cultured chondrocytes
may depend in part on active caspase-3 [36]. However, caspase-3 processing in re-
sponse to SNP was not detected by immunoblotting and a caspase-3-specific inhibitor
peptide failed to inhibit DNA degradation in cultured human chondrocytes [37]. It was
also observed that IL-1β-induced NO can partially inhibit internucleosomal DNA
fragmentation and caspase-3 processing induced by CD95 activation and simultaneous
treatment with proteasome inhibitors. This effect of endogenous NO was mimicked by
SNP. However, cell death was not blocked suggesting that NO specifically interferes
with apoptosis execution but does not prevent chondrocytes from undergoing a form of
cell death that does not require caspase-3 activation or internucleosomal DNA frag-
mentation [38]. In rabbit chondrocytes SNP induced p38 mitogen-activated protein
kinase-dependent cell death and this was associated with enhanced caspase-3 activity,
suggesting apoptosis as the cell death modality [39,40]. However, NO production as a
result of adenovirus-mediated overexpression of iNOS did not cause cell death in rabbit
chondrocytes [41]. There are no reports on the induction of apoptosis by endogenous
NO or NO donors in cartilage explants. An in vivo study in a canine model of OA
showed that oral application of the iNOS inhibitor L-NIL significantly reduced the
number of apoptotic cells in femoral condyles [42] but it is not clear whether this is
directly related to NO effects on cell survival or the result of protective effects of
L-NIL against cartilage degradation.
In certain cell types NO inhibits apoptosis through S-nitrosylation of cysteine resi-
dues present in the catalytic center of caspases as well as through a variety of additional
mechanisms while in other cell types exogenous or endogenous NO are proapop-
totic [43]. The mechanisms responsible for these dual actions of NO in regulating
apoptosis are poorly defined. In human chondrocytes the effects of NO-donors on cell
death are age-dependent: chondrocytes from older donors show an increased ratio of
oxidized glutathione to reduced glutathione when compared to cells from younger do-
nors. This may indicate that the cells from older donors are more susceptible to oxidant
stress causing a greater number of chondrocytes from older donors to die in response to
a nitric oxide donor [44]. Collectively, these studies illustrate that the impact of NO on
cell survival is strongly dependent on the context in which NO is generated. While
chondrocyte apoptosis is a feature of OA cartilage and often correlates with the expres-
sion of iNOS [45], a causal link has yet to be established.
NO and Extracellular Matrix
Most of the currently available data suggest that NO promotes cartilage extracellular
matrix degradation. Some discrepancies on the NO effects appear to relate to species
differences. Studies with human [46], rat [47] and rabbit cartilage [48] indicate that NO
donors and IL-1 induced endogenous NO inhibits proteoglycan synthesis. This was not
observed with bovine cartilage [49]. NO also activates matrix metalloproteinases [50]
and depolymerizes hyaluronan [51]. NO inhibits the chondrocyte response to the ana-
bolic growth factor IGF-1 [52] and shifts the cytokine balance towards proinflamma-
tory direction by reducing the synthesis of TGFß and IL-1 receptor antagonist [53].
Interaction Between NO and COX Pathways
Many of the extracellular stimuli that induce iNOS expression also increase pros-
taglandin production and in OA-affected joints there is simultaneous increase of
COX-2 and iNOS expression. However, the literature is divided with respect to
whether NO activates or inhibits PG production and COX activity. In mouse macro-
phages lipopolysaccharide causes an increase in the release of NO and PGE2. Produc-
tion of both NO2 and PGE2 was blocked by NOS inhibitors and this was thought to be
a direct NO interaction with COX to cause an increase in the enzymatic activity [54].
NO donors also increased cyclooxygenase activity in endothelial cells and intravenous
infusion of NO donors in vivo released 6-keto PGF1alpha, the stable metabolite of
PGI2 [29]. Besides the activation of COX function, NO donors also amplified IL-1
beta-induced PGE2 production and potentiated IL-1 beta-induced mRNA and protein
expression of COX-2 in macrophages and mesangial cells [55]. In chondrocytes NO
stimulated NF kB activation and this was required for COX-2 expression [56].
Nitric Oxide Inhibition in Animal Models
Development of bones and joints in iNOS knock-out mice appears normal. When ex-
perimental OA was induced by ligament transection and partial medial menisectomy
iNOS knock out mice showed an unexpected acceleration of OA development [57].
In contrast, experimental rheumatoid arthritis induced by injection of monoclonal
antibodies to collagen [58], zymosan-induced arthritis and collagenase-induced os-
teoarthritis were less severe in iNOS-deficient mice [59].
Inhibitors of NO synthesis have been evaluated in various models of inflammatory

arthritis [59–64] as well as in experimental OA [42,65]. The outcome of NO inhibition
in inflammatory arthritis depends on the specific model. Early work in adjuvant arthri-
tis showed antiinflammatory effects of L-NIL and L-NMMA [62,66]. In an acute
model of joint inflammation induced by intraarticular injection of carrageenan and kao-
lin the iNOS inhibitor L-Nil and a selective COX-2 inhibitor inhibited joint swelling
and greatest therapeutic benefit was observed with a combination of the two inhibitors.
In contrast, in the chronic adjuvant arthritis model L-NIL exacerbated joint inflamma-
tion and abrogated the antiinflammatory effect of the COX-2 inhibitor [67]. A profound
increase in joint destruction was also seen in response to L-NIL in streptococcal cell
wall-induced arthritis [68]. Furthermore, septic arthritis [69] and the acute phase of
antigen-induced arthritis [70] were more severe in iNOS-deficient mice. NO can also
have certain antiinflammatory effects. NO donors reduce cytokine induced endothelial
cell activation [71], inhibit endothelial-leukocyte interactions [72] and attenuate vascu-
lar inflammation [73]. These antiinflammatory actions may account for the increased
severity seen after iNOS inhibition in some of the animal models. Only one study ex-
amined an iNOS inhibitor in experimental OA. In dogs subjected to anterior cruciate
ligament transection, the administration of L-NIL reduced the severity of OA lesions
and this was associated with reduced chondrocyte apoptosis, MMP and cytokine pro-
duction [42,65].
Conclusions
Increased production of reactive oxygen and nitrogen species in articular cartilage has
been documented in human and experimental OA. A large body of literature is avail-
able on the in vitro effects of NO and other radicals on chondrocyte survival, extracel-
lular matrix and inflammation. The specific effect of a radical on a given cell function
is very much dependent on the experimental context. Specifically, the simultaneous
presence of superoxide which leads to the formation of peroxynitrite, a stronger and
highly reactive radical is likely to lead to cell and tissue damage. It is important to note
that NO does have certain protective effects in cartilage and other tissues. Inhibition of
NO in vitro or in vivo can lead potentially lead to undesired exacerbation of inflamma-
tion and tissue destruction under certain conditions. The pathophysiological role of
peroxynitrite is supported by multiple lines of evidence and consequences of its genera-
tion in arthritis-affected tissues have been documented. Selective scavengers of per-
oxynitrite may thus be more promising candidates for the treatment of osteoarthritis.
Compounds such as urate and polyphenolics decrease tissue nitrotyrosine formation.
Administration of uric acid but not of and iNOS inhibitor to animals with zymosan-
induced arthritis protected against cartilage degradation [74].
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IOS Press, 2007
XII
Mitochondria and Chondrocytes:

Role in Osteoarthritis
Francisco J. BLANCO ∗, MD, PhD, María J. LÓPEZ-ARMADA, PhD
and Ignacio REGO, PhD
Osteoarticular and Aging Research Lab., Biomedical Research Center,
Rheumatology Division, C.H. University Juan Canalejo, A Coruña, Spain
fblagar@canalejo.org
Abstract. Mitochondria are critical regulators of cell function and cellular sur-
vival. Many lines of evidence suggest that mitochondria have a central role in age-
ing-related diseases. Mutations in mitochondrial DNA and oxidative stress both
contribute to ageing. Osteoarthritis (OA) is a rheumatic disease associated to aging
and it is characterized by articular cartilage degradation and increases of chondro-
cyte death. Articular cartilage chondrocytes must survive and maintain tissue in-
tegrity in an avascular environment. Then chondrocytes from deep and superficial
zones may require adaptively increased anaerobic glucolysis and aerobic respira-
tion respectively to support ATP synthesis. Recent ex vivo studie reported dys-
function of mitochondrial human OA chondrocytes. The analysis of mitochondrial
electron transport chain activity in OA chondrocytes shows a significant decrease
in Complexes I, II and III compared to normal chondrocytes. This mitochondrial
dysfunction can mediate several pathways implicated in cartilage degradation such
as, oxidative stress, inadequacy of chondrocyte biosynthetic and growth responses,
up-regulated chondrocyte cytokine-induced inflammation and matrix catabolism,
pathologic cartilage matrix calcification and increased chondrocyte death (necrosis
or apoptosis). Mitochondrial dysfunction in OA chondrocytes may be originated
by somatic mutations in mtDNA or by direct effect of pro-inflammatory mediators
(cytokines, prostaglandin, ROS and NO) on mitochondrial activity.
Keywords. Mitochondria, chondrocytes, osteoarthritis, apoptosis
Introduction
Mitochondrion (plural mitochondria) (from Greek mitos, thread or khondrion, granule)

is a membrane-enclosed organelle, found in most eukaryotic cells. Mitochondria are
sometimes described as “cellular power plants,” because they convert food molecules
into energy in the form of ATP via the process of oxidative phosphorylation [1]. A
∗
Corresponding Author: Francisco J. Blanco. MD, PhD, Osteoarticular and Aging Research Lab., Bio-
medical Research Center, Rheumatology Division, C.H. University Juan Canalejo, A Coruña, Spain, E-mail:
fblagar@canalejo.org.
F.J. Blanco et al. / Mitochondria and Chondrocytes: Role in Osteoarthritis 193
Figure 1. Structure of Mitochondria.
typical eukaryotic cell contains about 2,000 mitochondria, which occupy roughly one
fifth of its total volume [2]. Mitochondria contain DNA that is independent of the DNA
located in the cell nucleus.
A mitochondrion contains inner and outer membranes composed of phospholipid
bilayers and proteins (Fig. 1). The two membranes, however, have different properties.
The outer mitochondrial membrane, which encloses the entire organelle, has a protein-
to-phospholipid ratio similar to the eukaryotic plasma membrane (about 1:1 by weight).
It contains numerous integral proteins called porins, which contain a relatively large
internal channel (about 2–3 nm) that is permeable to all molecules of 5000 daltons or
less [3]. Larger molecules can only traverse the outer membrane by active transport
through mitochondrial membrane transport proteins. The outer membrane also contains
enzymes involved in such diverse activities as the elongation of fatty acids, oxidation
of epinephrine (adrenaline), and the degradation of tryptophan.
The inner mitochondrial membrane contains proteins with four types of func-
tions [3]: 1) Those that carry out the oxidation reactions of the respiratory chain.
2) ATP synthase which makes ATP in the matrix. 3) Specific transport proteins that
regulate the passage of metabolites into and out of the matrix. 4) Protein import ma-
chinery.The inner mitochondrial membrane is compartmentalized into numerous cris-
tae, which expand the surface area of the inner mitochondrial membrane, enhancing its
ability to generate ATP. In addition, there is a membrane potential across the inner
membrane (mitochondrial membrane potential-Δψm).
Mitochondria possess their own genetic material, and the machinery to manufac-
ture their own RNAs and proteins. Although most mitochondrial proteins are encoded
194 F.J. Blanco et al. / Mitochondria and Chondrocytes: Role in Osteoarthritis
by the nuclear genome, mitochondria contain many copies of their own DNA. Human
mtDNA is a circular molecule of 16,569 base pairs that encodes 13 polypeptide com-
ponents of the respiratory chain, as well as the rRNAs and tRNAs necessary to support
intramitochondrial protein synthesis using its own genetic code. Inherited mutations in
mtDNA are known to cause a variety of diseases. One hypothesis has been that somatic
mtDNA mutations acquired during ageing contribute to the physiological decline that
occurs with ageing and ageing-related diseases [4].
Although it is well known that the mitochondria convert organic materials into cel-
lular energy in the form of ATP, mitochondria play an important role in many meta-
bolic tasks, such as apoptosis-programmed cell death, cellular proliferation, regulation
of the cellular redox state, heme synthesis and steroid synthesis. Production of ATP is
done by oxidizing the major products of glycolysis:pyruvate and NADH that are pro-
duced in the cytosol. This process of cellular respiration, also known as aerobic respira-
tion, is dependent on the presence of oxygen. When oxygen is limited the glycolytic
products will be metabolised by anaerobic respiration, a process that is independent of
the mitochondria. The production of ATP from glucose has an approximately 15–18
fold higher yield during aerobic respiration compared to anaerobic respiration.
Each pyruvate molecule produced by glycolysis is actively transported across the
inner mitochondrial membrane, and into the matrix where it is oxidized and combined
with coenzyme A to form CO2, acetyl CoA and NADH. The acetyl CoA is the primary
substrate to enter the citric acid cycle, also known as the tricarboxylic acid (TCA) cycle
or Krebs cycle. The citric acid cycle oxidizes the acetyl CoA to carbon dioxide and in
the process produces reduced cofactors (three molecules of NADH and one molecule
of FADH2), that are a source of electrons for the electron transport chain, and a mole-
cule of GTP (that is readily converted to an ATP) (Fig. 1).
Protein complexes in the inner membrane (NADH dehydrogenase, cytochrome c
reductase and cytochrome c oxidase) perform the transfer and the incremental release
of energy is used to pump protons (H+) into the intermembrane space (Fig. 2). This
process is efficient but a small percentage of electrons may prematurely reduce oxygen,
forming the toxic free radical superoxide. This can cause oxidative damage in the mito-
chondria and may contribute to the decline in mitochondrial function associated with
the aging protein [4].
The concentrations of free calcium in the cell can regulate an array of reactions
and is important for signal transduction in the cell. Mitochondria store calcium, a proc-
ess that is one important event for the homeostasis of calcium in the cell. Release of
this calcium back into the cells interior can initiate calcium spikes or waves. These
events coordinate processes such as neurotransmitter release in nerve cells and release
of hormones in endocrine cells.
In summary, mitochondria are critical regulators of cell function and cellular sur-
vival. Many lines of evidence suggest that mitochondria have a central role in ageing-
related diseases. Mutations in mitochondrial DNA and oxidative stress both contribute
to ageing. Osteoarthritis (OA) is a rheumatic disease associated to aging and it is char-
acterized by articular cartilage degradation and increases of chondrocyte death. Mito-
chondrial function in normal or OA articular chondrocyte has not been studied with
detail. However, recent investigations in mitochondria activity and function have
opened new exciting knowledge that we will summarize in this chapter.
Figure 2. Mitochondrial Respiratory Chain.
1. Mitochondria in Articular Cartilage Chondrocytes
Considering the structure of articular cartilage, it should be noted that there are no
blood vessels, lymphatic channels, or neural elements that enter or pass through adult
articular cartilage. Furthermore, the chondrocytes are separated from the blood vessels
of the underlying bone by a zone of dense calcification and the mature cortex of the
underlying subchondral bony end-plate (Fig. 3). Numerous studies have been per-
formed to assess whether diffusion from the underlying bone can provide nourishment
to the cartilaginous surface, and it is now well established that in the adult cartilage
transport of nutrients by this route does not occur [5]. Conversely, studies in which
nutrients have been injected into the joint cavity have demonstrated rapid transport
through the cartilage and the matrix to the cells, suggesting strongly that the major
source of nutrients is synovial fluid [6]. Since the synovial fluid is in itself an ultafil-
trate of plasma, it is apparent that chondrocytes receive their nutrition through a double
diffusion system. Nutrients must first diffuse across the synovial barrier into the syno-
vial fluid and then across the matrix of articular cartilage to reach the cell. It has benn
demonstrated that the matrix of articular cartilage is not freely permeable and that dif-
fusion of nutrients is in large measure dependent on size and charge [7].
Detailed information about the metabolic fuels required by cartilage is scanty. The
results of a considerable number of experiments indicate that while the TCA is active
in cartilage, as much as 80% of glucose is metabolized to lactate by anerobic glycoly-
Figure 3. Scheme of Joint (Modified from Dinarello C and Moldawer L, A primer for Clinicians, 3rd ed.
Thousand Oaks, Ca, USA, Amgen Inc, 2001.
sis [7]. Studies of cells in culture also indicate that isolated chondrocytes are dependent
of anerobic glycolysis. In this respect, chondrocytes are similar to other cell types. Re-
sults of many investigations clearly document that cultured cells generate much of their
ATP through glycolysis and produce lactate as an end-product.
However, evidence that cartilage cells can utilize oxidative metabolism to produce
chemical energy is supported by the observation that chondrocytes contain mitochon-
drial dehydrogenases; that isolated mitochondria can utilice TCA cycle substrates; and
that the mitochondria contain enzymes required for oxidative phosphorylatoin and elec-
tron transport [8,9]. In addition, in cultured human articular chondrocytes, the activity
of mitochondrial respiratory chain (MRC) shows an enzymatic activity similar to other
mesenchymal cells (Table 1) [10]. Furthermore, it has been reported that mitochondrial
oxidative phosphorylation may account for up to 25% of the ATP produce in carti-
lage [11,12].
In all tissues, oxygen serves as the final electron acceptor for mitochondrial cyto-
chrome oxidase. In addition, oxygen serves as a substrate for a very large number of
other enzymes (oxygenases and oxidases). Tissue oxygen tensions vary and the actual
value is dependent on a number of factors. These include the number of cells per unit
volume and the rate of cellular metabolism, oxygen diffusibility through the extracellu-
lar matrix and the vascular supply. Thus chondrocytes in articular cartilage receive
oxygen only by diffusion from the synovium (Fig. 3). Moreover, because of the asym-
metry of the oxygen supply, a gradient must exist across the tissue [13,14]. As a result
of the gradient, it is estimated that cells at the surface experience between 5% and 7%
Table 1. Values of mitochondrial respiratory chain (MRC) complexes in cultures of normal, OA and normal
chondrocytes treated with NO
Normal OA Chondrocytes
Chondrocytes Chondrocytes with SNP
Age, years 59.7 ± 21.8 (30) 68.5 ± 7.6 (53) 59.7 ± 18.9 (11)
Proteins, mg/ml 3.6 ± 1.3 (30) 3.3 ± 0.9 (53) 4.2 ± 1.4 (11)
CS enzymatic activity, 111.7 ± 29.8 (29) 124 ± 2.6 (51) † 106.6 ± 26.2 (11)
nmoles/minute/mg protein
Mitochondrial complex activity‡
Complex I 27.9 ± 13.6 (22) 22.5 ± 9.4 (46) 22.8 ± 19.1 (11)
Complex II 11.5 ± 5.7 (25) 9.2 ± 3.3 (47) † 10.2 ± 1.81 (11)
Complex III 54.2 ± 13.6 (25) 46.5 ± 9.7 (49) † 6.27 ± 9.7 (11)
Complex IV 53.6 ± 11.9 (29) 53.1 ± 13.2 (49) 40.2 ± 11.3 (11) †
* Values are the mean ± SD (n). CS = citrate synthase. † P ≤ 0.05 versus normal chondrocytes.
‡ CS-corrected complex activity is expressed as (nmoles/minute/mg protein) / (CS specific activity) X 100.
Complex I = rotenone-sensitive NADH-coenzyme Q1 reductase; Complex II = Succinate dehydrogenase;
Complex III = antimycin-sensitive ubiquinol cytochrome c reductase; Complex IV = cytochrome c oxidase.
O2, compared with 13% in arterial blood [15]. In contrast, cells in the deepest regions
of the cartilage are exposed to a low oxygen tension [16,17].
In summary, articular cartilage chondrocytes must survive and maintain tissue in-
tegrity in an avascular environment. In addition, oxygen and glucose concentration
supply to cartilage is characterized by asymmetry with a gradient from superficial to
deep zone. Then chondrocytes from deep and superficial zones may require adaptively
increased anaerobic glucolysis and aerobic respiration respectively to support ATP
synthesis.
2. Mitochondria and Osteoarthritis
Because articular cartilage chondroyctes are traditionally classified as cells highly gly-
colytic, mitochondrial mediated pathogenesis has not been previously investigated with
detail for OA [16,17]. However alteration in some mitochondria functions such as ATP
production, apoptosis and redox state could explain some mechanisms involving carti-
lage degradation during OA.
2.1. Mitochondria in OA Chondrocytes
Morphologic studies describe changes in OA cartilage tissue showing nuclear (pykno-

sis and karyorrhexis) and cytoplasmic changes (fat droplets, glycogen granules, and
microfilaments) have been reported [18]. In this study mitochondria were described as
“swollen” and authors concluded that the chondrocyte underwent “necrosis”. They
noted that these changes increase in extent and degree with an increased severity in the
arthroscopic stage classification.
Recent ex vivo studie reported dysfunction of mitochondrial human OA chondro-
cytes [10]. The analysis of mitochondrial electron transport chain activity in OA chon-
drocytes shows a significant decrease in Complexes II and III compared to normal
chondrocytes (Table 1). A dysfunction in Complexes II and III compromises the elec-
tron transfer pathway and this defect could be solved by overloading the electron trans-
port via Complex I, a pathway with little oxygen consumption. However, the dimin-
ished efficiency to transport electrons via Complex II does not enhance the activity of
Complex I (rather, activity of Complex I is reduced).
On the other hand, mitochondrial mass is increased in OA chondrocytes compared
with cells from normal cartilage, as demonstrated by a significant rise in citrate syn-
thase (CS) activity [10]. Therefore, an increase in mitochondrial mass could be a
mechanism of OA chondrocytes to compensate the electron transfer deficiency via
Complexes II and III and its resulting low production of ATP per mitochondrium. Re-
duction in chondrocyte respiration causes intracellular ATP depletion by 50–80%
[11,19]. Furthermore, OA cells showed a reduction in the Δψm as demonstrated by
using the fluorescent probe JC-1. Quantitative studies performed by flow cytometry
showed that OA chondrocytes have a lower red/green fluoresecence ratio that normal
chondrocytes, indicating depolarization of the mitochondria [10].
2.2. Mitochondria and OA Pathogenesis
Although OA chondrocytes have dysfunction in mitochondria activity, an important

aspect to know is the relevance of this finding to explain specific pathogenic pathways
implicated in OA. In this sense, some results show that mitochondrial dysfunction can
mediate several pathways implicated in cartilage degradation. These include oxidative
stress, inadequacy of chondrocyte biosynthetic and growth responses, up-regulated
chondrocyte cytokine-induced inflammation and matrix catabolism, pathologic carti-
lage matrix calcification and increased chondrocyte death (necrosis or apoptosis).
Several studies have reported that the in vitro use of specific inhibitors of mito-
chondrial electron transport suppressed the synthesis of proteoglycans and collagen by
human articular chondrocytes [11,19,20]. In addition, in vitro inhibition of complex
I with rotenone also reduced the proteoglycan content of the extracellular matrix in the
superficial and middle zones and it increased the release of GAGs from cartilage to
supernatant [20].
Several mechanisms can explain the effect of mitochondrial dysfunction on pro-
teoglycans and collagen depletion. For example, the inhibition of complex III or V is
able to reduce proteoglycan synthesis and to induce proteases synthesis such as
MMP-1, -3, -13 and ADAMS-5 [21,22]. Furthermore, the activity reduction of both
mitochondrial complexes increases the synthesis of cytokines (IL-1, IL-6 and IL-18);
prostaglandin E2 (PGE2) and chemokynes (IL-8 and MCP-1) [21,22]. Mitochondrial
respiratory chain is one of the most important sites of ROS production [23]. In human
articular chondrocytes, the inhibition of complex III and V activity with specific MRC
inhibitors (antimicyn-A and oligomicyn respectively) induced ROS synthesis [21]. Al-
though increase in ROS formation during hypoxia is difficult to explain, two factors
may contribute to an increase mitochondrial ROS formation in hypoxia [23]. Firstly,
under hypoxic conditions, low concentrations of NO: may still be produced since the
Km for oxygen of the mitochondrial nitric oxide synthase is around 30–40 microM.
Secondly, NO may bind and inhibit cytochrome oxidase, resulting in an increase in its
Km for oxygen and an increased reduction of electron carriers located upstream from
the terminal oxidase, favouring O2- formation at low oxygen concentrations. In this
sense, NO production by OA chondrocytes is increased and NO suppresses mitochon-
drial oxidative phosphorylation by reducing the activity of complex IV and decreases
the Δψm [24].
Some studies suggest that the chondrocyte mitochondria are specialized for cal-
cium transport and are important in the calcification of the extracellular matrix [25–27].
Mineral formation has been demonstrated in matrix vesicles (MV) and within mito-
chondria. Calcium and phosphorus are also clearly present in single mitochondrial
granules within growth plate chondrocytes and in certain extracellular particles distinct
from MV [25]. Pathologic hydroxyapatite (HA) deposition and calcium pyrophosphate
dihydrate (CPPD) crystal deposition are both common in OA, particularly in advanced
disease [26]. Significantly, the onset of HA deposition around hyperthophic endo-
chondral cartilage chodrocytes coincides with sharply defined changes in the redox and
metabolic states of chondrocytes, in association with focal loss of cell respiration [27].
Moreover, a marked decreases in MV PPi is obligatory for HA crystal deposition to be
initiated in the interior of MV in vitro.
Direct suppression of mitochondrial respiration promotes MV-mediated minerali-
zation in chondrocytes [11,12]. Previously described changes in mitochondrial function
that directly modulate mineralization include regulation of intramitochondrial calcium
accumulation and release of intramitochondrial calcium stores. Chondrocytes MRC
complexes also regulate mineralization at the levels of the MV content of PPi and the
calcium-precipitanting ability associated with MV. Then, the chondrocytes MRC activ-
ity could partly regulates differential deposition of HA and CPPD. Regulation of MRC
activity may be one of the signaling pathways by which NO modulates articular carti-
lage matrix biosynthesis and pathologic mineralization [11].
Interestengly, in vivo studies carried out in an animal model support these results.
Recently, it has been assessed chondrocytes for ATP depletion and for in situ changes
in mitochondrial ultrastructure prior to and during the evolution of spontaneous knee
OA in male Hartley guinea pigs [28]. Results showed that spontaneous NO release
from knee cartilage samples in organ culture doubled between ages 2 months and
8 months as knee OA developed. Concomitantly, chondrocyte intracellular ATP levels
declined by approximately 50%, despite a lack of mitochondrial ultrastructure abnor-
malities in knee chondrocytes. As ATP depletion progressed with aging in knee chon-
drocytes, an increased ratio of lactate to pyruvate was observed, consistent with an
adaptive augmentation of glycolysis to mitochondrial dysfunction.
2.3. Mitochondria and Chondrocyte Apoptosis
Histological studies of OA cartilages show a reduction in the number of chondrocytes

compared with normal cartilages [29]. Several authors have suggested that apoptosis is
the responsible of the hypo-cellularity in OA cartilage [30–32]. Apoptosis is a distinct
mode of cell death that differs morphologically from necrosis [33]. Apoptosis is con-
ceptually an evolutionarily conserved, innate process by which cells systematically
inactivate, disassemble, and degrade their own structural and functional components to
complete their own demise. “Programmed cell death” is often used interchangeably
with apoptosis, reflecting the fact that cell death occurs according to a sequential ‘pro-
gram’ of cellular, biochemical and molecular events. Apoptosis has been distinguished
from necrosis in vivo by the following features: 1) while necrosis is usually accompa-
nied by an acute inflammatory response with exudation of neutrophils and monocytes,
this phenomenon is absent in apoptosis; and 2) while necrosis involves groups of
neighbouring cells, apoptosis usually appears in discrete individual cells in a tissue.
3) morphologically, nuclear condensation may appear in necrosis, but it has poorly
defined edges and is irregularly scattered through the nucleus, while apoptotic nuclear
condensation reveals sharply defined masses of uniform texture. The reason that the
cellular phenomenon of apoptosis has become the focus of such great interest among
Figure 4. Mitochondria in the apoptosis of chondrocytes.
researchers is that apoptosis involves a signalling cascade that leads to the organized
disintegration of cells that, in contrast to necrosis, is potentially reversible and amena-
ble to therapeutic manipulation.
The reported percentages of apoptotic chondrocytes in OA cartilage range from
0–6% [34]. The discrepancy in the percentages by various reports was probably
deemed the result of the methodology employed to detect apoptosis [35]. In addition,
how it is very difficult to demonstrate typical apoptotic chondrocytes accompanying
apoptotic bodies even in advanced human OA articular cartilage; thus, nonapoptotic
programmed cell death due to other cell death mechanisms has been postulated (chon-
droapoptosis or paraptosis) [36].
Taking in mind these comments, abundant literature investigates the stimuli and
signalling mechanisms of chondrocyte apoptosis such as the death receptor and mito-
chondrial pathways (Fig. 4). Apoptosis through the mitochondrial pathway occurs after
cellular damage, which causes changes in the conformation and/or activity of the pro-
apoptotic Bcl-2 family members, such as Bak and Bax [37], in the outer mitochondrial
membrane. Cytochrome c and other polypeptides are subsequently released from the
intermembrane space of the mitochondria and bind to the cytoplasmic scaffolding pro-
tein, Apaf-1, causing an ATP-dependent conformational change that allows Apaf-1 to
bind to the prodomain of pro-caspase-9 [38]. This interaction enhances the proteolytic
activity of procaspase-9, resulting in the activation of executioner caspases, such as
caspases-3 and -7. The release of Smac/Diablo inhibits the effect of the inhibitor of
apoptosis (IAP), resulting in the inhibition of the interaction of IAP with caspase-9.
The role of NO has been one focus of interest in the chondrocyte death and apop-
tosis. Recent studies suggest that NO induces apoptosis in chondrocytes because, it
reduces the activity of complex IV and decreases the Δψm [24]. Apart from inhibiting
respiration, NO has additional effects on mitochondria such as induction of ROS and
induction of mtDNA damage which are related with cell death [39]. However, the pre-
cise role of NO in the induction of chondrocyte death is currently debated. Although
treatment with NO donors consistently induces apoptosis in cultured chondro-
cytes [40,41], the production of high levels of endogenous NO by the overexpression of
the iNOS gene in transfected chondrocytes was not found to cause cell death [42]. This
discrepancy might be the result of using chemical NO donors, which not only generate
reactive nitrogen species but also produce various secondary reactions depending on
the cellular milieu in in vitro experiments. A recent study that employed diazenium-
diolates, which have been shown to be reliable sources of NO, suggested that exoge-
nous NO is not cytotoxic to cultured chondrocytes per se, and can even be protective
under certain conditions of oxidative stress [41].
Nitrite was found to exert a protective effect upon hypochlorous acid-induced
chondrocyte toxicity, thus implicating NO in a novel cytoprotective role in inflamed
joints [43]. While recent data indicate that NO may not be the sole mediator of chon-
drocyte death, the role of peroxynitrite, a reaction product of NO and superoxide ani-
ons, is postulated [41]. Interestingly, it is therefore proposed that the balance between
intracellular NO and ROS may determine the type of chondrocyte death, with a low
concentration of ROS promoting apoptosis in the presence of NO and a high concentra-
tion of ROS promoting necrosis [40,44]. Finally, a recent study on peroxynitrite-
mediated chondrocyte apoptosis revealed that the predominant mode of cell death in-
volved calcium-dependent cysteine proteases, known as calpains, and that peroxynitrite
induced mitochondrial dysfunction in cells that leads to caspase-independent apop-
tosis [45].
In an “in vitro” experiment using human cartilage, chondrocytes from old donors
were found to be more susceptible to cell death induced by an NO donor; this suscepti-
bility was correlated with a higher ratio of oxidized glutathione to reduced glutathione,
providing evidence that increased oxidative stress with aging makes chondrocytes more
susceptible to oxidant-mediated cell death [46].
In summary, the pathways and patterns of chondrocyte death (apoptosis, chondrop-
tosis, parapotosis) are much more diverse than originally observed. Mitochondria activ-
ity plays an important role in chondrocyte survival. Furthermore, it should be noted that
the relative contribution of apoptotic cell death in the pathogenesis of OA is still diffi-
cult to assess because of the chronic nature of the disease process. However, recently
some in vivo studies analyzing the effects of intra-articular apoptosis inhibitors in
chondrocytes death and cartilage degradation have showed interesting results [47]. In-
tra-articular administration of the pancaspase inhibitor Z-VAD-FMK, Caspase-1 inhibi-
tor and the combination of caspase-3 and caspase-8 inhibitors reduced the severity of
cartilage lesions in experimental OA (ACLT in rabbits), suggesting that they may have
disease-modifying activity in human OA [47].
3. Mechanisms Modifying Mitochondrial Activity in OA
Several molecules with catabolic profile (NO, PGE2) and some pro-inflammatory cy-
tokines (TNFα and IL-1β) localized in the synovial fluid of OA joints at high concen-
trations may modify the mitochondrial activity (Fig. 3). Some results indicate that both
cytokines, TNFα and IL-1β, modified mitochondrial function by a mechanism involv-
ing decrease in the activity of complex I of CRM and ATP production, as well as a
reduction in Δψm in human chondrocyte cells [20]. Interestingly, in human articular
chondrocytes, NO suppresses mitochondrial oxidative phosphorylation because it re-
duces the activity of complex IV causing decrement of ATP synthesis and Δψm [11].
In addition, stimulation of OA chondrocytes with PGE2 decreases Δψm and ATP gen-
eration [48].
Mitochondria have been trough to contribute to ageing and pathology through the
accumulation of mitochondrial DNA (mtDNA) mutations induced by mutagens such as
ROS or NO. It is well established that mtDNA accumulates mutations with ageing,
especially large-scale deletions and point mutations [49]. The accumulation of dele-
tions and point mutations correlates with decline in mitochondrial function. In the
mtDNA control region, point mutations at specific sites can accumulate to high levels
in certain tissues: T414G in cultured fibroblasts, A189G and T408A in muscle and
C150T in white blood cells [50]. However these control region “hot spots” have not
been studied in chondrocytes. One hypothesis is that somatic mtDNA mutations ac-
quired during ageing contribute to the mitochondrial activity decline that occurs in OA
chondrocytes.
Accumulation of mtDNA mutations may be due to increases production of ROS or
to a defect in the mitochondrial anti-oxidant system [23]. One of the primary scavang-
ers of ROS is superoxide dismutase, which catalyzes the dismutation of superoxide to
hydrogen peroxide and then to water. Local deficiency of SOD can lead to the forma-
tion of peroxynitrite and other oxidizing species. Three distinct SODs are found in the
human body: SOD1 (Cu/Zn SOD) which is found in the mitochondria, which localized
primarily to the cytosol, SOD2 (MnSOD) and SOD3 or extracellular SOD (EC-SOD).
SOD2 is the enzyme that plays an essential role in oxidative stress mitochondrial pro-
tection [23]. The role of SOD2 in OA chondroyctes is unknown, however the levels of
SOD2 protein in human articular chondrocytes decreases with aging [51].
Interestingly, it has been reported other mechanisms to induce somatic mutations
in mtDNA. Several groups have addressed the issue of causation using a clever ap-
proach to generate mtDNA mutations experimentally. mtDNA replication is carried out
by mtDNA polymerase-γ (POLG), which has 3-to-5 exonuclease (proofreading) ac-
tivity in addition to its 5-to-3 polymerase activity [52]. If the proofreading activity of
POLG is eliminated and the polymerase activity preserved, mtDNA mutations accumu-
late because of uncorrected errors during replication. In mice with such proofreading-
deficient POLG (mtDNA-mutator mice), mtDNA mutations accumulate to high levels
in all tissues. By 8 weeks of age, homozygous Polg–/– animals had 9 point mutations
per 10 kb in cytochrome b. By contrast, normal mice had less than 1 mutation per
10 kb. This marked increase in mtDNA mutations resulted in decreased respiratory
enzyme activity and ATP production. To begin with, the mice appeared normal, but by
25 weeks of age began to exhibit pathology frequently seen in human ageing, including
weight loss, alopecia, osteoporosis, kyphosis, cardiomyopathy, anaemia, gonadal atro-
phy and sarcopaenia (presence of OA was not studied). Somatic mutations of mtDNA
have been detected in synoviocytes from Rheumatoid Athritis and OA patients [53].
Authors described that RA synoviocyte mtDNA had about twice the number of muta-
tions as the OA group. However they did not compare the number of mtDNA muta-
tions with healthy group. There are not published studies describing the mutations in
mtDNA from OA chondrocytes. In this sense, preliminary results obtained in our lab
using Temporal Temperature Gradient Electrophoresis (TTGE) showed that mtDNA
from OA chondrocytes has higher number of mutations than mtDNA from healthy
chondrocytes [54].
4. Conclusions
In summary, mitochondrial function (MRC activity and ATP synthesis) of OA chon-

drocytes is altered. Mitochondrial dysfunction may mediate several specific pathogenic
pathways implicated in OA. These include oxidative stress, inadequacy of chondrocyte
biosynthetic responses, up-regulated chondrocyte cytokine-induced inflammation and
matrix catabolism, increased chondrocyte death (e.g. apoptosis), and pathologic carti-
lage matrix calcification. Mitochondrial dysfunction in OA chondrocytes may be origi-
nated by somatic mutations in mtDNA. Another explanation is the direct effect of some
pro-inflammatory mediators (cytokines, prostaglandin, ROS and NO) on CRM and
ATP synthesis. The weight of evidence reviewed herein strongly supports chondrocyte
mitochondrial impairment as a mediator of the establishment and progression of carti-
lage degradation during OA. Thus, therapies targeting basic mitochondrial processes,
such as energy metabolism or free-radical generation, hold great promise to treat OA
diseases.
Acknowledgements
This study was supported by grants from Ministerio Educacion y Ciencia (SAF
2005-06211), Secretaria I+D+I Xunta Galicia (PGIDIT06PXIC916175PN and from
Fondo Investigación Sanitaria (CIBER- CB06/01/0040)-Spain, with participation of
fundus from FEDER (European Community). MJ López-Armada was supported by
Contrato Investigadores SNS (Fondo Investigación Sanitaria, Spain, CP06/00292).
Ignacio Rego was supported by Contrato de Apoyo a la Investigación-Fondo
Investigación Sanitaria (CA06/01102).
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IOS Press, 2007
XIII
Subchondral Bone and Osteoarthritis

Progression: A Very Significant Role
Johanne MARTEL-PELLETIER, PhD, Daniel LAJEUNESSE, PhD
and Jean-Pierre PELLETIER, MD
Osteoarthritis Research Unit, University of Montreal Hospital Centre,
Notre-Dame Hospital, 1560 Sherbrooke Street East, Montreal, Quebec,
Canada H2L 4M1
Abstract. Osteoarthritis (OA) is considered a complex illness in which cross-talk

between the different tissues of the joint plays a significant role in the evolution of
the disease. Although we may not yet completely know all the initiating factors in-
volved in the degeneration of the articular tissues, significant progress has been
made with respect to the proposal of new concepts regarding the etiopathogenesis
of this disease. For decades, the prevailing concept centered around the destruction
of articular cartilage as the focal pathological feature of OA. Consequently, it is
not surprising that investigators concentrated their efforts at identifying mecha-
nisms involved in the destruction of this tissue. There is now substantial evidence,
from preclinical and clinical studies, that changes in the subchondral bone metabo-
lism comprise an integral component of the disease process, and its key role in the
initiation and/or progression of cartilage degeneration may have been largely un-
derestimated. This concept as well as the complex pathophysiological mechanisms
taking place in this tissue during OA, are the focus of this chapter.
Introduction
Osteoarthritis (OA) is the most common disabling chronic condition in the western
world. It is not a single disease entity but, rather, involves several subgroups with dif-
ferent underlying pathophysiological mechanisms. OA is a disease of an entire organ
system, in which both anatomical and metabolic changes act together to bring about the
structural changes. The notion that the degeneration and erosion of cartilage is the pri-
mary pathophysiological mechanism of OA has recently been challenged, in view of
the identification of prominent changes in the subchondral bone which provide evi-
dence suggesting that this tissue may also play a key role in the etiology of the disease.
In fact, data strongly suggest that the subchondral bone could be a driving force behind
the cartilage degradation observed in OA.
It is common knowledge that, in OA animal models, changes in bone density os-
teoid volume and subchondral bone thickness are often more severe than cartilage
changes. The severity of cartilage fibrillation and loss generally exceeds bone changes
only in advanced OA in primate animal models [1,2].
J. Martel-Pelletier et al. / Subchondral Bone and Osteoarthritis Progression 207
Subchondral Bone as a Risk Factor in OA
Magnetic resonance imaging is ideal for assessing the structural changes that take place
in OA, particularly in longitudinal studies of cartilage and bone, and for identifying the
very early changes often overlooked by other, less sensitive, imaging technologies. It is
now possible to quantify the trabecular architecture, the volume of subchondral bone
marrow oedema and cyst lesions, as well as bone attrition and volume of cartilage. At
first, a number of studies focused on the exploration of structural changes in the sub-
chondral trabecular bone in knee OA patients. In a cross-sectional study, Beuf et al. [3]
found that, in OA, the loss of femoral trabecular bone was correlated to the severity of
the disease. For their part, Blumenkrantz et al. [4] showed that the loss of cartilage vol-
ume and the deterioration of the subchondral bone structure were interdependent, and a
positive correlation was established between the loss of cartilage and subchondral bone
sclerosis and osteopenia of the underlying trabecular bone. Moreover, other studies
have recently reported that bone marrow lesions, including oedema and cysts, have a
high prevalence in human knee OA [5–7], and a statistically significant correlation was
found for the medial compartment between the increase in oedema and cyst size over
time (2 years) and the loss of cartilage volume juxtaposed to the location of the lesions
[8]. These data underline the importance of the subchondral bone remodeling in OA
pathophysiology, and that bone marrow lesions are markers and strong predictors of
structural worsening of knee OA cartilage lesions.
Although these data suggest that subchondral bone alterations may be more inti-
mately related to the OA process than merely being a consequence of the disease, a
question that still remains is whether changes in subchondral bone induce or participate
in disease progression.
Studies have shown that in knee OA, the subchondral bone is stiffer and can in-
crease trabecular bone strain in the proximal tibial plateau and distal tibia [9–11]. Bone
strain could then lead to subsequent cartilage lesions. Hence, a steep stiffness gradient
in the underlying subchondral bone may be an initiating mechanism of OA, as the in-
tegrity of the overlying articular cartilage depends on the mechanical properties of its
bony bed. Inhomogeneities in density or stiffness of the subchondral bone may thus be
key factors that could modulate cartilage loss. Indeed, articular cartilage above a less
dense and more compliant bone will deform more than that above a denser and stiffer
bone. Such deformation can then stretch the articular cartilage at the edge of the joint
contact area, generating tensile and shear stresses [12].
OA Subchondral Bone Material Versus Mineral Density
It has been shown that in knee OA, the progressive joint space narrowing correlates
positively with the bone density of the tibial subchondral bone [13]. However, further
studies point to the fact that the higher density of this tissue in OA is due to an increase
in material density, not an increase in mineral density [14–16]. The higher density ob-
served in this diseased tissue appears to be due to an increased osteoid collagen matrix
and an increase in trabecular number and volume. However, in vivo and in vitro studies
have revealed the presence of an abnormal mineralization process in OA subchondral
bone, resulting in a hypomineralization of this tissue [15,17,18]. Thus, subchondral
bone sclerosis results from an increased stiffness, and not an actual increase in bone
mineral density. Some clinical studies have shown that the indices of bone resorption
208 J. Martel-Pelletier et al. / Subchondral Bone and Osteoarthritis Progression
Figure 1. Cartilage and subchondral bone. Osteoarthritic subchondral bone showed an increased resorption at
the early stage of the disease and sclerosis at the late stage. From reference [19].
are increased early in the disease, while subchondral bone sclerosis is a relatively late
occurrence (Fig. 1; [19]). Consequently, the acceleration of bone turnover in OA results
in the deposition of a hypomineralized bone, which reduces its stiffness for a given
apparent density but increases stiffness if this is offset by increased bone volume.
OA Subchondral Bone: Early Versus Late Stages
Several reports have indicated that the subchondral bone remodeling that occurs in OA
involves both bone resorption and bone formation. However, a major area understudied
is the characterization of specific changes that can distinguish advanced from early
disease. Studies from animal models that allow for a chronological evaluation of these
changes suggest that in the more advanced stage of the disease, bone formation is pre-
dominant [1,2,20,22]. In contrast, in the early phase there is a remodeling process that
primarily favors bone resorption. In experimental OA animal models representing an
early stage of the disease, it has been shown that the subchondral plate and the underly-
ing trabecular bone become thinner, indicating excess bone resorption (Fig. 2)
[21,23,24]. This agrees with the study of Bettica et al. [25] who demonstrated that gen-
eral bone resorption, as defined by the level of type I collagen N- and C-terminal te-
lopeptides biomarkers, is increased in patients with progressive knee OA. Similarly,
Messent et al. [26], with the use of fractal signature analysis, showed that bone loss
occurred in patients with knee OA, specifically in the medial compartment, and that
these changes were associated with an increase in the number and size of the remodel-
ing units.
Abnormal Biochemical Pathways
Many studies have demonstrated that the subchondral bone is the site of several dy-
namic morphological changes that seem to be part of the disease process. These
changes are allied with many local abnormal biochemical pathways, including the in-
creased synthesis of several bone markers, growth factors, cytokines, proteases and
inflammatory mediators. The levels of alkaline phosphatase, osteocalcin, collagen type
Figure 2. Morphometric analysis of subchondral bone in normal (n=6) and osteoarthritic (OA) (n=7) anterior
cruciate ligament dog model. OA subchondral bone specimens were selected at the lesional (L) and non-
lesional (NL) areas of the tibial plateaus. Right panel: Data are the bone surface at different depths starting
from the calcified cartilage 0–500 µm or 500–1000 µm. Morphometric data are presented as box plot, where
the boxes represent the first and third quartiles, i.e. line within the box represents the median and lines out-
side the box represent the spread of values. The statistical analysis data are the comparison made between the
normal and the OA groups using the Mann-Whitney U test. Left panel: Representative morphological sec-
tions of subchondral bone. From reference [23] with modification.
I, IL-6, IGF-1, TGF-ß, PGE2, LTB4, proteases including urokinase, cathepsin K and
metalloproteases (MMPs) have all been found elevated in OA subchondral bone os-
teoblasts [27–30].
An abnormal level of production of mature collagen type I [14,27,31] accompanied
by a significantly elevated level of the collagen type I α1 chain has also been found
in OA subchondral bone. Collagen type I is composed of heterotrimer α1 and α2
chains. In normal bone an average ratio of 2.4:1 is found. Yet, in OA this ratio varied
from 4–17:1 [14,27,31]. The abnormal production of the α chains is of great impor-
tance and could provide an explanation for the reduction in bone mineralization, as it
has been shown that the increase in α1 homotrimers in bone causes a 50% reduction in
the strength of this tissue in α2 knock-out mice [32].
The higher levels of osteocalcin found in the subchondral bone of OA patients,
even at non-weight-bearing sites, and the finding that in vitro subchondral human OA
osteoblasts produce an increased amount of osteocalcin, could also explain the abnor-
mally low bone mineralization in these individuals, as this factor has been suggested to
retard normal mineralization [33]. Thus, an imbalance in collagen and non-collagen
Figure 3. Immunohistochemical detection of osteoclasts staining positive for cathepsin K (arrows) in sub-
chondral bone in normal (n=6) and osteoarthritic (OA) (n=7) anterior cruciate ligament dog model. OA sub-
chondral bone specimens were selected at the lesional (L) and non-lesional (NL) areas of the tibial plateaus.
Right panel: Data are presented as box plot, where the boxes represent the first and third quartiles, i.e. line
within the box represents the median and lines outside the box represent the spread of values. The statistical
analysis data presented are the comparison made between the normal and the OA groups using the Mann-
Whitney U test. Left panel: Representative sections of the immunohistological detection. From refer-
ence [23] with modification.
protein production, such as osteocalcin, could contribute to an increase in bone volume

without a concomitant increase in the bone mineralization pattern.
Urokinase, through its wide proteolytic activity, seems to be a particularly impor-
tant factor in OA subchondral bone remodeling, since this protease has been shown to
be capable of activating factors involved in bone resorption and formation, such as
TGF-ß and IGF-1, and can also directly degrade the bone matrix [34–37].
The higher levels of PGE2, IL-6 and LTB4, which occur in OA subchondral bone
osteoblasts are capable of promoting bone formation and the deposition of a new ma-
trix, yet this matrix may be undermineralized. Indeed, PGE2, at low concentrations,
stimulates bone formation while it may have inhibitory activity at high concentrations
[38–40]. In addition, PGE2 stimulates collagen synthesis and can promote the prolifera-
tion of osteoblasts. Conversely, IL-6 and PGE2 may also be responsible for the in-
creased number of active osteoclasts found in OA subchondral bone. Lastly, LTB4
stimulates osteoclast differentiation and bone resorption [41].
Two other proteases, cathepsin K and MMP-13, known to be potent bone resorp-
tive factors, are also present in increased amounts in OA subchondral bone and calci-
fied cartilage [23,42]. Cathepsin K has been found to be present quite selectively in the
subchondral bone osteoclasts, and preferentially located in the zone where there is a
very active bone resorption (Fig. 3). MMP-13 has been found in chondrocytes from
calcified cartilage, in subchondral bone osteoblasts and also in osteoclasts. As this en-
zyme is known to work in conjunction with cathepsin K in the induction of bone re-
sorption, their combined effect is likely to be very potent in inducing the resorption of
subchondral bone. The activity of two other MMPs, MMP-2 and MMP-9, is elevated in
OA proximal cancellous bone tissue, a situation possibly linked to abnormal collagen

matrix deposition [16].
Some of the factors belonging to the TNF family, namely OPG and RANKL,
are of key importance in regulating bone metabolism [43]. RANKL is a factor synthe-
sized by osteoblasts, and is essential for osteoclast differentiation and bone resorption
[44–46]. It exists as a cell membrane or extracellularly in soluble form, both of which
stimulate osteoclastogenesis and osteoclast action after binding to and activating the
cell surface RANK, located on osteoclast precursors and osteoclasts. In addition, a
soluble decoy receptor for RANKL, OPG, has been identified [47]. This decoy receptor
blocks the binding of RANKL to RANK, thus blocking RANK activation and subse-
quent osteoclastogenesis and, as a result, inhibiting bone resorption. An abnormal pro-
duction of these factors by human OA osteoblasts has recently been identified [48].
Interestingly, a recent study reported a differential level of these factors according to
OA osteoblast metabolic state [49], suggesting that, in some cases, OA subchondral
bone tissues are enriched in factors promoting bone resorption, while others, con-
versely, have reached a different metabolic state which favors a reduction in resorptive
properties.
Some factors produced in the subchondral bone by cells derived from the bone
marrow were also suggested as being involved in the pathological process. Aspden
et al. [50] put forth the concept that OA is a metabolic disease in which systemic and/or
local factors induce changes in skeletal tissues by modifying the formation and activity
of mesenchymal precursor cells, suggesting a possible link between abnormal lipid
metabolism and OA subchondral bone structural changes. This hypothesis is supported
by the observation that osteoblast maturation from bone marrow stromal cells in OA
patients is enhanced while that of adipocytes and chondrocytes is blunted [51]. More-
over, OA patients have, in general, higher than average body weight, and obesity is a
major risk factor for OA [52–54].
In this context, leptin, a protein involved in lipid metabolism, has been suggested
as an important pathological factor in OA [55]. Leptin is the product of the obese gene.
Its protein level is increased in human OA cartilage [56], however, its expression is
greatest in OA subchondral osteoblasts [57]. A point of interest is the fact that within
the bone marrow, leptin favors the differentiation of mesenchymal stromal cells into
osteoblasts while impeding the maturation of adipocytes, a situation that is also ob-
served in OA [51]. It is noteworthy that leptin injections in rats stimulate the expression
of IGF-1 and TGF-ß, increase alkaline phosphatase activity, and the level of synthesis
of osteocalcin and collagen type 1 α1 chain [58], factors found at higher levels in OA
subchondral bone tissue. In addition, leptin was also found to be associated with in-
flammatory states and to stimulate PGE2 and leukotriene production [59–63]. Other
studies have shown that leptin can enhance the synthesis of endothelin-1 (a factor also
found to be involved in human OA cartilage), trigger nitric oxide production and acti-
vate the p38 MAP kinase, changes which are commonly found in OA tissues.
A clear link between circulating leptin levels and OA remains to be established. To
that effect, the role of serum leptin and obesity in the progression of knee OA has re-
cently been investigated. Data showed [64] that weight loss in the obese OA patients
resulted in a significant decrease in the level of serum leptin. Although speculative,
these findings could imply that a decrease in serum leptin may be one of the mecha-
nisms by which weight loss could possibly slow progression of the disease in OA pa-
tients. However, it would not preclude the involvement of other local factors, as leptin
might be a contributing factor that alone may not be sufficient, although necessary, to
promote joint damage in OA. On the other hand, and logically, the local levels of leptin
in the joint may be more relevant than circulating leptin toward OA progression.
On the subject of lipoprotein, recent genome-wide scans have revealed an OA sus-
ceptibility locus on chromosome 11q, in close proximity to the low-density lipoprotein
receptor-related protein 5 (LRP5). An altered haplotype of LRP5 was observed in indi-
viduals conferring a 1.6-fold increased risk of OA [65]. This gene product controls
bone mass, and therefore could explain the abnormal bone tissue mineralization and
remodeling observed in OA patients.
Subchondral Bone and Cartilage Cross-Talk
Although the initiating event responsible for the degradation of cartilage in OA patients
remains unclear, the concept whereby subchondral bone and cartilage should be con-
sidered as an interdependent functional unit is gaining strong support. The idea of a
biological link between bone and cartilage that was originally hypothesized a few dec-
ades ago, was halted for a while by the concept that the calcified cartilage was an im-
penetrable structure. This was challenged by independent groups that demonstrated the
presence of channels between the subchondral region and the uncalcified cartilage, and
the presence of microcracks in the articular cartilage [66–69]. The presence of both
microcracks and vascularization in the subchondral bone plate could then facilitate the
transfer of factors from the subchondral bone region to the cartilage by diffusion
through the basal layer of OA cartilage. These findings could explain that cytokines,
proteases, growth factors, and eicosanoids produced locally by subchondral bone tissue
seep through the bone-cartilage interface and stimulate cartilage breakdown. In that
respect, if the abnormal production of proinflammatory cytokines, PGE2 and LTB4 by
OA osteoblasts under in vitro conditions reflects in situ conditions, it could very well
be that these cells have a significant influence on the metabolism of the contiguous
cartilage.
Recent data have also shown that bone resorption pits in subchondral bone may be
an important factor in cartilage destruction via the release of proteases, thereby estab-
lishing a clear link between subchondral bone activity and altered metabolism, and
cartilage loss. Two of the enzymes that are produced in the resorption areas of the sub-
chondral bone, cathepsin K and MMP-13, may also contribute to the degradation of the
articular cartilage matrix macromolecules. The findings that, in situ in OA cartilage,
MMP-13 synthesis is located preferentially at the lower intermediate and deep layers
[70,71], support this hypothesis.
Hepatocyte growth factor (HGF), has recently been proposed as another possible
candidate for this cross-talk between these two tissues. First identified in the liver, this
multifunctional factor can induce proliferation, motility, morphogenesis, and it has
chemotactic properties. HGF was found to be preferentially located in the deep layers
of OA cartilage [72]. However, data have shown that chondrocytes do not express
HGF, but OA osteoblasts do and they produce higher levels when compared to nor-
mal [73]. From these findings, it would seem most likely that this factor originates
from the subchondral bone. The report that HGF induces MMP-13 production by
chondrocytes also adds credibility to the hypothesis [74]. In OA subchondral bone,
HGF could have a dual role, sclerosis and resorption. Indeed, as a growth factor HGF
was shown to increase bone mass, conversely in the presence of osteoblasts it stimu-
lates osteoclast resorption [75] and elevated serum concentration of HGF correlates
with markers of disease activity in multiple myeloma associated with bone destruc-
tion [76] while it directly increases osteoblastic function [77–79]. In addition, HGF
could act on the neovascularization of the subchondral bone, as it is known to induce it
in other tissues [77,80,81].
Another factor which is found in both cartilage and in subchondral bone, TGF-ß,
could very well be an important factor contributing to OA cartilage degradation. The
stimulation of normal cartilage explants with TGF-ß mimics the increased production
of MMP-13 found in situ in the lower zones of OA cartilage [71]. The level of TGF-ß
as well as its receptor level are increased in the low intermediate and deep zones of OA
cartilage [82], and in OA subchondral bone osteoblasts [28]. This particular distribution
of TGF-ß and its receptors in cartilage therefore suggests that bone-derived TGF-ß
could be responsible, to a certain extent, for the upregulation of MMP-13 in OA carti-
lage.
Vitamin A derivatives can promote subchondral bone proliferation whereas they
lead to progressive atrophy of articular cartilage at the same time that they initiate ec-
topic collagen type I production in cartilage, a feature reminiscent of OA [83]. It was
also shown that subchondral bone proliferates into the cartilage, and the affected carti-
lage produces a developmentally regulated matrix molecule, osteoblast-stimulating
factor-1, normally expressed by fetal and epiphyseal growth plate cartilage but not by
articular cartilage, indicating that vitamin A derivatives may promote de-differentiation
of chondrocytes as observed in OA.
The intimate link between the articular cartilage and subchondral bone is also re-
flected in the observation that autologous chondrocyte implantation for cartilage repair
is less effective than osteochondral cylinder transplantation [84].
Therapy Directed Against Subchondral Bone Remodeling
Joints affected by OA have an increased bone turnover, which consequently increases

the possibility of benefiting from drugs that alter bone metabolism, particularly the
antiresorptive agents such as bisphosphonates. There is a good rationale to use such
agents since they are safe for long-term administration (as they are used for osteoporo-
sis) and easy to administer. Carbone et al. [85] conducted a study in which they exam-
ined the association between use of medications that have a bone antiresorptive effect,
including estrogen, raloxifene, and alendronate, and the structural features of knee OA.
Although the women treated with both alendronate and estrogen showed significantly
less subchondral bone attrition and bone marrow oedema-like abnormalities than those
who had not received these medications, no significant effect was found on the pro-
gression of cartilage damage. Others [86] looked at the effect of risedronate, another
bisphosphonate, on joint structure and symptoms of OA patients. A definite trend to-
ward improvement was observed in a Phase II study in both joint structure and symp-
toms in patients treated with risedronate [85]. The results of this study, however, could
not be confirmed in a Phase III study [87]. Explanations for these disappointing data
are that patients selected for these studies had long disease duration upon study entry
and that the technologies used (X-rays) were not sensitive enough for the cartilage
structure. Before definitely eliminating this line of drugs from OA treatment, patients
with less advanced disease should be tested and a more reliable and sensitive imaging
technology, such as magnetic resonance imaging, should be used.
The most recent knowledge of the underlying molecular pathological mechanisms

leading to bone remodeling/resorption in OA such as the RANK, RANKL and OPG
will help to bring new therapeutic strategies to clinical practice. Clinical trials are al-
ready underway using OPG and RANKL antibodies to test their efficacy in the treat-
ment of osteoporosis and of bone erosions in rheumatoid arthritis patients [88]. The
potential of these agents in OA is also very appealing.
Conclusion
Several processes are altered in cartilage during the OA process. Recent data, however,
indicate that the subchondral bone plays a major role in this process, and is not merely
a secondary manifestation of the disease. This tissue is now considered part of a more
active component of the initiation and/or progression of OA. Some bone parameters,
including abnormal bone mineral density, osteoid volume, bone mechanical parameters
or indicators of bone turnover, are altered in OA patients. Similarly, several biochemi-
cal factors are also found elevated in the subchondral bone tissue in OA, and these may
seep through clefts or channels in the tidemark to invade the overlying cartilage and
promote its degradation. Therefore, even though the initiating event or effector in OA
has yet to be identified, subchondral bone can now be focused upon as a potential can-
didate.
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IOS Press, 2007
XIV
Osteoarthritis and Inflammation –

Inflammatory Changes in Osteoarthritic
Synoviopathy
Thomas AIGNER a,1, Peter VAN DER KRAAN b and Wim VAN DEN BERG b
a
Osteoarticular and Molecular Pathology, Institute of Pathology,
University of Leipzig, Germany
b
Experimental Rheumatology & Advanced Therapeutics,
Nijmegen Centre For Molecular Life Sciences, Radboud University Medical Centre,
Nijmegen, The Netherlands
Abstract. OA research traditionally focuses on understanding events that occur

within the degenerated articular cartilage whereas changes in the synovial mem-
brane are largely neglected. However, inflammatory changes do occur in the syno-
vium that may contribute to the overall effects observed in at least a subsets of OA
patients. This implicates that the inflammatory and degradative activities of syno-
viocytes represents an interesting (therapeutic) target in OA research.
Keywords. Osteoarthritis, Cartilage, Synovial membrane, Synovitis, Inflamma-

tion, Interleukin
1. Osteoarthritis and Inflammation
Osteoarthritis (OA) is a multifactorial disease, which results primarily in degeneration

of articular cartilage tissue. Disruption of the homeostatic anabolic and catabolic events
in articular cartilage is thought be the initiation point that causes this condition [1].
However, other joint structures such as the subchondral bone plate and the synovium
play their own roles within the OA disease process [2]. Thus, the importance of the
synovial membrane and joint capsule in terms of causing disease symptoms is apparent
and worthy of further investigation. The two main clinical symptoms of OA, pain and
joint stiffness, are both significantly related to synovial inflammation and capsular fi-
brosis. However, the role of synovial inflammation in the pathogenetic process of carti-
lage destruction is largely unknown.
The synovial (inflammatory) reaction observed in OA joint disease is primarily
considered to be a secondary effect resulting from the release of cartilage debris from
the damaged articular cartilage [3–7]. This is in contrast to the situation found in rheu-
1
Osteoarticular and Molecular Pathology, Institute of Pathology, University of Leipzig, Liebigstr. 26,
D-04103 Leipzig, Germany, E-mail: thomas.aigner@medizin.uni-leipzig.de.
220 T. Aigner et al. / Osteoarthritis and Inflammation
matoid arthritis, which is considered to originate from a synovial inflammatory auto-

immune reaction with secondary cartilage destruction. However, inflammatory reac-
tions in the synovial membrane do occur to some degree in all OA joints as discussed
below. Also, the fact that most OA patients display a minor elevation of C-reactive
protein within the serum [8,9] suggests that the inflammatory component plays some
role within the disease process.
In addition to local and/or general inflammatory responses within the synovial
membrane, the activation of inflammatory pathways within the chondrocytes them-
selves may also play a crucial role in disease progression. Activation of such processes
would be independent of direct inflammatory cell infiltrates, which are not present in
OA articular cartilage. In fact, inflammatory signaling pathways have been shown to
induce catabolic responses in chondrocytes, namely matrix degrading proteases such as
MMP-13, MMP-1 and others. One of the most prominent catabolic cytokines in OA is
the pro-inflammatory cytokine interleukin 1 (IL-1) [10,11]. Elevated levels of IL-1 are
found in synovial fluids of patients suffering from rheumatoid arthritis and, to a lesser
extent, in synovial fluid from OA patients [12]. An increase in IL-1ß levels in OA carti-
lage has been reported using immunolocalization technology [13]. Although own stud-
ies could not confirm an increased expression of IL-1 mRNA in OA chondrocytes by
sensitive PCR technology [14], this may still reflect increased levels of IL-1 protein
diffused into cartilage from the synovial space. IL-1 significantly affects gene expres-
sion patterns within articular chondrocytes [15] via multiple intracellular pathways,
particularly the MAPkinases and NFkB-pathways (Fig. 1) (for review see Saklatvala
2007 [16]). IL-1 down-regulates the expression of the major cartilage matrix compo-
nents, aggrecan and collagen type II [17–19] and, thus, counteracts the effects of ana-
bolic factors on matrix synthesis. Additionally, IL-1 induces the expression of matrix
degrading enzymes such as MMP-1, MMP-3, MMP-13 or ADAMTS-4, which are all
potential major players in the destruction of cartilage matrix components [18–21]. Be-
sides these direct effects, IL-1 also induces other cytokines with synergistic (catabolic)
effects such as IL-6 and LIF (leukaemia inducing factor) [14,22,23].
2. Physiology of the Synovio-Cartilage-Interaction
Joints are highly specialized organs that allow repetitive pain-free, frictionless move-
ments. These properties are provided by the articular cartilage and its extracellular ma-
trix which, under physiological conditions, is capable of sustaining high cyclic loading.
Joints are, however, complex composites of different types of connective tissue includ-
ing subchondral bone, cartilage surfaces, ligaments and the joint capsule. All the differ-
ent joint tissues together provide their own specific roles to permit correct functioning
of the joint.
The synovial capsule and, in particular, the synovial membrane (i.e. the synovial
lining cell layer (Fig. 2 a,b)) vastly contributes to the physiological functioning of the
articulating joints. It is the synovial capsule together with the ligaments that provide the
mechanical stability of the joints and ultimately determines the flexibility or range of
motion of the joint. The synovial membrane, containing high metabolically active sur-
face cells (synoviocytes) plays a crucial role in nourishing the chondrocytes as well as
removing metabolites and matrix degradation products from the synovial space. There-
fore, the synoviocytes maintain the normal metabolic milieu within the joints. Further-
more, the synoviocytes produce large amounts of hyaluronic acid and other factors
T. Aigner et al. / Osteoarthritis and Inflammation 221
IL1
IL1-R
MAPKKKK
NIK MAPKKK / TAK1 / TAB1
MAPKK
NFΚB JNK p38 ERK
c-jun, ATF, SAP-1, ELK,…
catabolic genes
catabolism
Figure 1. Schematic representation of the IL-1 signaling pathway.
such as lubricin/superficial zone protein [24], which provide the joint surfaces with its
lubrication capacity. In addition, the synovial fluid is composed of other substances
that diffuse between the articular cartilage and the synoviocytes including chondrocyte-
derived nutrients and metabolites as well as oxygen molecules.
3. Synovial Changes in OA – Histologic Reaction Pattern
We and others have shown that all cases of clinically relevant OA joint disease are as-
sociated with some sort of synovial pathology [25,26]. This reflects the notion that
there is a direct relation between clinical symptoms and the synovial reaction in OA.
This suggests also that changes in the synovial membrane are partly involved in the
progression of the disease [27].
In OA synovial specimens, in principle, four different types of OA synoviopathies
are found: hyperplastic, inflammatory, fibrotic, and detritus-rich synoviopathy (Ta-
ble 1) [26].
Figure 2. a,b: Histological appearance of normal synovial membrane with flat, non-activated synovial lining
cells at the surface (b: detail). c: Typical picture of hyperplastic synoviopathy with numerous synovial villi.
d,e: Typical picture of fibrotic synoviopathy with a very much thickened and fibrotic capsule (note at the
surface also some hyperplastic synovial villi; e: collagen stain (van Gieson´s stain)) f,g: Inflammatory syno-
vitis in osteoarthritic patients with a minor to moderate lymphocytic infiltrate, partly organized in lymph
follicles (g). h,i: detritus-rich synovitis, which is a typical feature of rapid-progressive end-stage disease with
numerous bone and cartilage particles intermixed with fibrin and partly incorporated into the synovial
stroma. Large fragments get successively degraded by osteoclast-type multinuclear giant cells (i: here labeled
with CD68, a marker of phagocyting cells).
Detritus-rich synovitis, which is found in end-stage OA disease [28,29], is due to

abundant macromolecular cartilage and bone detritus (i.e. bone and cartilage fragments
attached to or incorporated into the synovial membrane; Fig. 2h) in addition to abun-
dant molecular debris, not visible microscopically. Besides the debris, a significant
Table 1. Table listing the major histopathological features of the four pattern of osteoarthritis associated
synoviopathy in comparison to each other and to normal synovial membrane. Bold letters indicate key diag-
nostic criteria
normal hyperplastic inflammatory fibrotic detritus-rich

villous hyperplasia – ++(+) ++(+) ++(+) ++(+)
synovial lining – proliferation – + ++ ++ ++(+)
synovial lining – activation – + ++ + +
fibrinous exsudate – – (+) + ++(+)
capsular fibrosis – – (+) +++ +++
(macromolecular) cartilage and bone debris – – (+) – +++
granulocytic infiltrate – – – – +
lymphoplasmacellular infiltrate – diffuse – – ++ (+) +(+)
lymphoplasmacellular infiltrate –
– – ++ (+) (+)
aggregates/follicles
amount of fibrinous exudate is found either at the surface of the synovial membrane.
This exudate may be combined with incorporated fibrin reflecting longer ongoing fi-
brinous exudation already being organized (i.e. resorbed). Detritus-rich synoviopathy
usually contains a minor inflammatory cell infiltrate consisting of lymphocytes and
granulocytes as well as some foreign body giant cells (Fig. 2i).
Another form of OA synoviopathy found in late stage disease, fibrotic OA syno-
viopathy (capsular fibrosis) [26,30] (Fig. 2d,e), is mainly characterized by the shorten-
ing and thickening of the joint capsule, which is partly responsible for some symptoms,
in particular joint stiffness, seen in OA patients.
The most interesting of the OA synoviopathies in terms of pathogenesis is the in-
flammatory OA synoviopathy, which displays moderately extensive lymphocytic infil-
trates [26,31,32] (Fig. 2f,g). Histologically, this condition resembles a less severe case
of rheumatoid synovitis where dense infiltrates of B-lymphocytes, plasma cells and
T-lymphocytes (CD4- and/or CD8-positive) are detected. It is intriguing to speculate
whether this condition reflects some kind of autoimmune aspect that may be occurring,
at least in this subset of OA patients. In fact, this opens up the possibility that “overlap-
ping forms” of rheumatoid arthritis and OA might exist, but this has not been investi-
gated in any detail yet. Interestingly, the lymphocytic infiltrate in the subsynovial
stroma appears to correlate directly with Il-1ß in the synovial fluid as well as MMP-1
expression by synoviocytes [33,34] suggesting a direct stimulatory role of the inflam-
matory cells on the activity of the synovial lining cells. In any case, the presence of
inflammation in a significant portion of OA patients clearly points to the option of anti-
inflammatory therapy at least for some subsets of OA patients.
In early OA, mostly hyperplastic OA synoviopathy is found (Fig. 2c,d). This pat-
tern shows only moderate synovial hyperplasia with or without cellular activation, but
without significant capsular fibrosis and thickening and without significant inflamma-
tory infiltrates or macromolecular detritus [26,35]. Overall, three forms of alterations of
the synovial surface can be observed: 1) increased cytoplasmic volume of the usually
flat synovial lining cells. These cells may even become cuboidal or even cylindrical in
shape suggesting that they have been activated in some way; 2) the in normal condi-
tions single cell layer of synovial lining cells can proliferate to form as many as five
cell layers; 3) the whole synovial surface, including the underlying stroma, can become
hyperplastic and form the classical synovial villi. Synovial hyperplasia per se can be
found in all forms of OA synoviopathy and in chronic synovitis. Thus, villous hyper-
plasia is largely a non-specific feature of chronic synovial alteration and activation.
Synoviocyte activation and proliferation as well as synovial hyperplasia presuma-
bly all represent reactive changes responding to increased demands for clearance of
molecular debris in the synovial fluid of the joint [3,7,36]. This also explains the in-
crease in the amount of CD68-positive type A synoviocytes, which have phagocytic
capacity, in the synovial lining layer [5,7,26,37–40]. Of note, the highest percentage
(up to 60%) of type A synoviocytes is found in the inflammatory OA synoviopathy
suggesting that this subform is associated with a very significant matrix catabolic activ-
ity.
Although a cellular inflammatory component is missing in synovial hyperplasia,
the proliferation and activation of the synovial lining cells might generate significant
problems for the articular cartilage as these cells are able to secrete matrix-degrading
proteases (MMPs) and catabolic cytokines (IL-1, TNF-alpha) [34,41]. It is therefore
intriguing to speculate that the cartilage matrix catabolism may be partly induced by
catabolic mediators (e.g. Il-1ß and TNF-a) secreted by the activated synoviocytes. This
leads back to the notion discussed above where, in addition to the direct involvement of
inflammatory cell infiltrates, which do not occur in OA cartilage, inflammatory path-
ways induced in the articular chondrocytes themselves may have the potential to play a
critical role within OA cartilage destruction. Altogether, it appears that the production
of inflammatory mediators by the synoviocytes, in particular if they are activated, can
play a very important role in the OA disease process (Fig. 3).
4. Synoviopathy and Cartilage Matrix Degradation
The common perception of OA is a disease in which deranged processes in cartilage

and possibly bone are responsible for the destruction of the articular cartilage. Syno-
vium has been considered of minor importance and to contribute little to OA patho-
genesis. However, in addition to OA patients without obvious synovitis there has been
a subgroup of cases identified in which joint inflammation and synovial activation is a
major hallmark. It is apparent that OA synovial tissue shows typical activation markers
such as expression of transcription factor nuclear factor kappaB (NF-kappaB) [42].
Adenoviral gene transfer into osteoarthritis synovial cells of the endogenous inhibitor
IkappaBalpha showed that the synthesis of inflammatory and destructive mediators
from OA synovial tissue was NF-kappaB dependent [43].
The activated synovial tissue is proposed to contribute to degradation of the articu-
lar cartilage matrix. Synovitis has been demonstrated to be correlated with greater se-
verity and accelerated progression of structural damage in OA. Ayral et al found in
patients with knee osteoarthritis that activation of the medial perimeniscal synovium
was associated with more severe medial chondropathy [27]. Inflammation of the me-
dial perimeniscal synovium could be regarded as a predictive factor of succeeding in-
creased degradation of medial cartilage damage. In a study of Loeuille in patients with
knee OA, histologically scored synovitis was associated with chondral lesions in the
medial femorotibial compartment but not at other locations [44]. Synovitis scored with
Figure 3. Interaction action between synovium and cartilage in osteoarthritis. Molecular detritus from the
cartilage activates the synovial lining cells. The synovial lining cells produce cytokines, growth factors and
(latent) enzymes. Synoviocyte-derived cytokines and growth factors further activate the chondrocytes. En-
zymes produced by the synovial lining cells can directly degrade matrix molecules if not inactivated by in-
hibitors in the synovial fluid. Latent enzymes can be activated in the milieu of the osteoarthritic cartilage.
MRI could not discriminate patients with moderate cartilage damage from patients with
severe cartilage lesions demonstrating the lower sensitivity of MRI compared to histol-
ogy of tissue biopsies to detect inflammation [44].
Synovitis appears to play a role in the destruction of cartilage in part of the OA pa-
tients. This can be either a direct effect of factors produced by the synovial tissue or an
effect of activation of chondrocytes by mediators released by the activated synovium.
The cells in the synovium can function as a source of cytokines, growth factors, metal-
loproteinases (MMPs), reactive oxygen species and various other mediators. The role
of cytokines and growth factors in synoviopathy in OA will be discussed in the section
below.
Many studies have evaluated MMP levels in synovial fluid of OA patients but a
smaller number of studies have analyzed MMP expression in OA synovium. However,
already in the early 1980’s elevated synthesis of MMPs in OA synovium has been re-
ported [45,46]. Increased levels of collagenase and stromelysin were detected in syno-
vial tissue of OA patients [34,47,48]. Davidson et al. compared expression of MMP
and ADAMTS genes in synovium from patients with either hip OA or femoral neck
fracture [49]. Genes upregulated in OA synovium compared to synovium from the
femoral neck fracture patients were MMP-9, -11, -13, -16 and -28 and ADAMTS-2,
-10, and –16. For MMP-9, -10, -12, -17, -23, -28, ADAMTS-4, and -9, there was a sig-
nificant correlation between expression levels in the synovium and cartilage, suggest-
ing similar mechanisms of regulation [49]. In a zymography study of MMPs in knee
OA, latent and activated forms of MMP-2 and MMP-9 were found to be produced by
cultures of synovial tissue. Moreover, it was found that both protein and mRNA levels
in lesional cultures were significantly higher than those in paralesional ones [50].
Kanbe et al. performed a very interesting study on the effect of synovectomy on serum
MMP levels in OA patients. Levels of MMP-9 and MMP-13 decreased 7 to 9 fold in
serum after arthroscopic knee synovectomy [51]. This study indicates that inflamed
synovial tissue contributes significantly to systemic MMP levels and it can be expected
that it will have similar or even greater effects on local MMP levels in the knee joint. In
experimental OA we have detected elevated expression of MMP-13 by quantitative
RT-PCR in synovial tissue of OA prone STR/ort mice and C57Bl/6 mice with colla-
genase-induced OA. These results indicate that in OA synovium the synthesis of
MMPs is turned on. Before becoming biologically active MMPs have to be activated.
Plasminogen activator and plasmin have been found elevated in OA synovium and are
thought to play a significant role in the activation of MMPs [52].
Proteases (MMP and ADAMTS) produced in OA synovial tissue might contribute
to the progression of OA by damaging joint structures such as cartilage and ligaments.
However, MMPs produced by the activated synovium have to travel through the syno-
vial fluid to reach articular cartilage unless synovium and cartilage are in close contact.
In the synovial fluid MMP inhibitors such as Tissue Inhibitors of Metalloproteinases
(TIMPs) and alpha1-antitrypsine and alpha2-macroglobuline are present [53–55].
These factors limit the action of MMPs on articular cartilage. On the other hand, in
general only active forms of MMPs are inhibited by protease inhibitors and one can
envision that inactive MMP reach the articular cartilage and these are locally activated
leading to degradation of cartilage matrix components.
Not only proteases but also other factors are expressed by inflamed OA synovium.
OA synovium produces increased amounts of reactive oxygen species (ROS), such as
nitric oxide, peroxynitrite and superoxide anion [56–58]. Immunohistochemistry
showed that not only iNOS but also cNOS was expressed by cells in the synovial lining
and subsynovium of patients with OA [59]. These data indicate that NO is produced by
the activated synovial lining not only by iNOS but also by cNOS. The presence of
functional NO synthetases is confirmed by studies that demonstrate that cultured syno-
vial tissue of OA patients spontaneously produce nitric oxide [60]. Both fibroblast and
macrophages contributed to this production [60]. The presence of enzymes involved in
the synthesis of lipoxygenase products, prostaglandins and leukotrienes, has also been
demonstrated in OA synovium [61–63].
Reactive oxygen species can either directly damage matrix components or indi-
rectly by inhibiting matrix synthesis, inducing apoptosis or by activating latent metal-
loproteinases. Lipoxygenase products are known to activate chondrocytes and to stimu-
late the synthesis of catabolic factors by chondrocytes [64,65]. Inhibition of cyclo-
oxygenases and lipoxygenase pathways in chondrocytes inhibit MMP-13 production in
human osteoarthritic chondrocytes [64,65]. These data show that ROS and lipoxy-
genase products synthesized in inflamed OA synovium could be harmful in OA carti-
lage and contribute to the process of cartilage destruction in this disease.
Synoviopathy and Cytokine/Growth Factor Balance
In general, cytokines are defined as peptide factors that are produced by, and act on
cells habitually in close vicinity. In that respect, the definition includes the various
growth factors. To call a factor a cytokine or a growth factors is in most cases based on
historical grounds and not directly related the mode of action and activity of a particu-
lar factor. Various cytokines and growth factors are found in augmented quantities in
activated OA synovium and can have a direct impact on chondrocyte function. Even
though absolute levels of specific mediators may be indicative of their importance, the
net effect is for the most part determined by the balance of synergizing, counteracting,
and regulating mediators. Increased production of various mediators is found in syno-
vial tissues of OA and RA patients and differences are mostly quantitative and not
qualitative. In terms of their most distinctive effect on chondrocytes, cytokines and
growth factors can be broadly categorized in three classes: catabolic, anabolic and regu-
latory. However, a strict distinction is not always possible and several growth factors and
cytokines show so-called contextual actions on cellular behaviour [66].
Several studies have identified cytokines and growth factors presence in both OA
and RA synovial tissue using immunohistochemistry. Most studies were focused on RA
synovial tissue, and OA synovium was in general used as a control. Most cytokines and
growth factors were found in both OA and RA synovium. The differences found were
mainly quantitative and not qualitative. A study in patients with a range of disease stages
of osteoarthritis demonstrated that highest IL-1 and TNF-α expression was related with
the most severe cases of inflammation, the latter resembling RA synovial tissue [35].
It is generally accepted that both TNFα and IL-1 are the prevailing cytokines in
RA synovium. IL-1 is also synthesized in substantial quantities in OA synovial tissues
and this may be a major source of the increased IL-1 levels in OA synovial fluid
[67–69]. TNFα is less abundant and this is in line with the observation that TNFα can
only be found in a limited number of OA cases [67–69]. Of importance, IL-1 is regarded
to be the driving force for the production of MMPs and ADAMTS in OA synovial tis-
sue. This is demonstrated by the striking inhibition of enzyme production when culturing
OA synovium in the presence of IL-1 receptor antagonist (IL-lra) [70].
A cytokine resembling IL-1, IL-18, is found in low levels in OA synovial tissue
and the enzyme responsible for the activation of IL-1β and IL-18, ICE (Interleukin-
1beta-converting enzyme), has been identified in OA synovial tissue [71–75]. The ex-
pression of the proinflammatory cytokine IL-12 was found to be similar, both on the
mRNA and protein level, in RA and OA patients [76]. Remarkably, expression of both
Th1 and Th2 cytokines have been detected in OA synovium [77]. The number of inter-
feron-gamma positive cells was higher than the number of IL-4 positive cells, indicating
that Th1 cells dominate in the synovium of OA patients.
Members of the TGF beta superfamily are found in OA synovium. Synovial tissues
from patients with osteoarthritis express and secret TGF beta, mainly TGF beta1 [78].
Expression of BMP-2 and –4 was reduced in OA synovial tissue compared to con-
trols [79]. The latter suggests that diminished BMP levels in OA synovium can con-
tribute to a loss of joint homeostasis in this disease. The BMP antagonists follistatin,
gremlin, chordin are expressed by synovial fibroblast from OA patients at similar levels
as in fibroblast from controls while expression was reduced in OA cartilage [80]. In
experimental (collagenase-induced) OA in mice we have found an increased expression
of BMP-2 and –4 in synovium using Immunohistochemistry [81,82]. Depletion of
macrophages from the synovial lining using chlodronate-loaden liposomes resulted in
diminished expression of BMP-2 and –4 indicating that these cells are responsible for a
major part of BMP-2 and -4 production in activated synovial lining cells.
Vascular Endothelial Growth Factor has been detected in OA synovium and im-
munoreactivity increased with increasing histological inflammation grade. In the syno-
vial lining, VEGF immunoreactivity was localized to macrophages [83]. Synovial tis-
sue from patients with OA expressed basic Fibroblast Growth Factor (bFGF) mainly in
hyperplastic lining synoviocytes [84]. Arthroscopic synovectomy resulted in a five-fold

reduction in the serum levels of the chemokine stromal cell derived factor 1 (CXCL12)
in OA patients, indicating that synovium contributes significantly to the production of
this factor in these patients [43,85]. The parathyroid hormone-related peptide (PTHrP)
is more strongly expressed by RA synovium than by OA synovial cells [86,87]. How-
ever, synovial fibroblasts from OA patients have been shown to produce PTHrP after
incubation with inflammatory cytokines such as IL-1 [88].
The adipokines, such as leptin, adiponectin and resistin, could give a clue for the
relationship between obesity and OA development [89,90]. Leptin levels correlate with
body mass index [89,90]. Synovia from OA patients have been shown to be a major
source of leptin and adiponectin [91].
The cytokines and growth factors described in the previous section are produced in
the synovial tissue, the articular cartilage, or in both tissues. The factors produced in
the synovium are thought to be able to reach the articular cartilage and modulate in this
tissue the cytokine/growth factor balance. Changes in the availability of cytokines and
growth factors will alter the behaviour of the chondrocytes and his can lead to loss of
cartilage homeostasis and OA.
Catabolic Factors
A key example of a destructive cytokine is IL-1. A critical role of IL-1 in early stages
of OA seems unlikely. Inflammatory models demonstrate that chondrocyte proteo-
glycan synthesis is strongly reduced shortly after induction of inflammation and that
synthesis remains being suppressed during ongoing inflammation [92,93]. Blocking IL-
1 with neutralising antibodies and IL-lra provided persuasive evidence that IL-1 is the
key mediator of the inhibition of proteoglycan synthesis [94–96]. In marked contrast,
chondrocyte proteoglycan synthesis, and collagen synthesis, has been reported to be
enhanced in early stages of OA [97–99]. This appears to exclude an essential role for
IL-1, or at least suggests an overkill by anabolic factors. An OA phenomenon that may be
attributable to IL-1 is the alleged shift in chondrocyte phenotype during OA. It has been
shown that after prolonged exposure to IL-1, synthesis of cartilage specific collagen
types such as type II and type IX is reduced while synthesis of types I and III collagen
is increased [100,101]. This shift could account for the unsuccessful matrix repair in
OA. Moreover, IL-1 can play a major destructive role in later stages of OA, which is
characterized by reduced matrix synthesis and overt cartilage destruction.
The functioning of cytokines, such as IL-1, is regulated by cytokine-specific soluble
receptors. Additionally, IL-1 is counteracted by the IL-1 receptor antagonist (IL-lra).
Synovia from OA patients abundantly produce IL-lra but it must be kept in mind that a
1000-fold excess of antagonist over IL-1 is needed to entirely block the IL-1 activity. This
makes it unlikely that the levels of IL-lra produced in the synovial lining are suffi-
cient to fully counteract IL-1 effects on chondrocytes.
Anabolic Factors
In addition to overproduction of catabolic factors, OA pathology may be linked to lack

of anabolic growth factors. IGF-I is one of the most potent anabolic factors for normal
cartilage. There is no proof that IGF levels are limited in synovial fluid of OA patients.
However, as a result of joint inflammation chondrocytes become non-responsive to IGF-I.
Chondrocytes in OA cartilage are reported to be less stimulated by IGF-I due to enhanced
levels of IGF binding proteins, limiting the anabolic action of IGF [102]. This lack of
response may be overcome with high levels of IGF and could justify therapeutic ap-
proaches with high doses of IGF-I [103].
Other anabolic factors are FGF and PDGF, which demonstrate stimulation of pro-
teoglycan synthesis above the IGF-I effect and may contribute to cartilage repair by
stimulation of chondrocyte proliferation. However, it was demonstrated that previous
incubation of chondrocytes to FGF stimulates the protease release after IL-1 expo-
sure [104]. PDGF also stimulated IL-1 dependent protease release, but inhibited IL-1
mediated reduction of proteoglycan synthesis [105,106]. These data indicate that a number
of factors not only stimulate repair but enhance breakdown as well.
The members of the TGF beta growth factor superfamily are considered as ana-
bolic factors due to their role in cartilage formation during embryogenesis. We have
shown that repeated local injection of TGFβ markedly upregulated chondrocyte pro-
teoglycan synthesis and induced osteophytes in murine knee joints [107,108]. Both
features could be an indication of a role of TGFβ in early OA. Osteophytes are char-
acteristic features in OA and it was observed that repeated local injection with another
growth factor, IGF-I, does not induce these hallmarks. Moreover, our group recently
showed that blocking of TGF beta activity in the synovial lining by adenoviral overex-
pression of the TGF beta inhibitor SMAD7 significantly inhibited osteophyte formation
and synovial fibrosis [109]. Finally, prolonged exposure to TGF beta results in proteogly-
cans loss close to the tidemark in femoral cartilage of the murine knee joints. Both
suboptimal and supraoptimal level of TGF β appear to result in cartilage pathology and
ultimately osteoarthritis.
We have found profound synovial expression of Connective Tissue Growth Fac-
tors (CTGF/CCN2) in murine experimental models of osteoarthritis. A role for CTGF
in the repair of cartilage damage in a full thickness defect model and an osteoarthritis
model have been reported, suggesting that CTGF functions in regeneration of articular
cartilage [110]. In contrast, we have found that in normal murine knee joints overex-
pression of CTGF induced transient synovial fibrosis, as shown by extracellular matrix
accumulation and an increase in the number of procollagen type I-expressing
cells [111]. The fibrotic tissue showed elevated mRNA levels of MMP-3, MMP-13,
ADAMTS-4, ADAMTS-5 and TIMP-1. CTGF overexpression led to proteoglycan
depletion in the articular cartilage. The observed damage is either a direct effect
of CTGF on the articular chondrocytes or mediated by factors released by the
CTGF-induced fibrotic tissue. The discrepancy between our study and the study of
Nakao et al could be based on the recent observation that CTGF can modulate TGF
beta action [90]. Wahab et al showed that CTGF enhances TGF beta action by suppres-
sion of SMAD7 transcription and induction of the transcription factor TIEG11.
SMAD7 is a known specific inhibitor of TGF beta signaling while TIEG11 is a known
repressor of SMAD7 transcription. It can be expected that in the defect model and the
OA model used by Nakao et al TGF beta was present and that CTGF has amplified the
anabolic effects of TGF beta on cartilage repair.
Bone Morphogenetic proteins have been shown to stimulate matrix production in
normal and OA chondrocytes and are considered to be suitable activators of chondro-
cytes anabolism [112–117]. It has been shown that BMP-2 expression in OA cartilage
co-localizes with newly synthesized type-II procollagen [117]. Moreover, expression of
BMP-2 favored the expression of the differentiated-chondrocyte specific type II colla-
gen isoform IIB [113]. This indicates that BMPs derived from OA synovial tissue can
contribute to stimulation of matrix synthesis by chondrocytes and this might contribute

to delayed cartilage degradation.
5. Final Remarks
OA research traditionally focuses on understanding the events within the degenerated

articular cartilage, the major tissue where OA is presumed to commence. However,
changes occurring in the synovial membrane are largely neglected. The synovial cap-
sule and, in particular, the synovial lining cells represent an important portion of the
joint as an organ and must also play an important role in its normal physiology. Of im-
portance are the inflammatory changes observed in the synovial membrane, which oc-
cur to some extent in all OA joints. This supports a pathogenetic role of OA synoviopa-
thy in OA cartilage degeneration and implies that targeting the inflammatory and de-
gradative activities of synoviocytes may be an interesting target for research and ther-
apy.
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Part IV
Imaging and Clinical Applications
IOS Press, 2007
XV
Magnetic Resonance Imaging of

Cartilage: New Imaging and Clinical
Approaches
Daniel R. THEDENS a,∗ , James A. MARTIN b and Douglas R. PEDERSEN b
a
Department of Radiology, The University of Iowa, Iowa City, IA
b
Department of Orthopaedics and Rehabilitation, The University of Iowa
Abstract. Magnetic resonance imaging (MRI) remains the imaging method of

choice for depicting the morphological changes associated with osteoarthritis (OA)
and other diseases of cartilage, but the early stages of OA are characterized by tissue
level changes which are not evident with standard MRI protocols. Several emerging
MR-based techniques show promise for detecting changes in water, collagen, and
proteoglycans that are the hallmarks of cartilage degeneration. In this review, the
principles and application of several of these techniques, including T2 mapping,
T1ρ imaging, delayed gadolinium MRI of cartilage (dGEMRIC), and sodium MRI
are outlined and compared.
Keywords. Cartilage, osteoarthritis, magnetic resonance imaging, T1ρ imaging,

dGEMRIC, T2 mapping, sodium imaging
Introduction
As one of the most widespread causes of disability among adults, degenerative diseases
of articular cartilage such as osteoarthritis (OA) remain a significant public health con-
cern. OA can arise as a result of traumatic joint injury or may result from a general de-
generation of cartilage over time. The consequence of this cartilage breakdown is pain
and loss of motion in the affected joints. OA is generally a progressive disease. By the
time painful symptoms appear, the degenerative processes causing cartilage damage have
been long underway.
Currently available clinical imaging techniques such as magnetic resonance imaging
(MRI) provide superb depiction of cartilage anatomy and morphology noninvasively and
can readily show cartilage surface defects and thinning. But these imaging markers of
OA are also not exhibited until long after the onset of the disease process. The initial
steps in the disease process occur at the tissue level, and thus the lack of noninvasive
∗ Corresponding Author: Daniel R. Thedens, Department of Radiology, The University of Iowa, Iowa City,
IA 52242, E-mail: dan-thedens@uiowa.edu.

240 D.R. Thedens et al. / Magnetic Resonance Imaging of Cartilage
imaging methods that can detect such changes hampers effective diagnosis and treatment
of these conditions.
Among imaging modalities, magnetic resonance imaging (MRI) is one of the most
versatile as it can generate images sensitive to a wide range of intrinsic tissue parameters.
MRI is thus not limited to depictions of anatomical features, but can show changes in
functional and chemical characteristics as well. In the field of cartilage imaging specif-
ically, there are several emerging MRI-based methods that are showing promise for de-
tecting the tissue changes associated with the early stages of OA. The successful de-
velopment and clinical validation of such methods for assessing cartilage health would
create a valuable clinical tool for more effective diagnosis and treatment of progressive
cartilage diseases such as OA.
1. Cartilage Structure and Composition
Articular cartilage is a complex connective tissue consisting of a relatively small number

of cells (chondrocytes) that synthesize and maintain the extracellular matrix. The extra-
cellular matrix is composed primarily of water and macromolecules, including collagen
and proteoglycans. The collagen (type II being the most abundant component) acts as the
“scaffold” of the tissue, providing its shape and mechanical stability. The highly-charged
proteoglycans provide the ability of the tissue to take up water and swell, yielding its
cushioning and shock-absorbing properties. Cartilage is an avascular tissue, relying on
diffusion of required nutrients, which accounts for its poor ability to repair itself.
The primary purposes of articular cartilage are to provide a low friction surface over
which articulating bones can smoothly move and to act as a shock-absorbing cushion
for the joints. These functions depend on the tensile restraint provided by the collagen
fiber network and the osmotic pressure provided by proteoglycans. The precise compo-
sition of this extracellular matrix varies with depth [1,2] in a manner which affects the
distribution of stresses and strains in the tissue during loading. In the body the superfi-
cial zone experiences hydrostatic pressure, fluid flow, and tensile stress along with high
compressive strains. In response to this environment, chondrocytes appear to be flattened
and the region is relatively richer in collagen (type II) and poorer in proteoglycans [3,4].
Cells in the middle and deep zones experience more hydrostatic pressure but very min-
imal strain and fluid flow, resulting in synthesis and maintenance of large amounts of
glycosaminoglycan (GAG), uronic acid and type II collagen [5,6]. The superficial zone
has higher tensile modulus by a factor of 2, but lower compressive modulus by a factor
of 3 compared to the deep zone [2].
Healthy cartilage must be able to retain its mechanical stiffness and cushioning abil-
ities, which are in turn preserved by the integrity of the collagen matrix and proteoglycan
networks and maintained by functioning chondrocytes. A disruption in any of these com-
ponents can begin a cascade of degenerative processes. Deterioration of the articular car-
tilage extracellular matrix results in progressive changes in cellular response and compo-
sition of the tissue. The onset of degeneration charts a path from microscopic biochem-
ical events (proteoglycan loss, cell senescence, cell apoptosis and collagen degradation)
to irreversible morphological changes (cartilage thinning, clefts, lesions and fibrillations)
resulting in total disruption of the cartilage.
D.R. Thedens et al. / Magnetic Resonance Imaging of Cartilage 241
OA is one of the most common degenerative diseases of cartilage. OA may manifest

as a secondary effect of traumatic joint injury or other abnormal joint loading condition,
or it may arise from normal “wear and tear” on joints with no other primary cause. In ei-
ther case, the general progression of the disease is broadly similar as outlined above. The
initial changes in OA occur at the microscopic level with changes in collagen and macro-
molecular components of the extracellular matrix. These changes compromise the car-
tilage mechanical properties, leading to further disruption until the characteristic symp-
toms of pain and stiffness are experienced, and radiologic evidence such as joint space
narrowing and cartilage thinning appear. While these significant effects of cartilage de-
generation are readily appreciated, they are experienced in the later disease stages after
the responsible microscopic processes have long been underway. For effective diagnosis
and treatment, it is imperative to intervene at the onset of the disease to restrict if not re-
verse its progression. Noninvasive diagnostic procedures sensitive to these early changes
in tissue microstructure are needed if such early identification and treatment are to be
made possible.
2. MRI of Cartilage: Morphology
The exquisite soft-tissue contrast of MRI and the multiplicity of contrast mechanisms
available in a single exam have established MRI as the method of choice for clinical
imaging of cartilage and joint anatomy. A typical MRI protocol will acquire proton
density, T1, T2, and fat-suppressed images with rapid spin-echo-based techniques such
as fast spin-echo (FSE), which can give a comprehensive picture of the morphologic
changes associated with injury and subsequent degenerative processes [7]. T1-weighted
imaging provides visible distinction between cartilage and subchondral bone, although
contrast between cartilage and fluid is poor. For cartilage, fat suppressed T2-weighted
imaging is particularly valuable and routinely acquired as it yields good contrast with
synovial fluid at the cartilage surface, permitting identification of cartilage surface le-
sions. FSE acquisitions primarily produce two-dimensional (2D) data sets, with rela-
tively coarse resolution in the slice direction, limiting the possibilities for multiplanar
reformatting of the curved surfaces. Three-dimensional (3D) imaging with gradient echo
(GRE) acquisitions, particularly with fat suppression via water-selective excitation, can
generate isotropic image volumes with high cartilage signal relative to surrounding tis-
sues. The thinner slices and reformatting capabilities result in high sensitivity and speci-
ficity for identification of cartilage defects and the potential for accurate quantitative vol-
ume measurements [8]. The cost is a relatively lengthy scan time (10 or more minutes)
and the possibility of motion artifacts during that time.
At present, clinical practice for noninvasively assessing cartilage condition with
MRI in OA is primarily based on morphological characteristics and parameters such as
cartilage thickness and the appearance and identification of significant defects. Quan-
titative measures of cartilage volume are also possible with MRI. As noted previously,
however, these gross changes in cartilage appearance are seen long after the degenera-
tive processes have begun. Current standard imaging methods are thus not well suited to
identification of the early markers of OA. The lack of quantitative measures of cartilage
condition hampers treatment planning and meaningful and timely assessment of joint
injuries.
3. Functional MRI of Cartilage
Ultimately, it is biomechanical properties of cartilage that determine its functional state.

Direct assessment of biomechanical parameters is not presently possible with a noninva-
sive exam, and so there is no effective way to objectively assess this element of cartilage
health noninvasively. The next best alternative to determining biomechanical characteris-
tics of cartilage is to base assessments on the related biochemical composition of the ex-
tracellular matrix, especially in terms of proteoglycan and collagen content, the primary
components responsible for the mechanical strength of healthy cartilage.
The early stages of OA are characterized by numerous changes in the extracellular
matrix at the microscopic level, some of which may be detectable by imaging. As out-
lined previously, disruption of the organization of the collagen fiber network is initially
seen along with an increase in water content, causing the cartilage to swell. Proteogly-
cans are subsequently lost from the matrix as the disease progresses, increasing suscepti-
bility to mechanical stresses and continuing degradation. The magnetic resonance (MR)
signal characteristics of cartilage are affected by all three of the main components of the
extracellular matrix (water, proteoglycans, and collagen) that undergo changes in early
OA. The water content of tissue has a direct effect on T2 relaxation parameters. The
structure and orientation of collagen fibers further influences T2 relaxation in cartilage.
In particular, the regular structure of collagen restricts the motion of water molecules
and increases the interactions that are the basis of T2 relaxation. Molecular interactions
between proteoglycans (being highly charged macromolecules) and water or other mole-
cules also influence the MR behavior and thus the qualitative appearance and quantita-
tive relaxation characteristics of the magnetic resonance signal. Given the ability to de-
sign MRI acquisition techniques that are sensitive to and quantitative in these tissue re-
laxation changes, MRI-based methods show considerable promise for early detection of
these characteristic biochemical markers of OA.
Numerous MR-based methods have been proposed to noninvasively display and
quantitate changes in the composition and integrity of cartilage extracellular matrix in
vivo, including T2 MRI, T1ρ MRI, delayed Gadolinium Enhanced MRI of Cartilage
(dGEMRIC), and sodium MRI. In brief, quantitative mapping of T2 relaxation times is
sensitive to several processes involved in cartilage degeneration, including water content
and derangements in collagen fibril orientation. T1ρ MRI is most sensitive to interac-
tions between tissue water and macromolecules and thus correlates strongly with pro-
teoglycan content. dGEMRIC introduces an exogenous charged contrast agent that alters
T1 relaxation properties and whose distribution depends on the local concentration of
charged GAG molecules. Sodium MRI similarly probes the charge density (and thus con-
centration) of GAG by directly measuring the presence of the balancing concentration
of sodium in the tissue. Each of these methods has significant advantages and disadvan-
tages in terms of their sensitivity and specificity to relevant biochemical properties and
their implementation and practicality for in vivo and clinical work. Broadly, the methods
are ordered above by their specificity to relevant biochemical changes in cartilage (from
least to most specific) and their ease of translation to a clinical setting (from easiest to
most difficult).
3.1. T2 Mapping
Qualitative and quantitative T2-based MRI acquisitions are universally available and ma-
ture techniques in diverse areas of clinical MRI. The T2 relaxation times of many nor-
mal and diseased tissues are widely studied and T2-weighted images are routinely ac-
quired as part of virtually all musculoskeletal MRI protocols. The quantification of T2
relaxation times is also a readily available technique on most clinical scanners, produc-
ing pixel-by-pixel maps of T2 relaxation times and color maps that can highlight areas
of abnormality. Generation of this information requires only a standard spin-echo-based
acquisition protocol and fundamental data analysis tools for nonlinear curve fitting of
the variation of local signal intensity with the change in acquisition parameters (the echo
time or TE).
As noted previously, the T2 relaxation time can be altered by changes in water con-
tent and collagen fiber concentration, orientation, and condition. Specifically, degrada-
tion of the collagen matrix permits greater mobility of the water component of the extra-
cellular matrix, which results in longer T2 relaxation times. Several in vitro studies have
confirmed this effect in animal and cadaveric models, demonstrating a strong correlation
between T2 changes and histologic changes [9]. In contrast, proteoglycan content does
not appear to have a strong direct influence on T2 relaxation time [10,11].
Clinical studies have also begun to appear that show significant changes in T2 re-
laxation times in areas of cartilage degeneration. These changes may be seen in subjects
with OA in the absence of volume and thickness changes, indicating sensitivity to the
biochemical changes that are hallmarks of early OA [12]. The changes were significant
between subjects with and without OA, but did not demonstrate graded differences with
the severity of OA. Dependencies on the age of subject have also been observed [13]
suggesting sensitivity to the general deterioration of the cartilage over time.
The observed changes in T2 relaxation time in these studies were significant but rel-
atively small, and may result from multiple mechanisms including water content changes
and collagen concentration and orientation, with the latter having the greatest influence.
In particular, the dependence on collagen fiber orientation gives rise to spatial variations
in T2 relaxation within healthy cartilage which can confound the detection of abnormal-
ities. Fiber orientation effects can be diminished in in vitro studies, but may be problem-
atic when interpreting results from clinical exams. Thus, while T2 changes in cartilage
are seen in degenerative processes and may often be diagnostically valuable prior to vis-
ible morphologic changes, it can be unclear which of these mechanisms is the predomi-
nant cause, potentially limiting its sensitivity and specificity.
Nevertheless, T2 mapping may provide valuable information on the biochemical and
biomechanical properties of cartilage and is deserving of continued study. Its primary
(though complex) dependence on collagen condition and water content rather than pro-
teoglycans means it may also serve as a useful adjunct to one or more of the imaging
techniques described below, which are predominantly sensitive to proteoglycan content.
3.2. T1ρ Imaging
T1ρ describes an alternative relaxation characteristic in MR experiments, “spin-lattice re-

laxation in the rotating frame.” As the name implies, this relaxation mechanism is related
to T1 relaxation, but is measured in the presence of an externally applied radiofrequency
Figure 1. T1ρ pulse sequence. The T1ρ magnetization preparation step consists of a 90◦ tip-down, spin-lock
RF for variable T1ρ weighting, a 90◦ tip-up, and crusher gradient to eliminate any residual transverse magne-
tization. A standard fast spin echo pulse sequence follows for image acquisition.
(RF) magnetic field (the B1 field) applied in a direction perpendicular to the large main
magnetic field (the B0 field). This is the same type of magnetic field used to perform the
excitation that generates the MR signal. T1ρ relaxation has been experimentally shown
to be sensitive to changes in proteoglycan content both in tissue and in vivo studies, and
thus shows considerable promise for detecting some of the early biochemical changes
associated with OA both in tissue and in vivo.
In a common type of T1ρ acquisition, a standard 90◦ pulse is applied to tip the mag-
netization into the transverse plane, as shown in Fig. 1. Next, a constant low-power RF
pulse (the spin-lock pulse) is applied for some relatively long duration (several millisec-
onds). This pulse counteracts the usual effects of the interactions that cause T2 signal
decay, but other relaxation mechanisms similar to T1 relaxation still yield some char-
acteristic signal loss. Hence, the T1ρ relaxation time is necessarily longer than the T2
relaxation time (the signal loss that would occur in the absence of the spin-lock pulse).
At the completion of the spin-lock RF pulse, a –90◦ pulse restores the remaining magne-
tization back to the longitudinal (B0 ) axis. The signal available for a subsequent image
acquisition will be modulated by the signal loss during the spin-lock pulse due to T1ρ
relaxation.
The methodology for generating a quantitative measure of the T1ρ relaxation time
for a given tissue is then conceptually similar to that for quantitation of T2 relaxation
time. A series of images are acquired, each utilizing a spin-lock pulse of identical ampli-
tude but different duration, producing a sequence of signal intensities dependent on the
pulse duration. A nonlinear (decaying exponential) curve fit of the resulting signals to
the applied pulse durations provides a best-fit estimate of the relaxation parameter. Such
an analysis can be carried out on a pixel-by-pixel basis to generate a T1ρ map. The T1ρ
sensitizing step acts as a magnetization preparation step which can be applied to most
any type of subsequent imaging acquisition, such as FSE [14] or 3D GRE [15].
The application of T1ρ imaging to cartilage is predicated on its sensitivity to the
low-frequency interactions among water and macromolecular protons that contribute to
the relaxation (i.e. signal decay). In the case of cartilage, the macromolecules of interest
are proteoglycans. A depletion of proteoglycan content in cartilage will reduce these
interactions and signal decay and yield a corresponding increase in the measured T1ρ
relaxation time. While the complete set of dependencies of T1ρ relaxation in cartilage
is not fully known, experimental models with enzymatically degraded cartilage have
shown strong correlation of T1ρ with proteoglycan content and minimal dependency of
T1ρ on collagen content [16], though some experiments have noted an influence from
interactions due to collagen fibril orientation [17].
As it relies only on a set of magnetization preparation pulses as described above,
T1ρ MRI is a completely noninvasive method for generating quantitative information
about cartilage proteoglycan content without the need for special hardware or exogenous
agents [18,19], and can therefore be implemented on any clinical scanner with a variety
of imaging pulse sequences [20,21]. The primary factor limiting resolution and coverage
at high field strength is RF energy deposition, arising from the low-power but relatively
lengthy spin-lock pulse. These energy deposition factors scale as the square of the field
strength, so much of the initial clinically-oriented work has been done at a field strength
of 1.5 T. However, advances in parallel imaging capabilities are rapidly eliminating these
limitations [22], along with the use of extended readout techniques such as spiral and
echo-planar imaging [20]. As described, the image analysis techniques required to quan-
tify T1ρ are very similar to those required for T2 measurements and are straightforward
to implement at the scanner console.
The close ties between proteoglycan content and cartilage function make T1ρ MRI
a much more direct and valuable indicator of cartilage health and treatment efficacy than
purely anatomic imaging. Because of the dependence on proton exchange between water
and proteoglycans, quantifying proteoglycan depletion associated with early OA devel-
opment with T1ρ is a more discriminatory cartilage assessment than T2 mapping [23].
The changes in T1ρ parameters are of a considerably greater magnitude, offering the
likelihood of earlier and greater sensitivity to degenerative processes [24], though both
may be valuable as sources of complementary information. Thus, T1ρ MRI pulse se-
quences may be able to detect cartilage compromise occurring both shortly after acute
injury such as anterior cruciate ligament (ACL) rupture as well as early stages of de-
generative changes arising from OA. Both in vitro and in vivo studies suggest that the
relationship between T1ρ and proteoglycan content is sufficiently strong and sensitive to
detect harbingers of OA while being simple to implement in clinical settings.
Sample images from a recent in vitro study of the ability of T1ρ to assess changes
in proteoglycan content are shown in Fig. 2. A set of fresh cartilage cylindrical explants
taken from the tibial plateau of amputees with no known history of OA underwent three
one-hour episodes of cyclic mechanical loading over a period of 12 days in order to in-
duce proteoglycan depletion. The corresponding T1ρ maps derived from the subsequent
imaging experiments showed increased T1ρ relaxation time in explants with the high-
est loading and greatest loss of proteoglycans, as confirmed by biochemical assay [25].
Figure 3 shows the relationship between T1ρ relaxation and proteoglycan content for all
ten samples used in the study, demonstrating a highly significant correlation between the
imaging and biochemical assay, indicating the sensitivity of the T1ρ technique to this
marker of cartilage condition.
T1ρ imaging has seen initial clinical use for early OA cartilage degeneration [19,26],
and its ability to detect arthroscopically confirmed cartilage abnormalities not otherwise
seen on morphologic imaging has been recently demonstrated [27]. Figure 4 shows a
T1ρ map of cartilage along with T2-weighted imagery from a patient with an acute ACL
injury. The continued development and validation of T1ρ MRI as an objective and quan-
Figure 2. Cartilage samples and corresponding T1ρ maps for cartilage explants from tibial plateau of a
51-year-old male. Images were acquired at a magnetic field strength of 4.7 T. An increase in T1ρ corresponds
to a decrease proteoglycan concentration ([PG]).
Figure 3. Comparison of computed T1ρ relaxation time and GAG content from biochemical assay in ten
cartilage sample explants. Proteoglycan depletion was accomplished by a multiday schedule of mechanical
loading of the explants.
titative measurement standard of cartilage condition may provide a new and clinically
viable tool to improve the assessment and understanding of acute injury, diagnosis, and
treatment.
3.3. dGEMRIC
Both T2 and T1ρ mapping depend only on intrinsic tissue relaxation characteristics to de-
rive information as to water and collagen (T2) or proteoglycan content (T1ρ) in cartilage.
An alternative method to improve the specificity of measurements of biochemical com-
position is to introduce and detect the distribution of an exogenous contrast agent with a
dependency on tissue characteristics. In cartilage, GAG (components of proteoglycans)
acts as a source of fixed charge density in the tissue. Delayed Gadolinium Enhanced MRI
of Cartilage (dGEMRIC) is an imaging technique that utilizes a widely available anionic
MRI contrast agent, gadopentate dimeglumine (Gd-DTPA2− , commercially available as
Magnevist, Berlex Laboratories, Wayne, NJ). Gadolinium-based contrast agents reduce
Figure 4. In vivo T1ρ acquisition from a patient with acute ACL injury. A color-coded map of T1ρ relaxation
time is overlaid on a fat-suppressed T2-weighted image in the sagittal plane. The T2-weighted image shows
evidence of bone bruise, while the lengthened T1ρ relaxation of the underlying cartilage is suggestive of local
proteoglycan loss (decreased [PG]).
the T1 relaxation time of the tissues that they penetrate in proportion to their concentra-
tion in the tissue. This contrast agent is introduced intravenously and allowed to penetrate
the cartilage (usually helped along by a period of light exercise after administration).
Because of the negative fixed charge density of the GAG (which is linked to core
proteins to form proteoglycans) and the like charge of the contrast agent, the concen-
tration of the contrast agent that equilibrates in the cartilage will be inversely related to
the GAG concentration of the tissue [28]. As noted, the T1 relaxation time measured in
the tissue will be dependent on the normal (without contrast) T1 relaxation time of the
cartilage and the concentration of the Gd-DTPA2− agent that penetrated the joint. The
Gd-DTPA2− concentration can be quantified through measurement of T1 relaxation time
with standard imaging and processing techniques and application of a physical model
(such as an electrochemical equilibrium model) to quantify the fixed charged density
to yield a marker of GAG concentration and proteoglycan loss. Since the T1 relaxation
time of cartilage does not show a dependency on proteoglycan content in the absence
of contrast agent, the post-contrast T1 measurements provide a direct marker for the in
vivo GAG concentration. As with T2 and T1ρ mapping, the results can be presented both
qualitatively as a color-coded image overlay as well as quantitatively in terms of local
and regional relaxation parameters implicitly or explicitly linked to tissue composition.
The dGEMRIC protocol is currently the most frequently used and widely vali-
dated of the emerging methods for in vivo characterization of proteoglycan loss. Nu-
merous in vivo and in vitro studies suggest dGEMRIC is the most specific for GAG
concentrations among the described methods, demonstrating very direct correlations be-
tween dGEMRIC measurements and GAG concentration and even mechanical proper-
ties [29,30]. Figure 5 shows two samples of human cartilage comparing GAG concentra-
tion as derived from T1 measurements of the contrast-infused cartilage with histological
preparation [31].
When logistically feasible, dGEMRIC can be expected to yield more specific pro-
teoglycan loss information than T1ρ imaging. This specificity comes at a cost of con-
siderable complexity in implementing the exam in a clinical setting. The usual imaging
Figure 5. Comparison of quantitative GAG concentration maps with histological analysis (toluidine blue stain-
ing) in human cartilage samples. The image-derived GAG concentrations show excellent correspondence with
the histological standard. Courtesy of Martha Gray, Ph.D. (MIT/Harvard Division of Health Sciences and
Technology).
protocol begins with subjects receiving an intravenous injection of contrast agent, fol-
lowed by a period of mild exercise to distribute the agent. Image acquisition then takes
place 1–2 hours after injection. In some cases a pre-contrast exam is also desirable to de-
termine subject-specific baseline T1 measurements for robust quantitative measurement
of relaxation changes. Thus, while dGEMRIC shows strong abilities for accurate mea-
surements of proteoglycan depletion, the rigors of the examination require considerable
planning and logistical coordination (these issues have been considered and discussed
in Burstein et al. [32]). It requires some minimal invasiveness and is thus somewhat
more cumbersome to implement clinically than the previously described techniques, but
it yields results that are most directly correlated to proteoglycan content.
Motivated by the unique quantitative information that can be derived, dGEMRIC
has been successfully applied in several subject populations. Figure 6 shows an in vivo
example of a GAG concentration map generated for assessment of cartilage in the knee
joint [31]. Examples of its application include assessing GAG content in subjects with
differing levels of physical activity [33], subjects with cartilage changes confirmed on
arthroscopy [34], and correlation with pain and severity in early OA subjects with hip
dysplasia [35]. Comparisons of dGEMRIC quantification and a variety of disease state
parameters as determined radiographically have also been carried out [36]. In injury
models, dGEMRIC has been applied to assessment of cartilage and synovial fluid GAG
content in subjects with acute ACL injury [37] and in a case report of GAG changes in
posterior cruciate injury [38].
3.4. Sodium MRI
Sodium (23 Na) NMR spectroscopy has long been applied to investigate proteoglycan
depletion in cartilage [39]. The principle for this method is that loss of proteoglycans
with their negatively charged GAG causes a reduction of fixed charge density (FCD) and
a loss of charge-balancing sodium ions, as well as a change in sodium MR relaxation
parameters. While initially applied to (non-imaging) spectroscopic measurements, these
changes can also be seen and quantified from sodium-based MRI of tissue samples as
Figure 6. Example of in vivo mapping of GAG distribution in the human knee joint. The image-derived GAG
concentration is overlaid on the standard T1-weighted acquisition in sagittal and coronal planes, permitting both
qualitative and quantitative assessment of cartilage condition. Courtesy of Martha Gray, Ph.D. (MIT/Harvard
Division of Health Sciences and Technology). Reprinted from: Burstein D Bashir A, and Gray ML: MRI
techniques in early stages of cartilage disease. Invest Radiol, 35:634, 2000.
well [40]. The referenced study demonstrated changes in image characteristics of 23 Na

in cartilage with enzymatically depleted proteoglycan content.
Because the signal used for image formation arises solely from the presence of
sodium, 23 Na imaging presents a very direct correlation to proteoglycan content and
changes. Feasibility studies have also demonstrated that this technique can be success-
fully applied in vivo [41,42]. However, as sodium MRI is not proton based, special imag-
ing coils and signal processing hardware is required, both of which are rare in clinical
environments. The signal strength of 23 Na is much smaller than that in standard proton
MRI, and the T2 relaxation time is short, limiting the resolution, signal-to-noise ratio
(SNR), and image quality that can be achieved. Sodium MRI must thus be regarded as an
esoteric research-oriented application at present and is unlikely to translate to standard
clinical scanners in the near future. Nevertheless, its unique ability to directly measure
fixed charge density may be valuable for nondestructive evaluations in benchtop studies
and as a validation technique at well-equipped research sites.
3.5. Diffusion Imaging
Diffusion-weighted imaging (DWI) and diffusion-tensor imaging (DTI) apply extra

pulses that create signal differences based on the rate of diffusion in the tissue of inter-
est. The acquisition may be made sensitive to the local diffusion coefficient or to diffu-
sion anisotropy, which in turn reflects variations in tissue microstructure and potentially
early changes in OA. While diffusion-based techniques have been successfully applied
in tissue samples [43–45], the relatively short T2 relaxation time of cartilage necessi-
tates lengthy exam times for the needed resolution in vivo. This in turn exacerbates the
already high sensitivity to motion inherent in diffusion weighted imaging. Feasibility of
the technical aspects has been demonstrated in vivo [46], but not widely applied.
4. Discussion
As seen in the foregoing description, MRI is a flexible imaging modality capable of

providing numerous mechanisms to characterize cartilage tissue in vivo. T2 mapping,
T1ρ imaging, dGEMRIC, and 23 Na sodium imaging have all demonstrated sensitivity to
important biomarkers and feasibility in in vivo applications. At present, however, these
tools still need more widespread dissemination and more extensive validation in clinical
populations before they can become routinely available as part of the clinical MRI tool-
box. Nonetheless, the initial studies with all the methods suggest several observations
regarding their future use.
As a mature and already widely available tool in other application areas, T2 map-
ping has the most immediate potential to have a clinical impact. The majority of high
field scanners already have the necessary pulse sequences and analysis tools to gener-
ate these maps directly on the scanner. However, initial studies with T2 mapping of car-
tilage have shown it to depend on numerous tissue factors (including collagen content
and orientation as well as water content) that may be difficult to isolate into a single
measure of cartilage condition. Its higher sensitivity to these factors compared to T1ρ
and dGEMRIC may merit a place for T2 mapping as an adjunct to one of these other
techniques, however.
The dGEMRIC technique is highly attractive due to its specific sensitivity to GAG
via the use of a exogenous charged contrast agent. The image acquisition protocol (quan-
titative T1 measurement using inversion recovery pulse sequences) is also one that is uni-
versally available at the present time. While the use of contrast material may constitute
a (minimally) invasive step, the widespread use of MR contrast media in other applica-
tions suggests this is a relatively minor burden. The exercise and time delay requirements
between contrast administration and image acquisition is the most challenging aspect in
translating this technique to the clinical realm.
T1ρ MRI represents a promising “middle ground” between the ease of application
of T2 mapping and the specificity to GAG content of dGEMRIC. If properly validated,
T1ρ MRI may become the most preferred of the methods as it can generate much of the
same information on proteoglycan content as dGEMRIC and sodium MRI, but can be
applied on standard MRI hardware and has less complex protocol requirements. It does
require specialized software presently only available at research sites, though it has been
implemented on systems from all major vendors.
Sodium MRI may have a niche in tissue and research studies as a validating tool but
is unlikely to achieve a place in the clinical imaging toolbox because of its demanding
hardware requirements. Although diffusion imaging is widespread in other MRI applica-
tions, the obstacles to clinical imaging of cartilage remain to be surmounted, and it is too
early to determine if it will have a place for cartilage imaging and tissue characterization.
5. Conclusion
The appearance of techniques for robust and noninvasive assessments of cartilage com-
position, structure, and function would be invaluable for all phases of the disease process
in OA. Such information may yield detection of early OA markers before morphologic
changes or physical symptoms occur. It could also direct treatment options and assess
recovery in later stages of OA development. There is therefore a great need to study

and validate these noninvasive techniques in order to bring such tools toward fruition as
clinical tools.
Acknowledgments
Thanks to Martha Gray, Ph.D. (Edward Hood Taplin Professor of Electrical and Medical
Engineering and Director, MIT/Harvard Division of Health Sciences and Technology)
and collaborators for images taken from her Elizabeth Winston Lanier Award lecture
delivered at the 53rd annual meeting of the Orthopaedic Research Society.
Supported by NIH grant P50AR048939.
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IOS Press, 2007
XVI
Multimodality of Microscopy Imaging

Applied to Cartilage Tissue Engineering
D. DUMAS a, B. RIQUELME b, E. WERKMEISTER a, N.D. ISLA a and J.F. STOLTZ a
a
Groupe de Mécanique et Ingénierie Cellulaire et Tissulaire. – UMR CNRS 7563
LEMTA et IFR 111 CNRS –UHP-INPL-CHU
b
Faculté de Médecine, Nancy-Université. 54505 Vandoeuvre-lès-Nancy, France
Facultad. De Cs. Bioquímicas y Farmacéuticas,
Universidad Nacional de Rosario, Argentina
riquelme@ifir.edu.ar
Abstract. As a comparatively non-destructive imaging technique into living

specimens, fluorescence microscopy has a number of strong advantages over
alternative imaging modalities (X-ray, MRI, CT-scan, arthro-scan, etc.). The lim-
ited analysis in thick tissue has given rise to the development of other techniques,
multiphoton excitation microscopy in particular. A need for increased sensitivity
and resolution has been driving the development of new sophisticated fluorescence
techniques based on microscopies to study: the tissue microstructure in situ
(CLSM, SHG) on deeper thick sections of tissue (Multiphoton), molecular diffu-
sion (FRAP, FCS) with fluorescent protein variants and molecular interaction
(spectral, FRET, FLIM). In this paper, we have considered developments based on
near infrared (NIR) femtosecond excitation in the imaging of articular tissue and
discussed the technical limitations and perspectives.
Keywords. Multiphoton Microscopy, Second-harmonic Generation, Fluorescence

Correlation Spectroscopy, Fluorescence Lifetime Imaging, Spectral Imaging
Introduction
Osteoarthritis (OA) is a degenerative disease of the joint characterized by fibrillation

and erosion in cartilage, chondrocyte proliferation and osteophyte formation at the joint
margins and sclerosis of subchondral bone. Articular cartilage damage and eventual
loss is the primary pathological change and the capacity of articular cartilage to regen-
erate is very limited. Numerous studies have shown that mechanical load can affect the
biosynthesis, turnover and structure of the macromolecules produced by the chondro-
cytes [1]. Tissue engineering of articular cartilage could represent an interesting path-
way to solve the complex problem of cartilage regeneration. Several ways of biothera-
pies have been developed to induce regeneration, either by mobilization of native cells
under chemical and biomechanical conditions for the neosynthesis of a replacement
matrix (growth factors, cytokines, gene); either by delivery of viable cells (chondro-
cytes or mesenchymal stem cells). There is a growing interest for optical imaging
D. Dumas et al. / Multimodality of Microscopy Imaging Applied to Cartilage Tissue Engineering 255
methods in challenge due to the high level of autofluorescence on native unstained

samples for cartilage tissue engineering [2].
All current imaging modalities derive information using ultrasound methods or
electromagnetic radiation (nuclear, optical or magnetic resonance). The resolution and
sensitivity of classical imaging techniques based on conventional radiography (X-ray,
MRI, CT-scan, arthro-scan, etc…) are too low to collect any information about a spe-
cific cellular structure into a cellular scale (micrometer). Actually, clinical non-invasive
techniques such as magnetic resonance imaging [3] and ultrasound lack resolution
needed to distinguish complex structures of articular cartilage [4]. The minimally de-
structive nature of arthroscopy is very useful but this technique which requires anesthe-
sia and surgery only provides information about cartilage structure at articular sur-
faces [5]. With a low ability to distinguish among biological constituents, Optical Co-
herence Tomography (OCT) provides full thickness, high resolution (around 10 µm),
cross-sectional images of cartilage [6]. Even if these clinical non-invasive (optical bi-
opsy) methods offer promise in the early detection of cartilage degeneration, they are
mainly used to make a precise inventory of articular lesions in osteoarthritis (slow deg-
radation of cartilage) and to investigate the superficial and deep layers of cartilage.
Despite a considerable level of development, the major limitation of clinical techniques
remains that the field of view is strictly limited to the surface and restrict their applica-
bility to the study of large articular disease.
By using visible and near-infrared windows of the electromagnetic spectrum, the
increase in lateral and spatial resolutions is one of the major targets of research and
development in the field of optical microscopy applied to living tissue. But it was
mainly in the past decade that it became possible to shift from whole-specimen analysis
to smaller volumes, through microscopy techniques (far and near-field microscopies,
fluorescence correlation FCS and FRAP, etc.), down to almost single-molecule explo-
ration or interaction (FRET) [7]. In this situation, conventional light and fluorescence
microscopies have shown in situ particular application for improving the ability for the
characterization of organization within the structure of the extracellular matrix of carti-
lage. Among the physical methods available to investigate biological media, fluores-
cence, with its high analytical sensitivity and resolutions (spectral, spatial, temporal,
order) offers interesting possibilities for cell or tissue biological analysis [8].
At this time, fluorescence microscopy is the most rapidly expanding microscopy
method in both the biological and medical sciences. Molecular emission spectroscopic
techniques (fluorescence, phosphorescence, chemo- and bio-luminescence), which cor-
respond to excitation deactivation processes, are now widely used to study variably
complex media such cells or tissues. Fluorescence microscopy is exempt from a num-
ber of constraints normally attached to standard techniques (probe concentration, low
cellular density, exploration and display at the cellular scale). Furthermore, it is the
only methods of investigation currently available with a high enough resolution to
specify the distribution of the fluorescent probe, because it collects fluorescence signals
emitted at the probe incorporation site. After staining, the ability to image a specific
biological target or predictive molecular markers that could be associated with cartilage
disease will represent a complementary approach to the anatomical imaging performed
in clinical practice. There is a need for sensitive molecular imaging techniques dedi-
cated to articular tissue autofluorescence which can be resolved by using new organic
or inorganic fluorescent probes. Quantum Dots (QDots) are very small inorganic
fluorophores (< 10 nm) based on a cadmium selenide semi-conductor core surrounded
by a zinc sulphide shell. This core-shell complex can be finally used for immunohisto-
256 D. Dumas et al. / Multimodality of Microscopy Imaging Applied to Cartilage Tissue Engineering
chemistry (after antibodies or streptavidin coupling) with a great interest because they
have several unique optical properties (single excitation wavelength for multiplexing,
high level of brightness, photobleaching reduced, multilabeling available due to their
narrow and symmetric emission). In the same way, multicolor imaging of proteins to
study their function in the context of the living cells has become a fundamental
technique in cell biology during the last decade. Natural fluorescent proteins (GFP,
CFP, YFP, DsRed and variants) are also widely used as probes thanks to their
brightness, resistance to photobleaching, and potential to be fused with virtually any
gene product. For instance, these chimeric proteins have been used to determine a
novel chondroprotective modality by overexpressing HSP70 in chondrocytes by using
a vector carrying HSP70/GFP. To distinguish between implanted and host-derivate
cells, in diseased states such as osteoarthritis cartilage matrix, Grossin at al developed a
vector carrying HSP70/GFP, and transduced chondrocytes were thus more resistant to
cell death mainly due to an increase in apoptosis [9].
Scanning confocal microscopy has been confronted with the problem of using in-
tact living cells and phototoxicity for imaging hard tissues. Most tissues have reduced
absorption in the near-infrared part of the spectrum and nonlinear infra-red optical mi-
croscopy can exploit the “optical window” at 700–1000 nm. Multiphoton excitation is
mainly characterized by less light scattering and deeper penetration of the light into the
sample and usually entails less damage from photobleaching. This nonlinear optical
microscopy allows living cells to be probed in real time under physiological conditions
within the intact microenvironment and is beginning to emerge as a powerful contrast
mechanism in combination with second and third harmonic generation imaging (SHG,
THG) [10].
In this part, we have considered developments based on near infrared (NIR) femto-
second excitation in the imaging of articular tissue and discussed the technical limita-
tions and perspectives.
1. Optical Scanning Microscopy/Deconvolution
Conventional fluorescence microscopy was primarily used to determine the spatial dis-
tribution of fluorescent probes. One of the major limitations of fluorescence micros-
copy is the progressive bleaching of the fluorochrome during prolonged exposure. Un-
fortunately, when focusing at a particular depth within a transparent fluorescently la-
belled 3D specimens, fluorochromes molecules throughout the whole of its thickness
are excited. The diffraction light disrupts the image which reduces contrast and spatial
resolution, allowing few strategies to overcome these limitations. Contrary to confocal
microscopy, each acquired 2D image (X, Y) contains data of its focal plane (clear area)
and of all other planes (blurred area). The clear area of the image corresponds to data
pertaining to the focal plane, the blurred part of the image originating from data per-
taining to all other planes (Fig. 1). These interference data can be removed and images
run through a computationally intensive set of algorithms that permit reassigning the
photons from adjacent planes to the focal plane. These techniques are oriented towards
modeling degradation phenomena (defocusing, noise) and to the application of reverse
procedures (mathematical reversion or iterative deconvolution so as to obtain an ap-
proximation of the original scene (an estimate of the subject by using the transfer func-
tion image). The optical transfer function reflects the way in which a punctual source is
deformed when displayed via an optical system and determines the impulse response of
Figure 1. Before (A) and after (B) deconvolution process of isolated chondrocyte imaging with the appropri-
ate Point Spread Function (Green Fluorescent Protein, GFP). CellScan EPR TM optical scanning acquisition
system (IPLab-Scanalytics, Billerica, USA) equipped with a 12 bit CCD camera (Princeton Instruments).
Z spacing: 0.25 µm. 60X/w1.2 NA PSF.
the optical system or point-spread function [11]. Then using a detailed knowledge of
the degradation introduced by the optical system (convolution), images are corrected
by computer deconvolution image processing (Fig. 1).
2. Confocal Laser Scanning Microscope
The optical geometry of Confocal Laser Scanning Microscopy (CLSM) demonstrates

its undeniable advantage on conventional fluorescence microscopy by segregating the
planes outside the focussing plane. One of the first review in cartilage research using
digital imaging CLSM was published in 1997 [12]. Confocal fluorescence microscopy
has the ability to control the depth of focus by spatial filtering and as a result, it allows
for collection of a series of optical sections throughout the sample, with reducing back-
ground fluorescence originating from sections that are away from the focal plane. The
benefits of confocal microscopy are 1) Increased effective resolution 2) Improved sig-
nal to noise ratio 3) Clear examination of thick specimens 4) Depth perception in
Z-sectioned images 5) Magnification can be adjusted electronically 6) Reduced blur-
ring of the image from light scattering. The improvements were essentially aimed at
offering solutions to the problems posed by CLSM 1) by multiple marking in fluores-
cence (cross-talking) 2) the too low scanning rate to catch rapid biological events,
3) the fast photodegradation of the fluorescent probe as well as cytotoxicity (under UV
light mainly) 4) the low quantum efficiency of detector 5) the fluorescence emission
restricted by optical configuration (pinhole). In contrast to cartilage which is highly
scattering, a culture transparent medium such as alginate matrix, permits reflectance or
transmitted mode imaging without the need for tissue fixation or exogenous dyes
(Fig. 2). By this way, Yansen and al have followed the time-dependent formation of the
cartilage matrix from 2 to 15 days [13]. Fluorescence confocal microscopy has showed
significant variations in the shape, size and orientation of chondrocytes an chondrons
revealing flattened discoidal chondrons in the superficial zone, rounded chondrons in
the middle zone, and elongated chondron, multicellular chondrons in the deep
zone [14]. The three-dimensional chondrocyte cytoskeleton is involved in mecha-
notransduction in the response to mechanical loads. The optimal processing method of
fixation and permeabilization on the preservation of cytoskeletal structure has been
Figure 2. Confocal images of fluorescence and light transmitted detection from articular cartilage chondro-
cytes. A-B; C: Autofluorescence of chondrocyte clusters of knee human osteoarthritis cartilage.
recently described for confocal imaging [15]. However, CLSM posed certain problems
linked to the use of living cells due to the high density of incident light (laser source)
focussed on a small volume (femtoliter) and very small illuminated diameter of the
specimen (about 0.3 µm). To overcome these limitations, other 3D fluorescence tech-
niques have been developed, such as multiphoton microscopy.
3. Multiphoton Microscopy
Advances in multiphoton fluorescence microscopy continue to appear. Multiphoton

excitation occurs during the simultaneous absorption of two or more photons by a
fluorophore. This multiphoton process (simultaneous absorption of 2 photons) is made
possible by a very high intensity combined with time-related concentration of a very
brief (pico or femtosecond) and very high-frequency (about 80 MHz) laser flash [16].
Because in multi-photon mode spatial resolution is the result of absorption (and excita-
tion) confinement to the focal event (smaller of one femtoliter) [17], the photobleach-
ing patterns and photodegradation outside the focal plane are reduced [18]. According
to the pulse duration/peak power ratio, two red photons (700 nm) can excite a fluoro-
phore whose excitation spectrum is in UV (350 nm) for an emission spectrum in the
blue (420 nm). Since the red or near infrared illuminating light used for 2-P excitation
has approximatively twice the wavelength of that employed for 1-P excitation, scatter-
ing effects at the excitation wavelengths are greatly reduced allowing deeper penetra-
tion into tissues than with visible or UV excitation [19]. Fluorescence generated by
two-photon absorption corresponds to the concentration of fluorophores, principally
NADH, NADPH, and flavoproteins as a redox imaging without the need for exogenous
dyes [20]. The effects of the heat produced after high-frequency pulse illumination in a
restricted volume (approx. 1 µm3) and the optical aberrations are thought to be negligi-
ble and similar to those observed with single-photon confocal microscopy [21]. To
study how chondrocytes respond to alterations in their mechanical environment, nu-
merous in vitro studies have been performed using 2P-excitation either viable cartilage
explants or chondrocytes embedded in a gel matrix [22]. Figure 3 shows nucleus of
chondrocytes excited by 2P-excitation pulsed laser light according to transparence and
nature of culture material (500 µm native matrix or ∅ 2mm alginate bead). As shown,
depth penetration in thick specimen is greatly enhanced (from 219 µm to 1.6 mm) for
sodium alginate gel in the case of transparent medium.
Figure 3. Multiphoton microscopy images of nucleus stained with Hoescht and illuminated by 2-Photon
absorption light at 780 nm (Laser femtoseconde MIRA 900). A: Chondrocyte in rat cartilage head cap (depth
illumination: 219 µm). B: Chondrocyte encapsulated in alginate bead (depth illumination: 1.6 mm).
4. FRAP and FCS
Fluorescence Recovery After Photobleaching (FRAP) makes use of the fact that fluo-
rescent molecules lose their ability to emit photons when exposed to repetitive cycles
of excitation and emission. By monitoring the levels and rates of fluorescence recovery
with time, one can determine kinetic parameters such as the mobile fraction and the
diffusion coefficient. Molecular transport in avascular collagenous tissues such as ar-
ticular cartilage occurs primary via diffusion. New technique of FRAP have shown that
diffusional transport of macromolecules is anisotropic in collagenous tissues with
higher rates of diffusion along primary orientation if collagen fibers [23]. However,
various processes such as membrane flow, molecular interactions and trafficking may
simultaneously contribute to the overall recovery kinetics, which makes it difficult to
interpret the data obtained [24]. Recently, there has been rapidly increasing use of
Fluorescence Correlation Spectroscopy (FCS) for biological applications which is a
sensitive technique for measuring dynamic processes (number density, interaction frac-
tions and molecular dynamics) in a fluorescent signal on the nanosecond to second time
scales [25,26]. FCS is based on the fluorescence intensity fluctuations associated with
molecules passing through a diffraction-limited observation volume created by a focus-
sed laser beam. FCS The autocorrelation function obtained from the intensity fluctua-
tions gives the average diffusion time taken by the molecule to cross the observation
volume, along with the average number of molecules present [27]. Sanchez and Grat-
ton used two-photon microscopy in conjunction with fluorescence correlation micros-
copy [28]. Several works have recently shown that Mesenchymal Stem Cells (MSC)
collected from bone marrow can differentiate in vitro into cartilage cells under the ef-
fects of transforming growth factor-β (TGF-β), others growth factors or critical tran-
scription factors [29]. The effect of TGF-β, a multifunctional cytokine, has been de-
scribed as a potent stimulant of the synthesis of the matrix molecules [30]. The mobil-
ity of type II TGF-β receptor in the MSC membranes has been compared for MSC
TGF-β stimulated or unstimulated cells (Fig. 4). FCS may appear as a more appropriate
technique since it analyzes an ensemble of molecules diffusing in the detection volume
There is a an increase in the decay time of the FCS curves from before stimulation in-
dicating that receptor diffuse more quickly through the focal volume and are thus
shown to have higher mobility after stimulation.
Figure 4. Confocal image of indirect immuno-labelling of RII collagen receptor (A11017 Invitrogen, Al-
exa488TM) on Mesenchymal Stem Cells. Fluorescence autocorrelation curves on a single MSC before and
after stimulation with TGF-β1 (10 ng/ml TGF-β for 12h). Each curve is the average of five measurements on
a single focal spot (target laser) at 488 nm on a SP2-FCS2 Leica Microsystems workstation. Excitation and
emission were focused through a 63 x 1.2 NA water immersion. Excitation volume has been calculated as
0.48 ± 0.02 fl.
5. Spectral Microscopy
Unlike a fluorescence image, where each point represents the intensity at a different
locations (x,y,z) in space, a spectral image is a sequence of image representing the in-
tensity of the 2D plane of the sample at different wavelength (spectral information).
Combination of fluorescence spectroscopy with image cytometry is useful in biology
by using fluorescent probes to identify and map several fluorophores and to improve
the signal-to-noise ratio. The ability to identify a fluorophore signal based on spectral
characteristics rather than intensity has significant advantages when trying to separate
signals from undesirable background fluorescence and when trying to separate signals
from closely related fluorophore. For chondrocytes; spectral imaging has also been
used successfully for the detection of early apoptosis by using a JC-1 (5,5’, 6-6’ – tet-
rachloro-1,1’, 3,3’- tetraethylbenzimidazolocarbocyanine iodide) sensitive mitochon-
drial potential dye [31]. This ratiometric probe can be used on human cartilage to in-
vestigate events involved in programmed cell death as a consequence of disease arthri-
tis. Incubation of chondrocytes from human OA cartilage with JC-1 revealed a fluores-
cence shift from orange to green with a transmitochondrial potential collapse as ob-
tained during apoptotic processes (Fig. 5).
6. Lifetime Imaging Contrast
The fluorescence lifetime of a substance represents an average amount of time the

molecule remains in the excited state prior to its return to the ground state, while emit-
ting fluorescence. The advantages and disadvantages of measuring the fluorescence
signal in terms of intensity or lifetime have often been discussed [7]. The precise nature
of the fluorescence decay can reveal more details about the interactions of the fluoro-
phore with its close surrounding: this parameter can reveal the frequency of collisional
Figure 5. Mitochondrial transmembrane potential for normal and pathological human chondrocytes. Fluores-
cence spectra (from 502.5 nm to 632.5 nm, excitation at 488 nm) acquired with a SP2-AOBS-confocal mi-
croscope Leica Microsystems (63x/1.2 NA water immersion). of 5,5’, 6-6’ – tetrachloro-1,1’, 3,3’- tetra-
ethylbenzimidazolocarbocyanine iodide (JC-1) in normal chondrocyte (A, B) and knee human osteoarthritic
cartilage chondrocyte (C, D).
encounters with quenching agents, the rate of excited state reactions. Multiple decay
constants can be the reflection of several distinct surrounding of a fluorophore or of the
presence of several conformational states of a molecule. Factors such as ionic strength,
hydrophobicity, oxygen concentration, binding to macromolecules can all modify the
lifetime of a fluorophore, considered as an indicator of these parameters. Fluorescence
Lifetime Imaging Microscopy (FLIM) combines the advantages of lifetime spectros-
copy with fluorescence microscopy by revealing the spatial distribution of a fluorescent
molecule together with information about its microenvironment. FLIM has been ap-
plied with spectral fluorescence to study of articular cartilage and its arthritis disease.
By exploiting autofluorescence, the diseased tissue has been detected that were not
detectable with the conventional diagnosis [32]. A greater FLIM contrast between the
cells and the extracellular matrix has been shown (Fig. 6).
Figure 6. For lifetime imaging, SPC-730 TCSPC Imaging module (Becker&Hickl, Berlin) was interfaced
(signals, Pixel Clock, Frame Sync) to the scan controller of the Leica TCS-AOBS Multiphoton laser scanning
microscope. The decay analysis measured by time-correlated single photon counting was performed using the
SPCImage software (Becker&Hickl GmbH). (A) Lifetime image of knee human osteoarthritic cartilage
chondrocyte labelled with JC-1 (mitochondrial transmembrane potential probe) showing chondrocytes in
blue for shorter lifetime and matrix in green-red for longer lifetime (B). The fluorescence decay of JC1 con-
sisted in a minor component (bleu) used to generate a mask for segmentation and to contrast the JC1 fluores-
cence against the matrix autofluorescence.
7. FRET Imaging Contrast
Fluorescence Resonant Energy Transfer (FRET) is a powerful technique for measuring

intermolecular distances on a scale of a few nanometers, orientation and dynamic prop-
erties. FRET microscopy is typically used to determine binding partners, conforma-
tional changes, and proximity or interaction between two molecules. FRET utilizes a
pair of fluorophores (donor and acceptor) and takes advantage of long-range dipole-
dipole interactions, when the excitation spectrum of the acceptor overlaps with the
emission spectrum of the donor and the energy is nonradiatively transferred from the
acceptor of the donor. This phenomenon only occurs when donor and acceptor are in
proximity (1–10 nm) [33]. However, due to the spectral overlap of donor and acceptor
excitation/emission, the acceptor is often also excited directly by the donor excitation.
Most of pair of fluorophore used in FRET experiment are difficult to separate spec-
trally by currently available methods based on fluorescence intensity [34]. FRET in
vivo studies has remained relatively unexplored due to the thickness of tissue cross
sections which can be in part resolved by using two photon excitation [35]. Spectral
analysis and FLIM have been combined for better distinction [36]. Autofluorophores in
chondrocytes have been characterized by multiphoton, confocal, lifetime and spectral
microscopy image in view to subtract their contributions to obtain a corrected anti-
body-marker fluorescence signal, and (ii) measure the interaction between Filamin A
and B proteins by detecting the fluorescence resonance energy transfer (FRET) be-
tween markers of the two proteins [37].
8. SHG
From histological studies have been described different zones of cartilage with increas-
ing depth. Collagen fibers are oriented parallel to the articular surface, then fibers may
have many different orientations to progress into the radial zone and starting the bone
Figure 7. Two-Photon Fluorescence Image (green channel for autofluorescence) combined to Second
Harmonic Generation Image (white channel for organized network collagen) at 805 nm (Laser femtoseconde
MIRA 900) in osteoarthritic human cartilage (5 µm) showing morphological changes (hyaline, amorphous
collagen matrix) with depth near the articular surface (A) and the radial zone (B).
surface (separated by the tide mark) where fibers are oriented orthogonal to the joint
surface. Collagen fibers present a high second order non-linear susceptibility, and
therefore produced a second harmonic generation (SHG) when exposed to high enough
electric fields produced by a focal point of a femtosecond pulsed laser (multiphoton
microscopy). As processes involved in multiphoton fluorescence and SHG are intrinsic
properties of the constituent molecules, non-linear microscopy allow submicron resolu-
tion images without need for sectioning or staining with dye. By placing bandpass filter
at the second harmonic of the excitation wavelength in front of the transmitted light
detector, SHG signal with frequency doubled is isolated for a new SHG contrast imag-
ing. SHG is a coherent elastic process which is dependent on the polarization of the
incident light, and the greatest intensity for collagen signal is produced when the fibers
are oriented parallel to the laser polarization [38]. Both the nonlinear optical micros-
copy and SHG at 800 nm have been combined to investigate living cells from normal
and degenerative cartilage due to macroscopic arrangement of type II collagen [39]. In
healthy tissue the fibrous structure of collagen II fibers in the extracellular matrix has
been seen along with dark areas representing the sites of chondrocytes [40]. Moreover,
chondrocytes and membranes have been identified on the basis of native fluorescence
emission spectra and SHG signals from the collagen fibers [41]. Using SHG, it was
possible to achieve two-photon excitation images at great depths in strongly (light)
scattering collagen membranes (depth up to 300 µm) and cartilage samples (depth up to
460 µm).
The SHG images clearly map the distribution of the collagen II fibers within the
extracellular matrix while the multiphoton fluorescence mages show the distribution of
endogenous two-photon fluorophores in both the cells and the extracellular matrix
(Fig. 7). The SHG image clearly demonstrated lacuna occupied by chondrocytes.
Conclusion
In this work has been presented a panel of studies of microscopy imaging which had
tissue cartilage relevance. Conventional imaging with one-photon excitation process
and organic fluorophores poses several challenges for the visualization of articular tis-
sue, including fluorophore overlap (crosstalk), rate of photobleaching and autofluores-
cence. The methodological and technological advances of the last five years have been
fast evolving, especially with regard to the optimization of CLSM and deconvolution
process. Even if the deconvolution techniques require considerable computing power
capacities and extended computation time, they prove very useful in situations of low
intensity levels or restricted use of confocal microscopy to improve spatial resolution in
multiphoton microscopy. In the future, multiphoton imaging has established itself as an
important method for optical microscopy particularly for live cell studies. The feasibil-
ity of multi labelling intimal structures by exciting green fluorescent protein variants,
multicoloured QDots or organic fluorophores with only one laser wavelength (820 nm)
is very useful. In addition to lower photodamage, less background fluorescence is ob-
served since the photon flux is usually too low for nonlinear excitation outside the tiny
volume where the laser is focused. Recent technological advances in non-linear optical
microscopy have created more powerful imaging as the only strategy currently avail-
able for non-invasive, reliable detection (Spectral Microscopy), quantitative (Fluores-
cence Correlation Spectroscopy) and real time fluorescence (Fluorescence Lifetime
Imaging Microscopy) studies in living cartilage tissue. Near-infrared tomography may
allow for sufficient tissue penetration to image organized structure (Second Harmonic
Generation) combined or not with fluorescence.
Future imaging techniques will be designed to answer more specific research ques-
tions in tissue cartilage engineering, particularly multimodal approaches by potential
combination of scanning multiphoton and arthroscopy (which is actually the standard
for assessment for articular cartilage). With the use of green fluorescent protein, it
could be possible for long-term to monitor cartilage regeneration after delivery of
autologous or mobilization of native cells. The methodological advantages of multimo-
dality imaging (FLIM-MRI) may lead the development of modified probes with a fluo-
rescent or phosphorescent dye characterized with very long lifetime (µs-ms) as ob-
tained by FLIM. The same markers could be very efficient with magnetic resonance
imaging as contrast agent (europium, terbium..) [42].
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IOS Press, 2007
XVII
Biomarkers of Matrix Fragments,

Inflammation Markers in Osteoarthritis
Leonardo PUNZI *, Francesca OLIVIERO and Paolo SFRISO
Rheumatology Unit, University of Padova, Italy
Abstract. Molecular markers or biomarkers have recently received growing atten-

tion in osteoarthritis (OA), due to their potential usefulness in early diagnosis, as-
sessment of disease activity and severity, and evaluation of drug effects. In this
context, biomarkers are ideals, due to their characteristics of non-invasive and non-
expansive measures. Concerning the diagnosis, no marker seems able to satisfy the
needs to diagnose OA in pre-radiological stages and to identify different subsets of
OA. Instead, biomarkers are useful in the assessment of disease activity and the
prevision of its outcome. In the recent years, stimulated by the recent introduction
of high sensitive immunoassays, number of studies have suggested a role of
C-reactive protein (CRP) as marker of activity or severity of OA. Furthermore,
higher CRP levels predict those patients whose disease will progress over 4 years.
Since metalloproteases (MMPs) are highly involved in cartilage degradation, their
levels or activities have been investigated to obtain information on OA severity or
progression. Both in serum and synovial fluid (SF), the most abundant MMP is
MMP-3. It has been proposed that pro-matrix MMP-3 may act as a marker for
posttraumatic cartilage degradation. The molecular markers most useful in sug-
gesting synthesis or degradation of cartilage originate from different articular
sources such as cartilage, bone and synovial tissue. Serum hyaluronan (HA) is the
most commonly used marker of synovial proliferation and hyperactivity, which
may reflect the OA evolution. Other useful biochemical markers are serum keratin
sulphate (KS), cartilage oligomeric matrix protein (COMP), YKL-40, and urinary
C-terminal crosslinking telopeptides of collagen types I and II (uCTX-II). COMP
concentration in SF from lavage as well as in serum is an early indicator of radio-
graphic progression at follow up. Furthermore, COMP was the most sensitive test
for identifying affected subjects with the genetic form of premature OA. uCTX-II
is well correlated with radiological severity of both knee and hip OA and, in addi-
tion, the combined measurements of uCTX-II and serum HA seem the best predic-
tor of the structural progression of hip OA.
Keywords. Osteoarthritis, Laboratory investigation, Synovial fluid, Biomarkers,

Biochemical markers, Molecular markers, Markers of inflammation, Cytokines
Introduction
The progress in the knowledge of the pathophysiology of osteoarthritis (OA) has con-
tributed to clarify the role of some substances as putative markers of this process [1]. In
*
Corresponding Author: Leonardo Punzi, MD, PhD, Rheumatology Unit, Department of Clinical and
Experimental Medicine, University of Padova, Via Giustiniani 2, 35128 Padova, Italy, E-mail:
punzireu@unipd.it.
268 L. Punzi et al. / Biomarkers of Matrix Fragments, Inflammation Markers in Osteoarthritis
OA they may be utilised for several objectives, including diagnosis, assessment of the
disease activity, prevision of the outcome, and evaluation of drugs effects. Recently a
multidisciplinary group, the NIH-funded Biomarkers Network, has proposed to classify
OA markers as “BIPED”, which stands for Burden of Disease, Investigative, Prognos-
tic, Efficacy of Intervention and Diagnostic [2]. However, although a great number of
substances are continually proposed, only few among these may be considered as true
“disease marker” in OA [1–6].
Biochemical markers or biomarkers available in OA may be classified in “direct”
and “indirect” markers, according to Thonar’s suggestion [7]. Direct markers are mole-
cules or fragments which originate principally from joint structures while indirect
markers are found in many tissues and produced by many cell types. They have the
potential to influence the metabolism not only of chondrocytes but also of synovial
cells and other joint cells.
Biomarkers may be determined in three different biological fluids: blood, synovial
fluid (SF) and urine. Obviously, since serum and urine are commonly available, deter-
minations in these fluids are easier performed than in SF that however, offers informa-
tion which better reflects local changes occurring in joint affected by OA. To appropri-
ately evaluate the significance of the substances determined in serum or in urine, it
should be remember that they may originate from many tissues outside the joints, in-
cluding non articular cartilage. Furthermore, molecular markers are smaller and have
shorter half-lives in the blood circulation than in joint fluid and in addition, due to their
complex metabolism, are difficult to be adequately interpreted. Marker concentration in
serum or urine may be influenced by the function of organs mainly responsible for the
elimination of the molecular fragments, in particular lymph nodes and liver [5,7]. For
example, the concentration of a marker of cartilage matrix degradation in SF may de-
pend not only on the rate of cartilage matrix degradation, but also on the rate of clear-
ance from the joint. Furthermore, some biomarkers vary diurnally, thus suggesting that
serum and urine sampling need to be carefully standardised [8].
Among the most ambitious objectives of OA markers there is the possibility for an
early diagnosis. Since main diagnostic hallmarks in OA are still represented by radio-
graphic changes, which are only evident when disease is established, biochemical
markers would be ideal in order to diagnose OA in pre-radiological stages and identify
different subsets of OA. In addition, these substances could be also potentially utilised
in assessing the value and the significance of new imaging techniques very sensitive in
detecting cartilage changes, such as magnetic resonance (MR) and sonography [9].
Unfortunately, no evidences have been until now demonstrated an established utility of
markers in satisfying these needs, although interesting studies are in progress. In diag-
nostic perspectives, the more traditional laboratory features are still helpful. These may
be utilised in a “routine” approach, to exclude inflammatory arthropathies or other con-
ditions causing arthritis, and in more specific aims, to investigate the causes mainly
responsible for OA or OA-like arthropathies, which may in turn allow to subdivide OA
in two classical categories: primary or secondary. In a “routine” fashion, two biological
fluids may be analysed: blood and SF, obviously when available (Table 1). Among
blood or serum determinations, although no features may be considered as a diagnostic
“marker”, erythrocyte sedimentation rate (ESR) and C reactive protein (CRP) may be
useful in the basic but essential task of identifying subclinical inflammatory conditions.
In fact, although some inflammatory reactions are important in the pathogenesis of OA,
this disease is usually considered, at least for classification purposes, as a non inflam-
matory arthropathy. For this reason, an ESR < 20 mm/h has been included among ACR
L. Punzi et al. / Biomarkers of Matrix Fragments, Inflammation Markers in Osteoarthritis 269
Table 1. Aspects to be considered in the evaluation of laboratory markers in osteoarthritis
Biological fluids in Comments

which markers are
detected
Serum a) Due to their complex metabolism, molecular markers may be difficult to
interpret adequately
b) Frequently molecular markers are smaller and present shorter half-lives in
blood circulation than in synovial fluid
c) Substances may originate from tissues outside the joints
d) Marker concentration in serum may be influenced by the function of the
organs mainly responsible for the elimination of the molecular fragments, in
particular lymph nodes and liver
Synovial fluid a) The concentration of a marker of cartilage degradation may depend not only
on the rate of cartilage matrix degradation, but also on the rate of clearance
from the joint
b) The volume of fluid within the joint cannot be accurately measured
c) The volume of an effusion can changes rapidly
d) Joint aspiration may be performed easily only a few joints
e) This determination gives information concerning a single joint
Urine a) The marker could be metabolised before detection
Table 2. Synovial fluid findings in osteoarthritis. Personal experience on 432 patients (3)
Findings Common features Observations

Aspect Clear Cartilage fragments, cells, crystals or
particles may cloud the synovial fluid
Colour Yellow pale or dark In some cases, severe OA may be associated
with hemartrosis
Viscosity Normal Usually decreased in relation to local
inflammation
Volume, median (range) 32 (3–130) Volume exceeding 100 ml is rare
3 3
WBC number (x 10 /mm ), mean 0.4 (0.7) (0.1–2.7) Most fluids contain WBC < 1,000/mm3;
(± SD) (range) very rare are those with WBC > 2,000/mm3
PMN, % range 2–10 Value higher than those indicated may be
found in patients with concomitant disease
Total protein, mean (± SD) 3.5 (2.7) Simultaneous serum determination is
recommended
PMN=polymorphonuclear cells
criteria for the classification of OA of the hip [10]. Although no OA criteria include
CRP, this test may be useful in detecting an inflammatory condition, in which it is usu-
ally found at levels above the limit of normal value commonly fixed at 6 mg/L. How-
ever, in rare cases of OA, such as erosive OA, ESR and mainly CRP may be increased,
with a value slightly above the normal [11–13].
SF analysis may be useful in OA even in a “routine” approach (Table 2). In par-
ticular, among the various possible determinations, white blood cell (WBC) number is
crucial, helping to differentiate the “non-inflammatory” SF typical of OA, characterised
by a number of WBC < 2,000/mm3, from the “inflammatory” SF of arthritis which ex-
hibit a WBC number > 2,000/mm3 [14]. Thus, the feature of “non-inflammatory” SF
was included in the classification criteria of OA of knee by the ACR [15]. However,
the approach to this useful finding offered by the SF analysis should be prudential, due
to number of mild inflammatory conditions possibly found in the range between 1,000
and 2,000 WBC/mm3. Thus, while a number of WBC < 1,000/mm3 indicates almost
always a “non-inflammatory” SF, a number of WBC between 1,000 and 2,000
WBC/mm3 need to be interpreted according with the clinical context.
The assessment of disease activity and the prevision of its outcome are essential
for a rational therapeutic approach of OA. To these purposes, several molecular mark-
ers are thought to be useful, due to their characteristics of non-invasive and non-
expansive measures.
1. Markers of Inflammation
Since the inflammation plays a key role in OA [16], markers of this process may be
useful also in OA. They include both systemic markers, such as ESR and CRP, or local
measured in SF, such as WBC number and concentrations of total proteins and various
enzymes. Cytokines or their receptors, in particular interleukin (IL)-1β, IL-6, tumor
necrosis factor (sTNF) and soluble receptors of TNF (sTNFR) and IL-2 (sIL-2R) may
be included in this category.
C-Reactive Protein
In the recent years, stimulated by the recent introduction of high sensitive (hs) immu-
noassays, a growing number of studies has been dedicated to the role of C-reactive pro-
tein (CRP) as marker of activity or severity of OA. Conrozier et al. showed that mean
hsCRP was significantly higher in the rapidly destructive than in the slowly progressive
hip OA, thus suggesting that rapidly destructive hip OA may be associated with some
degree of inflammation [17]. According with this study, Sharif et al. found that low
level increases in CRP were associated with progression of hip OA and knee OA [18].
By examining 655 consecutive patients with OA of the knee or hip, Wolfe observed
that CRP was elevated in OA compared to healthy individuals and in addition, CRP but
not ESR was correlated with clinical severity in patients with OA of the knee or hip
[19]. Furthermore, he found that CRP was significantly associated with functional dis-
ability, joint tenderness, pain, fatigue, global severity, and depression. In a study by
Spector et al., women with knee OA have hsCRP values increased, in comparison with
normal population, in a population based, cross sectional study of 845 women in
Chingford [20]. Furthermore, higher hsCRP levels predict those patients whose disease
will progress over 4 years, suggesting that low-grade inflammation may be a significant
aspect of early OA. Stürmer et al. assessed the association between hsCRP and severity
and extent of OA in patients with advanced OA, by measuring preoperative hsCRP in
serum samples from 770 consecutive patients with hip or knee joint replacement [21].
They observed that hsCRP was associated with severity of pain, but not with the bilat-
eral or the generalised extent of OA. In a study by Punzi et al., hsCRP was increased in
erosive OA in comparison with nodal OA and in addition, it was correlated with dis-
ease activity, as evaluated by the number of active joints [13].
Cytokines
Although cytokines have tight relationships with CRP and an important role in carti-
lage metabolism, their determination in blood of OA patients is not useful as disease
marker. This may be explained, at least in part, by technical difficulties, including the
sensitivity of the assays. Most cytokines are undetectable in serum of non inflammatory
arthropathies, with the exception of the receptors sTNFR, sIL-6R and sIL-2R, which
however are always found at levels lower than inflammatory arthropathies and thus
difficult to interpret [22–27]. Interestingly serum levels of sIL-2R have been found to
be increased in erosive OA of the hand, thus suggesting a possible immune disregula-
tion in affected patients [27].
Most proinflammatory cytokines, including IL-1, IL-6, IL-8, TNF and its receptors
sTNFRs have been found in SF of OA, even if at low levels, much lower than in rheu-
matoid arthritis (RA) [23–26,28,29]. IL-1β and TNFα are highly involved in the me-
tabolism of human cartilage [16]. At low concentrations they are thought to inhibit the
synthesis of aggrecan and collagen type II; at higher concentrations, they stimulate the
production of proteolytic enzymes responsible for the degradation of cartilage matrix.
Thus, at low levels found in OA, IL-1 and TNF are probably differently involved in the
inhibition of the synthesis and in the promotion of the degradation [16]. However, the
clinical significance of their determination is doubtful also in SF.
Proteolytic Enzymes
In inflammation associated with OA, proteolytic enzymes are probably the mainly re-
sponsible for degradation processes seen in OA cartilage. In particular, a central role
seems to be played by the metalloproteases (MMP) family, which contains the only
mammalian proteinases that can specifically degrade triple-helical collagens at neutral
pH [30]. The most classic collagenases MMP-1, MMP-8, and MMP-13 have differing
substrate specificities for types I, II, and III collagen, with MMP-13 showing a prefer-
ence for type II collagen [31]. It has been suggested that MMP-1, produced in the
synovium, is the primary collagenase in RA, while MMP-13, produced by chondro-
cytes, is the most important collagenase in OA. However, several other members of the
MMP family have been localized to cartilage or synovium in the arthropathies [32,33].
Another group of proteinases thought to be relevant in the extracellular matrix metabo-
lism is the ADAMTS (a disintegrin and metalloproteinase domain with throm-
bospondin motifs) family, which contains 19 members involved in collagen biosynthe-
sis as procollagen propeptidases (ADAMTS-2, ADAMTS-3, and ADAMTS-14) [34].
Other members of this family are the so-called aggrecanases (ADAMTS-1, ADAMTS-
4, ADAMTS-5, ADAMTS-9, and ADAMTS-15), able to degrade the interglobular
domain separating G1 and G2 of aggrecan [35,36]. Although it has been recently sug-
gested that aggrecanases are active early in the disease process, followed by increases
in MMP activity, the enzyme actually responsible for cartilage aggrecan destruction at
any stage in arthritis is unclear. MMP activity is also regulated by a family of 4 specific
inhibitors, the tissue inhibitors of metalloproteinases (TIMPs) [37], which may also
inhibit ADAMTS. The local balance of MMP and TIMP activities is pivotal in regulat-
ing cartilage homeostasis, and that disturbance of this balance, resulting in an excess of
MMPs over TIMPs, underlies pathologic cartilage destruction. The relative contribu-
tion of any MMP or TIMP to this balance is largely unknown. The most relevant in OA
are collagenases 1 (MMP-1) and 3 (MMP-13), and stromelysine (MMP-3). Proinflam-
matory cytokines stimulate chondrocytes to synthesise MMPs, which are usually pro-
duced in non-active forms and are subsequently activated by number of sub-
stances locally produced in the joint, including enzymes from the serine- and
cysteine-dependent protease families. In particular, a strong activator is the complex
plasminogen activator (PA)/plasmin [16]. PA and its inhibitor (PAI) are correlated with
IL-1β in the SF of patients with OA [38]. This is not surprising, due to the influence of
IL-1β on PA, and in particular on its urokinase-type receptor (uPAR) [39]. This recep-
tor plays an important role in OA cartilage degradation by regulating pericellular prote-
olysis mediated by serine proteinases. By immunohistochemical analysis Schwab et al.
have observed an enhanced expression of uPAR on chondrocytes derived from OA
human cartilage compared to non-OA controls [40]. They found an IL-1β-mediated
expression of uPAR on chondrocytes and a functional co-localization between uPAR
and MMP-9 on IL-1β-stimulated chondrocytes.
Due to the key role of MMPs in OA, number of studies have investigated the sig-
nificance of their determination in the serum or in the SF of OA [41–43]. However, no
clear information derived from these studies. One of the reasons could be that the value
of MMP determination is relatively limited without the knowledge of the levels of their
inhibitors TIMPs, since it has been observed that the effects of MMP mainly depend on
the net molar ratio between MMP and TIMP [44]. Both in serum and SF, the most
abundant MMP is MMP-3, considered as a cytokine-driven MMP, due to its strong
relationships with pro-inflammatory cytokines, in particular TNFα [45]. Marini et al.
have studied the amount and activity levels of MMPs and their inhibitors TIMP-1 and
TIMP-2 in SF from 56 patients with different degrees of either chondral lesions or knee
arthritis identified and classified by arthroscopy [46]. They observed that the degree of
cartilage degradation, as seen by arthroscopy, was strictly related to the activity of
MMP-2 and MMP-13 and on reduced inhibitory effect of MMP-2 by TIMP-2. In addi-
tion, a serine protease weighting about 125 kDa appears only in patients with severe
cartilage degradation [46]. In another study, it has been proposed that pro-matrix
MMP-3 may act as a marker for posttraumatic cartilage degradation [47]. Since the
development of posttraumatic OA is a relatively slow process, the early prevision of
OA changes would be crucial for the therapeutic intervention. To this purpose Bo-
bacz et al. investigated the significance of proMMP-3 in SF and serum samples from
259 patients of trauma clinic at the time of arthroscopy [47]. Serum proMMP-3 levels
of the total cohort were markedly increased compared to healthy controls. However, the
grade of cartilage damage did not correlate with enzyme concentration neither in pa-
tients’ serum nor in SF samples, thus suggesting that although proMMP-3 SF concen-
tration was increased early after trauma, its measurement in serum or SF did not reflect
the cartilage damage. That of post-traumatic OA is one of the more fascinating field of
application of laboratory markers, and in particular molecular markers in OA.
2. Molecular Markers
Tissue joint products or molecular markers most useful in suggesting synthesis or deg-
radation of cartilage, originate from different articular sources (Table 3), such as syno-
vial tissue, cartilage and bone. Some derive only from synovial membrane, such as
hyaluronan (HA), while some others are markers of both synovial and cartilage me-
tabolism, such as human cartilage glycoprotein-39 (YKL-40).
Table 3. Matrix markers in OA
SOURCE MARKERS of SYNTHESIS MARKERS of DEGRADATION
BONE
Serum PINP, PICP, bone specific ALP, DPD, PYD, NTX, CTX, ICTP,
osteocalcin BSP
Synovial fluid ND BSP
Urine HP CTX, DPD, PYD, NTX
CARTILAGE
Serum CS846, CS3B3, CS7D4, CPF, KS5D4, KSAN9P1, COL2-
PIICP,PIIANP,YKL-40, TIMPs 3/4m, COL 2-1/4N1, CTX-II, 2B4,
COMP, MMPs
Synovial fluid CS846, CS3B3, CS7D4, PIICP, CPF, KS5D4, KSAN9P1, COL2-
PIIANP, YKL-40, TIMPs 3/4m, COL 2-1/4N1, CTX-II, 2B4,
COMP, MMPs
Urine ND CTX-II, HELIX-II, TIINE
SYNOVIAL MEMBRANE
Serum PICP, PIIINP, HA, YKL-40, PYD, Glc-Gal-PYD, CTX
COMP, MMPs, TIMPs, Cytokines
Synovial fluid PICP, PIIINP, YKL-40, COMP, PYD
MMPs, TIMPs, cytokines
Urine ND CTX-II, Glc-Gal-PYD, PYD
BSP: bone sialoprotein; Col2-1/4n1: type II collagen denaturation product; Col2-3/4m: type II collagen dena-
turation product; CS3B3: chondroitin sulphate epitope 3B3; CS7D4: chondroitin sulphate epitope 7D4;
CS846: chondroitin sulphate epitope 846; CTX: C-terminal cross-linking telopeptide of type I collagen;
CTX-II: C-terminal crosslinking telopeptide of type II collagen; Glc-Gal-PYD: glucosyl-galactosyl-
pyridinoline; HA: hyaluronan; HELIX-II: type II collagen helical peptide I; HP: hydroxyproline; ICTP: car-
boxy-terminal telopeptide of type I collagen; KS5D4: keratan sulphate epitope 5D4; KSAN9P1: keratan
sulphate epitope AN9P1; NTX: N-terminal cross-linking telopeptide of type I collagen; PICP: Procollagen I
carboxyterminal propeptide; PIIANP: N-propeptide of type IIA procollagen; PIICP: procollagen type II
carboxy-terminal propeptide; PIIINP: type III collagen N-propeptide; PINP: collagen I amino-terminal
propeptide; TIINE: collagene type II neoepitope.
Serum HA is the most commonly used marker of synovial proliferation and hyper-
activity, which may reflect OA evolution [48,49]. Bruyere et al. observed that HA lev-
els were significantly correlated with 3-year progression in mean joint space width of
the femorotibial joint in a 3-year longitudinal study of patients with knee OA [50]. In
this ability, HA was similar to serum osteocalcin, but superior to other biochemical
markers, such as serum keratin sulphate (KS) and cartilage oligomeric matrix protein
(COMP), and urine pyridinoline (PYD) and deoxypyridinoline (DPD). These data were
subsequently confirmed by Pavelka et al. [52] who studied the prognostic value of dif-
ferent biochemical markers for morphological progression of early knee OA, by a fol-
low-up period of 2 years. They found that patients with higher basic serum levels
of HA had a faster radiological progression. Other biochemical markers, including
MMP-9, TIMP and COMP, had no statistically significant prognostic value. HA recep-
tor CD44H and v5 and v6 were studied in SF from patients with primary OA of the
knee joint with and without synovitis [53]. SF from OA with synovitis showed signifi-
cantly higher levels of CD44H and v6, but not v5, than OA patients without synovial
inflammation. However, CD 44 concentrations do not reflect the OA stage in the Kell-

gren grading scale. In ECHODIAH study, performed by French authors to determine
whether systemic markers of bone, cartilage, and synovium can predict structural pro-
gression of hip OA, 10 markers were evaluated: N-propeptides of collagen types I and
III, COMP, YKL-40, HA, MMP-1 and MMP-3, CRP, urinary C-terminal crosslinking
telopeptides of collagen types I and II (uCTX-II) [54]. Combined measurements of
uCTX-II and sHA were the best predictor of the structural progression of hip OA.
YKL-40 is a protein with an apparent MW of 42 kDa, originally described as a ma-
jor gene product of chondrocytes and synovial cells [55–58]. YKL-40 messanger RNA
seems expressed in cartilage from RA or OA but not in healthy adult cartilage [59]. In
SF of OA, YKL-40 was found to be increased, and correlated with disease sever-
ity [60–62]. It has been proposed that another related molecule, chitinase 3-like protein
2 (YKL-39, chondrocyte protein 39) also abundantly secreted by chondrocytes in vitro
being about 4% of all secreted proteins, may be a more accurate marker of chondrocyte
activation than YKL-40 [63].
Among markers assessing cartilage metabolism, the most interesting are COMP
and uCTX-II. It has been suggested that COMP concentration in SF from lavage as
well as in serum is an early indicator of radiographic progression at follow up [64–68].
Furthermore, COMP was the most sensitive test for identifying affected subjects with
the genetic form of premature OA [69,70]. In this context, a recent study on heritable
determinants of COMP was performed on 160 monozygotic and 349 dizygotic twin
pairs showed that heritable factors influence serum levels of COMP [71]. Another in-
teresting “direct” marker is AgKS, found almost exclusively in aggrecan, the main
noncollagenous constituent of articular cartilage. When the aggrecan molecules are
cleaved by proteolitic enzymes, AgKS fragments rapidly diffuse out of the tissues and
appear in the body fluid, where they can be measured. AgKS is elevated in the serum
of a high percentage of patients with polyarticular OA and is thought to precede clinical
evidence of degenerative changes [72]. Furthermore, destabilisation of the knee after
injury to a ligament or meniscus leads within hours to a marked increase in the SF con-
centrations of AgKS, aggrecan core protein and COMP, which often remains elevated
for several years [73]. These changes are not usually seen in individuals who present
with knee pain without evidence of damage to at least one internal supporting element.
Other recent studies have compared many putative markers of OA disease activity and
severity in humans [74–77]. In the Garnero’s study, a panel of biochemical markers
were measured in a group of 67 patients with knee OA and in 67 healthy controls, and
correlated with pain and physical function (WOMAC index) and with quantitative ra-
diographic evaluation of the joint space using the posteroanterior view of the knees
flexed at 30 degrees [76]. By univariate analyses, increased urinary glucosyl-galactosyl
pyridinoline (Glc-Gal-PYD) and decreased serum osteocalcin were associated with a
higher total WOMAC index. Increased urinary CTX-II and Glc-Gal-PYD, and serum
type III collagen N-propeptide PIIINP were the only markers which correlated with
joint surface area. By multivariate analyses, urinary Glc-Gal-PYD and CTX-II were the
most important predictors of the WOMAC index and joint damage [76]. More recently,
the same group has proposed the urinary type II collagen helical peptide (HELIX-II) as
a new biochemical marker of cartilage degradation in patients with OA and RA [77].
They developed a specific ELISA for HELIX-II which was subsequently utilised to
determine the urinary levels in patients and controls. Median urinary HELIX-II levels
were increased in knee OA and early RA, compared to controls. In a study by Otter-
ness, 14 serum and urine molecular markers were investigated for an association with
particular clinical end-points [78]. Thus, baseline clinical assessments were correlated
with urinary hydroxylysyl pyrididinoline (HP), swelling of the signal joint was corre-
lated with serum CRP, and change in clinical assessment over the 1 year evaluation
with TGFβ. The crosslinks HP, derived from bone and cartilage, and lysylpyridinoline
(LP), derived from bone, were both increased in urine from OA patients, in comparison
with age-matched controls [78]. Recently, new serum biochemical markers, Coll 2-1
and Coll 2-1 NO2 have been proposed for studying oxidative related type II collagen
network degradation in patients with OA and RA [79]. In these disorders, both markers
were found to be significantly increased compared to controls, and in RA were higher
than in OA. However, no relationship was found between radiological OA severity and
the levels of Coll 2-1 and Coll 1-2 NO2 in serum. Interestingly, this latter marker but
not Coll 1-2, was correlated with CRP in the sera of OA and RA patients [79].
Influence of Drugs on OA Markers
Laboratory findings may be proposed to monitor the therapeutic response in OA. In

this context, the most useful seem markers which are better related with the degree of
local inflammation or with cartilage degradation. Due to the possible influence of some
drugs on the metabolic processes of many molecular markers, SF determinations seem
more suitable than those in serum. The most frequently tested drugs are non steroidal
anti-inflammatory drugs (NSAIDs) and intra-articularly injected drugs, in particular
HA. Among NSAIDs, it has been demonstrated that etodolac reduced the SF levels of
prostaglandin (PG)E2 and IL-6 while increasing those of TNFα [80]. This observation
is interesting, because confirms in vivo the possibility of a modulation of TNF produc-
tion by PGE2. In vitro studies have demonstrated that PGE2 may induce elevation of
cAMP in mononuclear cells which in turn inhibit TNF and augment IL-6 expres-
sion [81,82]. The authors suggest that PG inhibition by etodolac could have conse-
quently lowered cAMP levels, resulting in augmented TNFα and lower IL-6 lev-
els [80]. Our studies on the influence of NSAIDs on SF cAMP are in contrast with such
hypothesis, because NSAIDs increased cAMP levels in all SF tested [83,84].
Molecular markers seems unaffected by NSAIDs, as suggested by the observation
that they are unable to modify serum level of AgKS when administered orally to pa-
tients with OA [7]. To investigate the effects of HA on inflammatory cytokines in SF
of patients with knee OA, Sezgin et al. have performed a single blind, placebo-
controlled, randomised study [85]. They administered intra-articular HA to 22 patients
in the study group and placebo to 19 in the control group. Both HA and placebo caused
a significant decrease in IL-6 levels, although more significantly in the study group,
while IL-8 and TNF-alpha levels did not change in either group. The ability of HA in-
jections to influence some aspects of SF inflammation, was confirmed by other stud-
ies, showing the SF decrease of PGE2, PGF1 alpha and F2 alpha, IL-1β, and MMPs
[86–89]. Furthermore, HA seems also to act on chondrocyte metabolism, as suggested
by the significant reduction in SF levels of proteoglycan monomers, and an increase in
COMP concentration and osteocalcin levels [90]. Among the other intra-articular
drugs, a single intra-articular injection of prednisolone markedly reduce serum and SF
levels of AgKS [7], and the bisphosphonate clodronate decrease SF levels of pros-
taglandin E2 [91].
Another category of drugs which have recently received attention in the treatment
of OA is that of the so called “chondroprotective” drugs or “structure modifying drugs
in OA”. Among these drugs, previously known as “symptomatic slow active drugs for
OA (SYSADOA)”, the most studied was the glucosamine sulphate, especially after the
demonstration-still under discussion-of its ability to reduce symptoms and progression
of knee OA in a long term (3 years) study [92]. The clinical effects of these drugs par-
allel corresponding changes in molecular markers [93]. During their clinical follow-up
study for evaluate the effects of glucosamine sulphate, these authors have found that
measurements of uCTX-II, enables the identification of OA patients with high cartilage
turnover who at the same time are most responsive to therapy with structure modifying
drugs. Concerning the possible mechanisms of action of these drugs, McCarthy et al.
suggested that glucosamine therapy can improve SF HA content in OA [94].
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IOS Press, 2007
XVIII
Cartilage Engineering
J.F. STOLTZ a, M. LOTZ b and J. BUCKWALTER c
a
Groupe d’Ingénierie Cellulaire et Tissulaire et thérapie, UMR CNRS 7563, Faculté de
Médecine (Université Henri Poincaré), 54 500 Vandoeuvre Lès Nancy, France
(jf.stoltz@chu-nancy.fr)
b
Scripps Research Inst, Div MEM 161, 10550 N Torrey Pines Rd, La Jolla, CA 92037
(mlotz@scripps.edu)
c
Department of Orthopaedics § Rehabilitation, University of Lowa Hospitals, 200
Hawkins Drive, Iowa City, IA 52242 (joseph-buckwalter@uiowa.edu)
Abstract. The rapid development of tissue engineering today allows us to

envisage the clinical use of grafts of chondrocytes, autologous stem cells,
biocartilage preparations or gene therapy . However, in spite of the high stakes for
orthopedic surgery as well as rhumatology or sports medecine, the answers remain
unclear. Clinical research on cell therapy and preparation of biocartilage must
continue to be developed in order to better determine the choice of a support
matrix (scaffold) the local mechanical forces on the cells (chondrocyte, MSL...).
1. Introduction
1.1. Normal Cartilage
Hyaline cartilage in adults is a non-vascularized, non-innervated and highly-specialized

type of connective tissue [1]. Its principal function is to protect underlying bone from
mechanical and traumatic stresses, to absorb shocks and to provide a virtually fric-
tionless articulating surfacing [3]. It has biochemical and biophysiological properties
which allow it to provide elasticity and resistance to the forces of compression. Knee
cartilage, with a thickness of between 2 to 4 mm, is thus able to withstand a force of up
to 5 times the body’s weight.
This tissue consists of a single type of cell, the chondrocyte (approximately 1% of
its volume), which is dispersed throughout an extracellular matrix base composed of
water (70–80%), a network of collagen fibers (90 to 95% of which are Type II collagen
fibers) surrounding a large concentration of proteoglycans (agrecans), the latter being
grouped in aggregates of a very high molecular weight. Proteoglycans have particular
biochemical and biophysiological properties (negatively charged and attractive of water
molecules), creating a strong osmotic pressure which ensures the hydration of the carti-
lage and maintains the tension within the collagen network. It is the qualitative and
quantitative maintenance of this three-dimensional structure that provides cartilage
with its functional properties. In this manner, the chondrocytes, nourished by the syno-
J.F. Stoltz et al. / Cartilage Engineering 281
vial liquid dispersed throughout the matrix, conserve their phenotype. The regeneration
of the matrix is constant but slow: 1000 days for proteoglycans and 200 years for col-
lagen. Collagen synthesis is even slower and is not significant except in very young
subjects. In adults, it appears that collagen synthesis may even decrease with age, caus-
ing collagen tissue to deteriorate faster than it is replaced.
1.2. Destruction of Cartilage
Cartilage is vulnerable to trauma and diseases which can produce irreversible tissular
lesions. The resulting disorganization of the collagen network and the proteoglycans
modifies the chondrocytes’ capacities to resist the mechanical stresses to which they
are subjected. When cartilage is damaged, its reparative capacity is weak and the le-
sions irreversible in the majority of cases. Deep losses of cartilage can naturally be re-
placed by fibrocartilaginous tissue originating from medullary stem cells, but this new
tissue is not functional [6]. The evolution of cartilaginous lesions over time is not well
known. Nevertheless, according to the majority of published works, the progression
towards osteoarthritis seems probable, especially when the lesions are located in weight
bearing regions.
The prevalence and incidence of losses of cartilage are unknown and a large vari-
ety of pathologies could be involved. Cartilaginous lesions which later may require the
implantation of chondrocytes are those which occur in patients with osteochondritis or
which are secondary to an instability in the knee due to a ligamentary lesion or a prob-
lem with the meniscus [3–5]. In young subjects, the most frequent cause is a sports-
related injury.
Cartilaginous lesions are difficult to diagnose because there is no correlation be-
tween the symptomology and the state of the cartilage. Because the cartilage is devoid
of nerve tissue, there are no early warnings signs of lesions. The symptomology in-
cludes pain, swelling, and mobility problems all of which can have a significant nega-
tive impact upon the quality of life. The arthroscopy is the standard examination to
diagnose and assess the loss of cartilage; at the same time, it can allow its treatment.
The use of the MRI remains more controversial. According to some, however, it allows
the diagnosis of an increasing number of lesions. For others, its performance is too
variable. In addition, there is currently a lack of consensus on which sequences to use.
2. Autologous Chondrocyte Grafts
The loss of cartilage in the knee, a weight-bearing zone, in young subjects poses a
therapeutic problem. Cartilaginous lesions, most frequently of traumatic origin, are for
the most part irreversible due to the weak ability for spontaneous regeneration of carti-
lage and the probability of a progression of these lesions towards osteoarthritis.
Autologous chondrocyte implantation is a recent therapeutic option [6,7]. This
method requires an expertise in the culture of chondrocytes on the one hand and good
surgical skills on the other. Recent clinical data appears to show encouraging signs of
clinical improvement, but these results have not passed the test of time and have been
obtained using heterogenous populations and pathologies [4,8–10]. Due to a lack of
exhaustive information on undesirable effects, tolerance is difficult to assess. It is not
currently possible to evaluate either the risk/benefit relationship of chondrocyte grafts
282 J.F. Stoltz et al. / Cartilage Engineering
or their place in the treatment of deep and isolated chondral lesions. In fact, this new
technique is can still be considered as within the domain of clinical research.
It is important to note that chondrocyte implantation is has been recognized by the
FDA since 1997 as a tissue engineering approach whereas in Europe, the absence of a
scaffold results in it being considered as an act of cellular therapy.
The potential indications for a chondrocyte graft are:
– The patient is between 15 and a 50 years of age (maximum) taking into ac-
count the level of physical activity and presenting with a traumatic and symp-
tomatic loss of cartilage, preferably of the femoral condyles;
– after period of non-surgical treatment;
– the size of the lesion (beyond 8 cm2 the implantation of chondrocyte tissue is
not recommended);
– a healthy mechanical environment (ligaments, meniscus, alignment).
The strict contraindications are: all synovial and inflammatory pathologies and le-
sions with a diameter of less than 1 cm2.
3. Surgical Alternatives to Autologous Chondrocyte Grafts
Various conservation or reparation techniques have been proposed, either by the elimi-
nation of microscopic debris (lavage) or by the formation of fibrocartilage by stimulat-
ing the stem cells of sub-chondral bone tissue. Other options include the transplantation
of autologous osteochondral tissue from a healthy, non weight-bearing region or the
antologous transplant of osteochondral cells from a tissue banks, however the latter
presents the risk of secondary immunological reactions or the risk, even slight, of the
transmission of disease (viruses, prions…).
4. Cartilage Engineering: Towards Biocartilage
Currently, numerous research projects are being conducted to develop new treatments
for osteochondral lesions. The most innovative ones involve the concept of tissue engi-
neering, which consists of seeding a biomaterial with chondrocytes or progenitor cells
in order to prepare a reimplantable cartilaginous matrix in vitro [11–14]. In this tech-
nique, the biomaterial must be biocompatible with the cartilage located in the surround-
ing lesional site. It thus serves as a scaffold for the synthesis of reparative tissue. Even
so, the functionality of the newly formed tissue is determined, on the one hand, by the
exogenenous supply or endogenous presence of the growth factor at the lesional site,
and on the other hand, the mechanical properties of the biomaterial to resist the forces
of compression imposed by the movement of the articulation. This new therapeutic
direction should eventually provide solutions to such problems as the lack of donors or
even the risks of viral transmission.
The ideal support matrix, or scaffold, should be biocompatible, non-toxic, biofunc-
tional, biodegradable and easy to use. The inclusion of chondrocytes cultivated in
three-dimensional matrix provides an environment that will not only promote cellular
differentiation but will also maintain the cells in the lesion. Different supports of syn-
thetic, organic or even hybrid origin have been proposed. Examples of synthetic com-
ponents include polylactic and polyglycolic acid. Supports of organic origin are com-
posed of fibrin and of collagen fibers, with the collagen fibers being used either in the
form of hydrogels or in the form of a sponge. Research has shown that collagen
sponges are superior to collagen hydrogels in promoting the proliferation of cells as
well as in the synthesis of collagen and proteoglycans. Finally, there is one last type of
biomaterial of organic origin which can be used: polysaccaride polymers such as the
hydrogels. Hydrogels are materials composed of reticulated polymers which have the
particularity of being able to absorb large quantities of water. From a mechanical point
of view, the hydrogels present the advantage of using water in the same way as carti-
lage [15]. Under the force of compression, water pushed out of the hydrogel, which
allows the latter to absorb the shock. Then, once the compressive force is released, the
water is returns back to its place in the hydrogel, thus allowing it to return to its initial
volume. From a biological point of view, the hydrogels provide a three-dimensional
environment that is sufficiently porous to allow the proliferation of cells as well as the
transportation of nutrients. Among the hydrogels, those containing of sodium alginate
constitute the model of reference not only in terms of studies of cellular morphology
and the synthesis of collagen and proteoglycans [16], but also in terms of mechanobi-
ology [17,18]. Although sodium alginate is not a natural component of the extracellular
matrix, it has a structure similar to that of the glycosaminoglycans of cartilage. It has
also been shown that this type of hydrogel ensures the maintenance of chondrocytary
phenotype [19].
Biomechanic al parameters are important for the in vitro development of biocarti-
lage as well as for the in vivo fate of implanted cellular scaffold constructs. Reaction to
local forces can be conceptualised as:
– Reaction of loads to the non living materials used (scaffold).
– Reaction of cells towards physical signal in the microenvironment of tissue.
Cartilage biomechanics varies within the different subsets of cartilage. The devel-
opment and maintenance of cartilage structure and mechanical characteristics are
closely correlated to the effect of mechanical loading. The impact of load on cartilage
structure and function is of outer most importance in hyaline cartilage. The histology
structure of articular cartilage is deeply influenced by the local mechanical loading of
chondrocytes in the different zones, but there is limited information on the physiologies
in vivo mechanical environment. Stresses in a normal joint are difficult to determine
but evidence from experimental studies indicate that stresses may range from 5 to
20 MPa in animal and human joints. Loads tend to deform cartilage but compression
and subsequent deformations are resisted by generation of fluid pressure and restriction
of tissue deformation. In other respect intermitted loads created by the movements of
joints transfer cyclic hydrostatic pressure in the interstitial fluid. Chondrocyte reactions
in response to loading are genes activation that determine the remodelling and repair.
Several types of cells have been described for their potential application in the en-
gineering of cartilage tissue such as mature cells (chondrocytes) or immature cells
(mesenchymal stem cells). The utilization of each type of cell has its advantages and its
disadvantages due to intrinsic biological properties as well as ethical issues.
For example, the utilization of autologous chondrocytes in the repair of cartilage
can present the problem of their dedifferentiation during the course of their amplifica-
tion ex-vivo. The necessity of harvesting these cells in a single layer in order to multi-
ply can result in the loss of the chondrocyte phenotype to a fibroblaste phenotype. In
order to avoid this phenomenon of dedifferentiation, recent studies have used cells that
are less mature such as the mesenchymal stem cells (MSC). These cells appear capable
of differentiating into numerous tissues including bones, cartilage, tendons, muscles
etc. [20]. The mechanisms behind the differentiation of the MSC are poorly under-
stood. However, several studies have demonstrated the importance of using growth
factors such as TGF-β1 in single layer cultures as well as the BMP-2, 6 et 7 [21,22]. In
addition, several authors have shown the beneficial effects on chondrogenesis and in
the quality of the newly-formed matrix when mechanical forces are applied in vitro to
mesenchymal stem cells [23–25].
5. Gene Therapy of Cartilage Defects
The development of methods for gene transfer has been the object of numerous studies
over the last 10 years. However, if the concept of gene therapy for the repair of articu-
lar cartilage seems appealing, recent research shows that the application of this tech-
nique remains extremely difficult and requires an optimization of the transfer of genes
into the cartilage [26–29]. Indeed, the therapeutic success of gene therapy for cartilage
involves the delivery and the expression of therapeutic factors at the lesional site. The
determining factors are the cellular density (cellularity) of the repaired tissue and the
production and maintenance of a matrix rich in Type II collagen and in proteoglycans.
The ideal therapeutic agent must also be capable of inducing chondrogenesis, stimulat-
ing cellular proliferation and promoting the synthesis of the matrix. Growth factors
would appear to satisfy these criteria and thus are ideal agents to be applied during
gene therapy (the super family of transforming growth factor beta (TGF – β 1 and 2),
bone morphogenetic protein 2 (BMP2), and the fibroblast growth factor family such as
FGF-2.
Another strategy concerns the applications of transcription factors such as matrix
proteins and inhibitors of articular cartilage degeneration. In this case, the transcription
factors modulate the expression of the genes involved in chondrogenesis. As such, ex-
perimental models have shown the chondrogenic properties of transcription factors
such as S0X-9 [30] and cart-1 [31].
Finally, another alternative can be found in the induction of the inhibition of the
chondrocyte catabolism. Potential targets in this case would be IL-1β , tumor necrosis
factor (TNF) and IL-17 [55]. It is thus a question of inhibiting the production of matrix
degradation, inflammation mediators and other mechanisms which lead to cellular
death.
Treatment requires the ability to deliver therapeutic agents, in the optimal concen-
trations, over the course of a period that is sufficiently long to allow for the induction
of a chondrogenic reponse.
Different target cells for gene transfer have been proposed: differentiated chondro-
cytes, synoviocytes and/or progenitor cells.
Chondrocytes are the key target cells for gene transfer. It has thus been proposed to
use adenoviral or retroviral vectors, however, their efficacy was only moderate [32].
Other transduction (vector) systems have also been proposed such as the lentivirus or
the baculovirus [33].
The synoviocytes are also a target cell of interest because these cells can differen-
tiate into chondrocytes and fill in chondral defects [34]. However, the majority of the
studies involving synoviocytes have been conducted in vitro.
Finally, the utilization of progenitor cells represents an attractive target for gene
transfer. The possibility of using stem cells [35–38] and adenoviral or retroviral vectors
has been well documented in vitro.
It is important to mention that embryonic stem cells have also been considered. In
fact, these cells differentiate into chondrocytes in the presence BMP-2 [39].
6. Conclusion
The regeneration of cartilage is and will remain a challenge for the development of cell
therapy, of tissue engineering and of gene therapy. However, to this day many prob-
lems remain to be solved:
– Technical problems regarding the definition of supports (scaffolds), cells used
current gene transfer techniques and transient expression. In particular, the
impact of the biomaterial used remains to be defined.
– Legal issues with respect to the different regulations in the USA, Europe, etc.
Cartilage engineering can be introduced via cell implantation, biocartilage trans-
plantation or gene therapy. Complementary scientific approaches remain to be devel-
oped. Nevertheless, current knowledge permits a certain optimism for the future.
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IOS Press, 2007
XIX
Therapeutics and Osteoarthritis

J. BUCKWALTER, M. LOTZ and J.F. STOLTZ
This whole chapter on therapeutics in osteoarthritis involves a large field that must be
subdivided according to the therapeutic class of agents and it would be quite presump-
tuous to summarize in a few pages the whole action of these drugs in osteoarthritis at a
time where numerous publications in many top journals have cast some doubt on the
tolerance of some of these drugs.
We used simple methods to retrieve most of the information about therapeutics
used in osteoarthritis (OA). We extracted data from MEDLINE and from the private
databases of the companies marketing the main drugs used in OA. We crossed on a 1:1
comparison all the current used drugs in OA with the following items: inflammation,
osteoarthritis, IL-1 beta, prostaglandins, Nitric Oxide, iNOS, cyclooxygenase-2, metal-
loproteinases, transcription factors and others items such as IL-6, IL-8, PPAR, NF
kappa and AP-1, MAPK P38, JNK, MMP-3, MMP-9 and MMP-13, ICAM and
VCAM, HSF-1 and HO.
As several drugs were used concomitantly in the same study, we considered that
all the data were respectively attributable to each of the drug provided the results were
individually consistent. We excluded all the data concerning the relation between
COX-2 and coxibs as the relevancy of this association seemed quite obvious. We at-
tributed a quotation to each selected peer-reviewed journal including both clinical and
fundamental journals. With this method, we included in our report data coming from 70
articles and we excluded 26 journals from our final report.
In a first part, we will try to show that among non steroidal anti-inflammatory
drugs (NSAIDs), different mechanisms of action and sometimes different outcomes for
the same drug were observed.
Thus, although NSAIDs have been used for many years in osteoarthritis and glob-
ally against all forms of inflammatory processes, their impact in osteoarthritis is the
matter of a continuing debate which has been lately extended beyond efficacy to the
safety margins of these drugs in this indication.
The second part of this chapter was centered on a heterogenous group of molecules
called disease-modifying drug for osteoarthritis (DMOADs), which encompasses both
DMOADs with a validated mechanism of action and nutraceuticals the action of which
is not clear enough to consider this latter group of molecules as fully efficient drugs
with a validated mechanism of action. Among these products, many of which are found
as OTC drugs, such different dosages are observed that it is barely difficult to find evi-
dence of a therapeutic efficacy and of a scientific reproducibility.
288 J. Buckwalter et al. / Therapeutics and Osteoarthritis
Non Steroidal Anti-Inflammatory Drugs (NSAIDs)
NSAIDs consist of a large group of agents with different mechanisms of action and the
pharmacological studies in this class present an inconsistent quality.
These molecules have a demonstrated anti-prostaglandins effect. This action is
however highly variable according to the given molecule and some NSAIDs:
– Have several mixed mechanisms of action,
– Have been evaluated by studies of inconsistent quality,
– Have different mechanisms of action according to the species,
– Have for most of them no pharmacological selectivity but the COX-2.
However it seems that NSAIDs reduce PGE-2 release through various mecha-
nisms:
– Inhibition of COX-1 and COX-2,
– Inhibition more centered on COX-2 than on COX-1, such as meloxicam,
– Selective inhibition of COX-2 such as coxibs or nimesulide,
– Inhibition of the COX/LOX pathways such as licofelone.
They have a more inconsistent effect on the other factors involved in the inflam-
matory process in osteoarthritis. It seems that they do not play a role against inflamma-
tory cytokines and some could even increase IL-1 and TNF alpha synthesis. However
the outcomes of many studies are sometimes contradictory. Thus, while tiaprofenic
acid exhibits an inhibiting effect of IL-1 in one study (Pelletier JP et al. 1993), it has a
stimulating action on IL-1 in two other studies (Weithman KH et al. 1997, Vignon E
et al. 1998). Piroxicam would have a stimulating effect on IL-1 in human (Hernvann A
et al. 1996) but naproxen would induce an inhibition of IL-1 in animal models (Ci-
cala C et al. 2000). Thus all these data do not yield a consistent conclusion.
Other NSAIDs could intervene in inhibiting IL-6 production such as aceclofenac
or indomethacin, and aceclofenac could stimulate IL-1Ra while piroxicam and aspirin
could have a reverse action. Therefore, the global mechanism of action of NSAIDs on
inflammatory cytokines is difficult to apprehend and it would be possible that accord-
ing to the type of molecule, the species and the used pharmacological model, the out-
comes were significantly different.
NSAIDs can have an effect on the transcriptional factors, mainly NF kappa B
(such as meloxicam), but there are very few studies tending to demonstrate an impact
on these factors. Celecoxib would inhibit NF kappa B while stimulating AP-1, an ac-
tion which could explain partly the renal side effects of the coxibs in comparison with
meloxicam which inhibits NF kappa B and AP-1.
NSAIDs can inhibit or not NO according to the type of molecule. Aceclofenac and
aspirin decrease NO production, as well as meloxicam and celecoxib. However other
studies reported contradictory results.
NSAIDs would have a stimulating effect on MMP1 production (such as naproxen),
but other studies report that NSAIDs would inhibit MMPs production through suppres-
sion of NF kappa B and AP-1 such as meloxicam. All these mechanisms of action still
remain non conclusive.
At last, PPARs have been seldom fully evaluated although nimesulide could de-
crease COX-2 synthesis through an effect on PPARs.
In conclusion, there are many contradictory studies with NSAIDs which have a
definite anti-prostaglandins action. They also have an impact on COX-1 and COX-2
J. Buckwalter et al. / Therapeutics and Osteoarthritis 289
which is more or less selective according to the evaluated agent. However, it seems
they tend to stimulate anti-inflammatory cytokines and metalloproteases which could
explain their potential for maintaining a chronic inflammatory process and therefore a
slow degradation within the joint.
Table 1 summarizes the mechanisms of action of NSAIDs.
Table 1. Effects of NSAID on the inflammatory chain reaction in osteoarthritis
IL- TNF
AINS PG NO Cox-2¶ MMP TF Others Study Type Authors
1bêta alpha
Animal in Patten C et al.
Celecoxib –
vitro 2004
Human in Takahashi T et al.
Celecoxib ++ + +
vitro 2004
Human in Takahashi T et al.
Celecoxib +++ + +
vitro 2004
Human in Mastbergen SC
Celecoxib +
vitro et al. 2005
Animal in Niederberger E
Rofécoxib # + +/– +
vitro et al. 2003
Diclofenac –
vitro 2004
Human in Sanchez C et al.
Diclofenac +
vitro 2002
Human in Smith RL et al.
Diclofenac +
vitro 1995
Human in Borderie D et al.
Diclofenac + /–
vitro 2001
Indomethacin –
vitro 2004
Indomethacin +
vitro 2002
Human in Lindsey HB et al.
Indomethacin +
vitro 1990
Human in Hernvann A et al.
Indomethacin –
vitro 1996
Human in Massicotte F et al.
Indomethacin* + +
vitro 2002
Human in Lader CS et al.
Indomethacin +
vitro 1998
Animal in Yin H et al.
Indomethacin –
vitro 2005
Human in Herman JH et al.
Indomethacin +
vitro 1994
Human in Mathy-Hartert M
Aceclofenac – + –
vitro et al. 2002
Aceclofenac +
vitro 2002
Human in Maneiro E et al.
Aceclofenac + +
vitro 2001
Aceclofenac** + +
vitro 2001
Human in Blanco FJ et al.
Meloxicam +
vitro 1999
Rainsford KD
Meloxicam – + H/A in vitro
et al. 1997
Table 1. (Continued.)
IL- TNF
1bêta alpha
Animal in Yin H et al.
Meloxicam +
vitro 2005
Human in Asano K et al.
Meloxicam*** + +
vitro 2006
Human in Li LC et al.
Meloxicam § +
vitro 2002
Animal in Engelhardt G
Meloxicam + +
vitro et al. 1996
Animal in Engelhardt G
Meloxicam + +
vivo et al. 1996
Nimesulide +
vitro 2002
Human in Fahmi H et al.
Nimesulide +
vitro 2001
Human in Di Battista JA
Nimesulide + +
vitro et al. 2001
Human in Manicourt DH
Nimesulide || –
vivo et al. 2005
Human in Kalajdzic T et al.
Nimesulide §§ + +
vitro 2002
Human in He W et al.
Nimesulide +
vitro 2002
Animal in Futaki N et al.
Nimesulide + +
vitro 1994
Piroxicam +
vitro 2001
Human in Lindsey HB et al.
Piroxicam +
vitro 1990
Piroxicam –
vitro 1996
Piroxicam –
vitro 2001
Human in Herman JH et al.
Piroxicam +
vitro 1994
Naproxen +
vitro 1996
Human in Massicotte F et al.
Naproxen* + +
vitro 2002
Animal in Cicala C et al.
Naproxen +
vitro 2000
Human in Martel-Pelletier J
Naproxen +
vitro et al. 2004
Human in Smith RL et al.
Ibuprofen +
vitro 1995
Human in Pelletier JP et al.
Tenidap +
vitro 1993
Animal in Griswold DE
Tenidap +
vitro et al. 1993
Tiaprofenic Human in Pelletier JP et al.
+
acid vitro 1993
Tiaprofenic Weithman KH
– – H/A in vitro
acid et al. 1997
Tiaprofenic Human in Vignon E et al.
– +
acid vitro 1998
IL- TNF
1bêta alpha
Animal in Jarvinen TA et al.
Ketoprofen + –
vitro 1996
Licofelone +
vitro et al. 2004
Animal in Lajeunesse D
Licofenone + + +
vivo et al. 2004
Human in Marcouiller P
Licofenone §§§ +
vitro et al. 2005
Red: stimulating effect of the study drug on the evaluated factor

Blue: inhibiting effect of the study drug on the evaluated factor
Green: intermediate effect of the study drug on the evaluated factor
Grey: effect non evaluated in the corresponding study
+ Diminution of IGF-1 with licofenone.

++ Diminished production of MAPK p38 with celecoxib.
+++ Diminished production of MAPK p38 and p44/42 after induction by NO.
* Diminution of IL-6 with indomethacin.
** 46-fold increase of IL-Ra with aceclofenac and of IL-1R with tenidap.
*** Inhibition of NF kappa B and AP-1 with meloxicam.
§ Inhibition of NF kappa B and AP-1 with meloxicam and diminution of ICAM expression.
§§ Inhibition of PPAR agonistic stimulation on COX-2 expression and synthesis.
§§§ Effect of licofen both on COX and LOX pathways.
# Rofecoxib induced opposite results on transcription factors; inhibiting effect on NF kappa B and
stimulation of AP-1, with stimulation of iNOS.
|| Nimesulide induced a diminution in serum levels of MMP-3 and MMP-13 opposed to ibuprofen.
¶ In some studies, the COX-2 have not been the main target of the study. Therefore they are not indi-
cated as COX-2 inhibitors.
Disease-Modifying Drug for Osteoarthritis (DMOADs)
DMOADs represent a difficult field to apprehend, as all molecules have not been sub-
jected to the same constraints of development and if some have followed the gold stan-
dard of development for a pharmaceutical agent, the vast majority has not been prop-
erly evaluated. When they have been studied, they have been evaluated as nutraceuti-
cals, which implies they have not gone through the long process of developing a phar-
maceutical agent.
Therefore the quality of the results suffers from a lack of consistency of the phar-
macological studies. Some of these studies have only been recently conducted in order
to justify a recent use of these products as DMOADs.
Chondroitin sulfate was pharmacologically evaluated only recently. The different

studies, both in human and in animal models, showed an inconstant inhibitory effect on
prostaglandins, NO and metalloproteases. However, this agent would only have an im-
pact on stromelysin (MMP-3) and MMP-9. To date, the few completed studies cannot
conclude definitely this agent has an inhibitory effect on any factors of the inflamma-
tory chain in osteoarthritis.
The avocado/soybean unsaponifiable mixtures have more consistent effects on
prostaglandins (PGE-2) and metalloproteases (MMP-3), an irregular effect on inter-
leukin-1, interleukin-6 and interleukin-8. However, when considered separately, these
compounds have no more any positive impact. A lack of proper pharmacological stud-
ies might explain these data.
As far as hyaluronic acid products are concerned, only one formulation has been
able to exhibit a positive effect on some components of the inflammatory chain in os-
teoarthritis. However all these studies yield different results depending on what type of
model has been studied, human or animal, as in man only an inconstant inhibitory ef-
fect on interleukin-6 has been shown, this specific study presenting scarce information
on the animal model to be fully conclusive (Sezgin M et al. 2005). In animal models a
positive effect on ICAZM-1 and VCAM-1 has been found, with again a lack of infor-
mation to conclude to something else than a pure mechanical effect of these products in
osteoarthritis.
Glucosamine hydrochloride has an effect on prostaglandins, NO, metalloproteases
(MMP-3) and transcriptional factors. These studies have been conducted both in human
and in animal models. However, the suppressed metalloproteases production in one of
these studies has only been obtained in normal chondrocytes without any induction of
pharmacological or mechanical stress. In another study, despite the positive comments
of the authors in their conclusion (Gouze JN et al. 2002), no data were given and the
effect on the transcriptional factors was only observed for NF kappa B without effect
on AP-1. In such experimental conditions, drawing a conclusion about glucosamine
hydrochloride having any positive impact on inflammation in osteoarthritis is difficult.
On the contrary, diacerhein, or its metabolite, rhein, have been fully evaluated by
many pharmacological studies, consisting of different models, both in animal and in
human. Its consistent and well established anti-interleukin-1 effect has been observed
across species.
This effect would be associated to an inhibitory effect all along the inflammatory
chain in osteoarthritis. Thus it was shown that:
– diacerhein had no anti-prostaglandin effect,
– diacerhein was able to inhibit transcriptional factors through a potential
JNK-dependent effect, in particular during AP-1 inhibition,
– diacerhein was able to decrease NO production in inhibiting iNOS (in a stimu-
lated-chondrocytes model),
– diacerhein could reduce metalloproteases production,
– diacerhein would present an anti-TNF alpha effect through HSF-1 and HO-1,
i.e. through the heat shock proteins pathway.
This targeted mechanism of action could explain its positive effect on the low-
grade inflammatory mechanisms as observed in osteoarthritis models.
DMOADs effects are summarized in Table 2.
Table 2. Effects of DMOAD on the inflammatory chain reaction in osteoarthritis
IL- TNF
Drugs PG NO Cox-2 MMP TF Others Study Type Authors
1bêta alpha
Chondroitin Animal in Neil KM et al.
sulphate vitro 2005
Chondroitin Human in Monfort J et al.
+
sulphate * vitro 2005
Chondroitin Human in Chan PS et al.
+ +
sulphate vitro 2005
Chondroitin Animal in Chou MM et al.
+
sulphate ** vivo 2005
Human in Henrotin YE et al.
AS § *** +/– + + +
vitro 1998
Human in Henrotin Y et al.
AS §§ + + + +
vitro 2003
Hyaluronic Animal in Greenberg DD
+ +
acid # vitro et al. 2006
Animal in Monfort J et al.
Hyaluronic acid +
vitro 2005
Animal in Diaz-Gallego L
Hyaluronic acid +
vivo et al. 2005
Human in Fioravanti A et al.
Hyaluronic acid +
vitro 2005
Hyaluronic Human in Karatay S et al.
+
acid ## vitro 2004
Hyaluronic Human in Sezgin M et al.
+
acid || vitro 2005
Human in Nakamura H et al.
Glucosamine ||| + + +
vitro 2004
Human in Largo R et al.
Glucosamine & + + +
vitro 2003
Animal in Fenton JI et al.
Glucosamine + + +
vitro 2002
Animal in Gouze JN et al.
Glucosamine + +
vitro 2002
Animal in Gouze JN et al.
Glucosamine ++ + + +
vitro 2001
Animal in Fenton JI et al.
Glucosamine + +
vitro 2000
Diacerein +
vitro et al. 1997
Diacerein +
vitro et al. 1998
Gigant-
Human in
Diacerein + Huselstein C et al.
vitro
2002
Animal in Martin G et al.
Diacerein + + +
vitro 2004
Diacerein +
vitro et al. 1997
Animal in Lin S et al.
Diacerein +++ +
vitro 2003
Animal in Mendes AF et al.
Diacerein + +
vitro 2002
Animal in Tamura T et al.
Diacerein +
vitro 2001
Animal in Tamura T,
Diacerein +
vitro Ohmori K 2001
IL- TNF
Drugs PG NO Cox-2 MMP TF Others Study Type Authors
1bêta alpha
Human in Yaron M et al.
Diacerein + +
vitro 1999
Stoltz JF et al.
Diacerein ++++ + + + A/H in vitro
2006 (in press)
Human in Mistry D et al.
Diacerein // + +
vitro 2006 (in press)
Diacerein + +
vitro 2003
Red: stimulating effect of the study drug on the evaluated factor

Blue: inhibiting effect of the study drug on the evaluated factor
Green: intermediate effect of the study drug on the evaluated factor
Grey: effect non evaluated in the corresponding study
* Inhibitory effect of CS on MMP-3 (stromelysin).

** Inhibitory effect of CS on MMP-9.
§ AS means avocado/soybean unsaponifiable mixtures.
*** AS: strong inhibitory effect of AS on MMP-3, IL-6, ILM-8 and PGE-2; however each isolated com-
pound of this mixture had no effect. Partial effect on IL-1.
§§ This study showed an effect on nearly any element of the inflammatory chain in osteoarthritis. No
confirmation was obtained in any other study.
# Only one formulation of hyaluronic acid exhibited an effect (Hyalgan).
## Inhibitory effect on ICAZM-1 and VCAM-1.
|| Inhibitory effect on IL-6; however, lack of information on this model.
||| Of note, MMPs production was only suppressed in normal chondrocytes.
& No numbers were indicated in this study.
+ Effect only on NF kappa B. No effect on AP-1.
++ Inhibitory effect on MMP-3.
+++ Inhibitory JNK-dependent effect of rein on AP-1.
++++ Inhibitory effect of diacerein on iNOS production in stimulated chondrocytes.
// Inhibitory HSF-1 via HO-1 induced effect of diacerein on TNF alpha.
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IOS Press, 2007
Author Index
Aigner, T. 219 López-Armada, M.J. 192
Berenbaum, F. v, 163 Lotz, M. 182, 280, 287
Bianchi, A. 77 Malemud, C.J. 99
Blanco, F.J. 192 Martel-Pelletier, J. 3, 206
Buckwalter, J. 280, 287 Martin, J.A. 239
Ding, L. 56 Oliviero, F. 267
Dumas, D. 254 Otero, M. 43
Gabay, O. 163 Pedersen, D.R. 239
Galteau, M.-M. 77 Pelletier, J.-P. 3, 206
Goldring, M.B. 118 Piera-Velazquez, S. 143
Gómez, R. 43 Punzi, L. 267
Gómez-Reino, J.J. 43 Rego, I. 192
Gosset, M. 163 Riquelme, B. 254
Gualillo, O. 43 Sandell, L.J. 118
Guo, D. 56 Sfriso, P. 267
Homandberg, G.A. 56 Smith, R.L. 14
Isla, N.D. 254 Stoltz, J.F. vii, 254, 280, 287
Jimenez, S.A. 143 Terkeltaub, R.A. 31
Jouzeau, J.-Y. 77 Thedens, D.R. 239
Kirchmeyer, M. 77 van den Berg, W. 219
Lago, F. 43 van der Kraan, P. 219
Lago, R. 43 Werkmeister, E. 254
Lajeunesse, D. 206

Osteoarthritis, Inflammation and Degradation

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OSTEOARTHRITIS, INFLAMMATION AND

Amsterdam • Berlin • Oxford • Tokyo • Washington, DC

Distributor in the UK and Ireland Distributor in the USA and Canada

PRINTED IN THE NETHERLANDS

Aigner Thomas Blanco Franscisco J.

Ding Lei New York, NY 10021

Guo Danping Kirchmeyer Melanie

Lopez-Armada Maria J. Oliviero Francesca

Piera-Velazquez Sonsoles Sfriso Paolo

Thedens Daniel R. Nijmegen

This monograph was published in appreciation of OARSI and of European Society on

Part I. Extra Cellular Stimuli

I. Inflammatory Factors Involved in Osteoarthritis 3

Part II. Signalling Mechanisms

VII. MAP Kinases 99

Part III. Effectors and Different Pathways

X. Prostaglandin E2 and Osteoarthritis: The Role of Cyclooxygenases,

Part IV. Imaging and Clinical Applications

XV. Magnetic Resonance Imaging of Cartilage: New Imaging and Clinical

Author Index 299

Inflammatory Factors Involved in

Considerable evidence has accumulated to indicate that the proinflammatory cytokines

Nitric Oxide and Eicosanoids

In addition to proinflammatory cytokines, other inflammatory mediators could also

Other major inflammatory factors involved in OA pathophysiology are the pros-

OA is mediated by a multitude of complex autocrine and paracrine anabolic and cata-

Mechanical Loading Effects on Articular

Abstract. This chapter examines the effects of mechanical loading on cartilage

Osteoarthritis (OA) is a disabling disease that is estimated to impact 1 in 3 individuals

1. Mechanical Loading and Cartilage Homeostasis

2. Articular Cartilage: Biochemistry and Biomechanics

3. Cartilage Explants: Mechanical Loading and Metabolism

Effects of mechanical loading on articular cartilage metabolism have been investigated

unconfined compression produces a weakening of the collagen network and an increase

4. Isolated Chondrocytes: Mechanical Loading and Metabolism

transport limitations for maintenance of an organized load-bearing matrix. Studies on

5. Cartilage Metabolism and Matrix Degradation

metalloproteinases are elevated during periods of active joint destruction [119–121].

Transmittal of the mechanical forces to chondrocytes localized within an extracellular

Aging, Inflammation, and Altered

Abstract. Extracellular matrix calcification with calcium pyrophosphate dihydrate

Keywords. PTH, PTHrP, NPP1, ANKH, PPi, transglutaminase, chondrocyte hy-

Scope of the Problem of Joint Cartilage Calcification

Extracellular matrix calcification commonly develops in osteoarthritic (OA) cartilage.

CPPD Crystal Deposition and Prognosis in OA

cinosis by modulating chondrocyte maturation to hypertrophy, (ii) chondrocyte re-

Altered Chondrocyte Differentiation and Matrix Calcification in OA:

Inflammatory Mediators in Extracellular Matrix Modification for Calcification

Alteration of the articular cartilage extracellular matrix by hypertrophic chondrocytes

tion [26–28,38]. Specifically, we treated TG2 knockout mouse chondrocytes with

mediated manner, associated with rapid phosphorylation of p38 kinase dependent on

Role of PTH, PTHrP, and the Calcium Sensing Receptor (CaR)

PTHrP is a central regulator of spatial and temporal aspects of chondrocyte develop-

tissue inhibitor of metalloproteinase (TIMP)-3, effects shared by elevated extracellular

Potential Translational Relevance of PTH in Chondrocalcinosis

Though chondrocalcinosis is highly prevalent in elders in Western countries, there have

Dysregulated Chondrocyte ATP Metabolism and PPi Generation in OA and

NO is a central mediator of the pathogenesis of OA, and NO suppresses mitochondrial

Dysregulated Responsiveness to the Chondrocyte Growth Factors TGFβ and

Dr. Terkeltaub’s research on OA is supported by the Department of Veterans Affairs

Leptin, the Prototype of Adipokines:

Abstract. The prevalence of obesity and obesity-related diseases focussed an in-

White Adipose Tissue: A Surprising Biochemical Factory

Leptin and Its Receptors