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Regenerative Medicine and Biomaterials for the Repair of Connective Tissues
Azioni libro
Inizia a leggereLunghezza: 1,014 pagine11 ore
- Editore:
- Elsevier Science
- Pubblicato:
- Jan 12, 2010
- ISBN:
- 9781845697792
- Formato:
- Libro
Descrizione
Regenerative medicine for the repair of connective tissues is a fast moving field which generates a lot of interest. Unfortunately the biomaterials and biomechanics for soft tissue repair has been under-represented in the past. Particularly the natural association between cartilage, tendons and ligaments is often not made.
Regenerative medicine and biomaterials for the repair of connective tissues addresses this gap in the market by bringing together the natural association of cartilage, tendons and ligaments to provide a review of the different structures, biomechanics and, more importantly, provide a clear discussion of practical techniques and biomaterials which may be used to repair the connective tissues.
Part one discusses cartilage repair and regeneration with chapters on such topics as structure, biomechanics and repair of cartilage. Chapters in Part two focus on the repair of tendons on ligaments with particular techniques including cell-based therapies for the repair and regeneration of tendons and ligaments and scaffolds for tendon and ligament tissue engineering. Addresses the natural association between cartilage, tendons and ligaments which is often not made Provides a review of the different structures, biomechanics and practical techniques which are used in the repair of connective tissues Chapters focus on such areas as cartilage repair and regeneration, the repair of tendons and ligaments, investigating techniques including scaffolds and cell-based therapies
Regenerative medicine and biomaterials for the repair of connective tissues addresses this gap in the market by bringing together the natural association of cartilage, tendons and ligaments to provide a review of the different structures, biomechanics and, more importantly, provide a clear discussion of practical techniques and biomaterials which may be used to repair the connective tissues.
Part one discusses cartilage repair and regeneration with chapters on such topics as structure, biomechanics and repair of cartilage. Chapters in Part two focus on the repair of tendons on ligaments with particular techniques including cell-based therapies for the repair and regeneration of tendons and ligaments and scaffolds for tendon and ligament tissue engineering. Addresses the natural association between cartilage, tendons and ligaments which is often not made Provides a review of the different structures, biomechanics and practical techniques which are used in the repair of connective tissues Chapters focus on such areas as cartilage repair and regeneration, the repair of tendons and ligaments, investigating techniques including scaffolds and cell-based therapies
Informazioni sul libro
Regenerative Medicine and Biomaterials for the Repair of Connective Tissues
Lunghezza: 1,014 pagine11 ore
Descrizione
Regenerative medicine for the repair of connective tissues is a fast moving field which generates a lot of interest. Unfortunately the biomaterials and biomechanics for soft tissue repair has been under-represented in the past. Particularly the natural association between cartilage, tendons and ligaments is often not made.
Regenerative medicine and biomaterials for the repair of connective tissues addresses this gap in the market by bringing together the natural association of cartilage, tendons and ligaments to provide a review of the different structures, biomechanics and, more importantly, provide a clear discussion of practical techniques and biomaterials which may be used to repair the connective tissues.
Part one discusses cartilage repair and regeneration with chapters on such topics as structure, biomechanics and repair of cartilage. Chapters in Part two focus on the repair of tendons on ligaments with particular techniques including cell-based therapies for the repair and regeneration of tendons and ligaments and scaffolds for tendon and ligament tissue engineering. Addresses the natural association between cartilage, tendons and ligaments which is often not made Provides a review of the different structures, biomechanics and practical techniques which are used in the repair of connective tissues Chapters focus on such areas as cartilage repair and regeneration, the repair of tendons and ligaments, investigating techniques including scaffolds and cell-based therapies
Regenerative medicine and biomaterials for the repair of connective tissues addresses this gap in the market by bringing together the natural association of cartilage, tendons and ligaments to provide a review of the different structures, biomechanics and, more importantly, provide a clear discussion of practical techniques and biomaterials which may be used to repair the connective tissues.
Part one discusses cartilage repair and regeneration with chapters on such topics as structure, biomechanics and repair of cartilage. Chapters in Part two focus on the repair of tendons on ligaments with particular techniques including cell-based therapies for the repair and regeneration of tendons and ligaments and scaffolds for tendon and ligament tissue engineering. Addresses the natural association between cartilage, tendons and ligaments which is often not made Provides a review of the different structures, biomechanics and practical techniques which are used in the repair of connective tissues Chapters focus on such areas as cartilage repair and regeneration, the repair of tendons and ligaments, investigating techniques including scaffolds and cell-based therapies
- Editore:
- Elsevier Science
- Pubblicato:
- Jan 12, 2010
- ISBN:
- 9781845697792
- Formato:
- Libro
Correlati a Regenerative Medicine and Biomaterials for the Repair of Connective Tissues
Anteprima del libro
Regenerative Medicine and Biomaterials for the Repair of Connective Tissues
dosgohj@nus.edu.sg
1
The structure and regenerative capacity of synovial joint tissues
A.-M. Säämänen, University of Turku, Finland
J.P.A. Arokoski, University of Kuopio and Kuopio University Hospital, Finland
J.S. Jurvelin, University of Kuopio, Finland
I. Kiviranta, University of Helsinki, Finland
Abstract:
This chapter provides an introduction to the structure, function, and biomechanical properties of synovial joint and its tissues with special emphasis to articular cartilage. Structural elements are described at the cellular level. Major extracellular matrix components, their organization and relationship with biomechanical properties are described. Also, a short introduction to basic methodology to measure biomechanical parameters is presented. In addition, the studies demonstrating presence of human endogenous multi-potent mesenchymal stem/stromal cells (MSCs) and mesenchymal progenitors in synovial joint and associated tissues are reviewed. Possible implications of endogenous MSCs in tissue repair potential are discussed.
Key words
synovial joint
biomechanics
multi-potent mesenchymal stromal cell
tissue regeneration
1.1 Introduction
The purpose of this chapter is to introduce the reader to the structure and function of synovial joint and associated structures. First, the macroscopic structure of synovial joint compartments, cellular composition, tissue organization, and description of the major extracellular components will be reviewed in articular cartilage, subchondral bone, tendon and ligaments, synovial membrane, and meniscus. Second, the interrelationship of extracellular matrix composition with biomechanical properties of the tissues will be discussed. In addition, the basic methodology used for measuring the biomechanical parameters of joint tissues, with emphasis on articular cartilage, will be introduced. Third, the presence of resident mesenchymal stromal cells (MSCs) and progenitors in synovial joint tissues will be described, and differences in properties of MSCs derived from different intra-articular and extra-articular tissues will be discussed. MSCs represent the intrinsic repair potential in these tissues, but they also have a significant input in regulating tissue homeostasis by secreting several growth factors, cytokines and bioactive factors. The progress of stem cell research during the last ten years has increased our understanding of their function in tissue regeneration. However, knowledge of the role of MSCs in the repair processes of many joints tissues is still deficient.
1.2 Structure and function of synovial joint
The synovial joint is a functional unit with mechanically interacting structural components (Fig. 1.1). The development of synovial joints arises from the mesenchymal cells (Archer et al. 2003; Khan et al. 2007). Hyaline cartilage itself forms the cartilaginous model of the developing skeleton. It is replaced by bone in a process known as endochondral ossification (Mackie et al. 2008). Articular cartilage (AC) covers the ends of the bones and synovial fluid lubricates and nourishes the cartilaginous tissue. Ligaments bind the skeletal elements together and a fibrous capsule encapsulates the joint. The synovial joint (e.g. knee joint) may also contain meniscal structures internally. Each joint tissue, including bone, muscle, AC, ligaments, and tendons, has its unique structure and functional properties, and changes in any component may lead to anabolic or catabolic responses in another joint component.
1.1 Schematic presentation of the anatomy of the knee joint. A sagittal view.
The knee joint, joining femur and tibia in the lower limb, is the biggest synovial joint in the body. It consists of three articulating bones (femur, tibia and patella) covered by hyaline cartilage, the quadriceps and hamstring muscles, collateral and cruciate ligaments that hold the joint together, patellar tendon and the menisci. In principle, the knee is constructed of two joints, i.e. the patello-femoral joint and the tibio-femoral joint. The knee joint structures enable compression, rolling and sliding between the contacting bones. Also, the joints transmit loads of the upper body, reaching tibio-femoral loads of eight times the body weight (Kuster et al. 1997) and patello-femoral joint loads seven times the body weight (Nisell 1985) during normal daily activities, such as downhill walking and jogging. Based on the experimental analysis during simulated walking cycle, maximum tibio-femoral contact stresses of 14 MPa were recorded (Thambyah et al. 2005). This could be considered potentially dangerous for AC, knowing that there appears to be a critical threshold stress (15–20 MPa) that causes cell death and rupture of collagen network in vitro (D’Lima et al. 2001; Torzilli et al. 2006). It is suggested that the amount of load-induced cell death is a function of the duration and magnitude of the applied load.
In a healthy synovial joint, the friction coefficient between contacting articular surfaces is low; typical values between 0.01 and 0.04 have been estimated in a human hip (Unsworth et al. 1975). Several lubrication theories have been proposed, including hydrodynamic, squeeze film, and weeping and boosted mechanisms for lubrication. Each of them specifically addresses the role of intrinsic fluid and synovial fluid. Based on the operational demand, more than one lubrication mechanism is needed to provide the low friction within the synovial joint. For lubrication, the highly important mechanism is the interstitial fluid pressurization within the cartilage matrix (Ateshian 2009). However, during static loading, the boundary type of lubrication is facilitated by the molecules such as hyaluronan (HA), glycoproteins, and surface active phospholipids found in the synovial fluid (Katta et al. 2008).
Functional adaptation is known as conditioning of the structure, composition and functional properties of the joint tissues to mechanical loads they are exposed to (Hyttinen et al. 2001; Tammi et al. 1987). In a healthy joint, this will lead to optimized joint function. However, mechanical conditioning may fail, leading to overloading of joint structures and, subsequently, to harmful changes in the tissues. Further, this will create an imbalance between tissue properties and functional demands, leading potentially to progressive degeneration of the structures in question. In osteoarthritis (OA), the pathological process may be triggered by changes in any joint component and no consensus has been found for the initial pathological mechanism. However, mechanical factors have been considered critical in the initiation and progress of OA. Changes in cartilage, such as early superficial depletion of proteoglycans (Arokoski et al. 2000; Helminen et al. 2000) has been considered as a primary mechanism for the OA process, but alternative theories address the role of initial subchondral changes, including bone stiffening (Radin and Rose 1986; Burr and Radin 2003), in the pathological degeneration of cartilage tissue.
1.3 Joint tissues and their biomechanical properties
1.3.1 Articular cartilage
Articular cartilage is highly specialized connective tissue, and it is aneural, alymphatic, and generally considered to be avascular. Its nourishment depends on the synovial fluid and subchondral bone. The thickness of AC varies from some micrometers to a few millimeters in different cartilage areas within the joint, in different joints, and animal species. Biomechanically, the primary function of the AC is to provide a covering material that protects the subchondral bone and provides a smooth, lubricated surface that facilitates movements with little friction between the articulating surfaces. Cartilage has the ability to reduce the nominal contact pressures and to increase joint congruence.
Traditionally, AC is divided into three pseudo-stratified zones that are separated by a tidemark from the deepest calcified zone (Fig. 1.2). This classification is based on collagen fibril orientation and the organization and morphology of chondrocytes at different depths throughout AC. The zones can be characterized as follows:
1.2 A schematic presentation of the structure of articular cartilage illustrating the different zones and regions of articular cartilage and subchondral bone. Collagen fibrils are oriented tangentially in the superficial zone and radially in the middle and deep zones of the articular cartilage. Chondrocytes in the superficial zone are discoidal and in the deep zone spheroidal and often form columns. Tidemark at the mineralization front separates deep zone and calcified zone. Subchondral bone plate and trabecular bone provide support to the articular cartilage and is vascularized.
• Zone 1: Superficial (tangential) zone; adjacent to the joint cavity occupying 5–10% of the matrix volume. It is characterized by relatively low proteoglycan (PG) content. The collagen fibrils are closely packed and the orientation of fibrils is predominantly tangential (parallel) to the surface. The cells are discoidal with their long axes parallel to the surface.
• Zone II: Middle (transitional or intermediate) zone; occupies up to 45% of the matrix volume. It is characterized by a significant increase in the PG content. The collagen fibrils are randomly oriented. The cells are spheroidal and equally spaced.
• Zone III: Deep (radial) zone; occupies up to 45% of the matrix volume and has the highest PG content. The distance between collagen fibrils increases and they are arranged perpendicularly (radially) to the articular surface. Spheroidal cells often form columns aligned with the radial collagen fibrils.
• Zone IV: Calcified zone; adjacent to the subchondral bone occupies 5–10% of the matrix volume. There are a few chondrocytes, the matrix is mineralized with crystals of calcium salts and the PG content is low. The collagen fibrils are radially aligned. A borderline, called the ‘tidemark’, separates the calcified zone from the deep zone.
This zonal pattern is present in adult AC of most species, although the relative proportion of each zone varies. The extracellular matrix surrounding each chondrocyte can be subdivided into three discrete zones, which are the pericellular, territorial, and interterritorial matrices.
Chondrocytes
Cell volume, 2–10% of the total tissue volume, in AC is low in comparison with other tissues. Chondrocytes are the only cell type in AC. As being entrapped within this highly organized matrix and isolated in the lacunae that is called as chondron, mature chondrocytes have essentially no migratory potential, although some in vitro motility has been shown (Poole 1997; Morales 2007). Chondrocytes are ultimately responsible for the integrity, organization, and maintenance of the extracellular matrix (ECM) (Bhosale and Richardson 2008).
Three subpopulations of chondrocytes with different morphologies have been identified in AC (Kouri et al. 1996). Type 1 cells found in superficial and upper middle zones of AC represent typical chondrocytes. Type 2 cells represent secretory, fibroblast-like cells that are more abundant in the non-fibrillated osteoarthritic samples than in healthy cartilage. Type 3 cells are degenerating chondrocytes found throughout the OA cartilage although more frequently in the fibrillated tissue.
Extracellular matrix
Articular cartilage consists of two principal phases: a solid organic ECM, which is predominantly composed of collagen fibrils and PGs, and a mobile interstitial fluid phase. Water and inorganic salts make up about 70–80% of the tissue wet weight. The distribution of the chondrocytes and ECM constituents varies throughout the thickness of the cartilage. The structure of cartilage ECM is reviewed in more detail in Chapter 3.
Type II collagen is the most abundant protein in the ECM of AC, where it forms a three-dimensional fibrous network of alpha1 (II) collagen homotrimers assembled into copolymeric bundles with type IX and XI collagens (Eyre 2002). Also minor amounts of type III, VI, XII and XIV collagens are found in cartilage. Type X collagen is normally restricted in the hypertrophic zone in AC cartilage and growth plate.
The three-dimensional network of collagens is filled with PGs that are macromolecules consisting of a central core protein to which one or more glycosaminoglycan (GAG) chains and oligosaccharides are attached (Knudson and Knudson 2001; Roughley 2006). On mass basis, aggrecan is the most abundant PG in cartilage (Fig. 1.3). The core protein of the aggrecan can be divided into N-terminal HA binding region with a G1 globular domain, small interglobular domain, another N-terminal globular domain G2, keratan sulfate attachment domain KS, two chondroitin sulfate attachment domains CS1 and CS2, and a lectin binding C-terminal globular domain G3 (Fig. 1.3). G1 domain interacts with link protein to stabilize the binding of aggrecan molecules into large aggregates with HA. The GAGs are unbranched carbohydrates made up of repeating disaccharide units with negatively charged sulfate and carboxyl groups responsible for the water binding properties of the aggrecan molecule that regulate the elastic properties of the cartilage (Cowin and Doty 2007a).
1.3 Schematic presentation of the major extracellular matrix components of articular cartilage, (a) Aggrecan molecules attach to hyaluronan with link protein to form large (> 1 × 10⁶ Da) proteoglycan aggrecates which are entrapped within the network of type II collagen fibrils, (b) Characteristic structural domains of aggrecan. Hyaluronan (HA); two interglobular domains (IGD) G1 and G2; keratan sulfate binding region (KS), chondroitin sulfate binding region 1 (CS1), chondroitin sulfate binding region 2 (CS2), and a C-terminal globular domain (G3) (adapted from Qu 2007).
Chondrocytes synthesize also numerous other non-collagenous ECM components (Roughley 2001, 2006). Small leucine-rich repeat proteoglycan (SLRP) family members decorin, biglycan, fibromodulin, lumican, and others have functions in regulating the collagen fibrillogenesis, activity and distribution of different cytokines and growth factors, as well as in cell signaling (Schaefer and Iozzo 2008). Several other PGs and glycoproteins are found in the extracellular space, e.g., cartilage oligomeric protein (COMP), versican and tenascin, or on the chondrocyte surface, e.g., perlecan, syndecans, integrins and growth factor receptors (van der Kraan et al. 2002; Chiquet-Ehrismann and Tucker 2004; Melrose et al. 2008; Shakibaei et al. 2008). These factors and their relationship in tissue function, homeostasis and degeneration have been reviewed by, for example, Gentili and Cancedda (2009), Goldring et al. (2008) and Goldring and Marcu (2009), and are more thoroughly reviewed in the other chapters of this book.
The poor intrinsic repair capacity of adult AC results from avascular nature of the tissue. Lacking the blood vessels, the damage in AC produces no inflammatory reaction, which would lead to chemotactic recruitment of the repair cells, typical in the repair process of other tissues. Also, synthesis and turnover of type II collagen in adult AC are exceptionally low, the half-life of the collagen molecules being over one hundred years (Maroudas et al. 1992; Verzijl et al. 2000). Hence, any marked injuries in the collagen network are likely to initiate a cascade leading to progressive degradation of AC (Aigner and Stöve 2003).
1.3.2 Subchondral bone
Subchondral bone is formed via endochondral ossification of the cartilage template at the secondary ossification centers of the bone epiphyses during joint development (Burr 2004; Mackie et al. 2008). Subchondral bone provides support to AC, and biomechanically it has an essential role on cartilage homeostasis.
Subchondral bone consists of the subchondral bone plate (SBP) to which the subchondral trabecular bone (STB) is attached (Fig. 1.2) (Imhof et al. 2000; Burr 2004). Both SBP and STB are formed of bone lamellae. Subchondral bone is composed of two types of lamellae: concentric lamellae around the osteons and flat lamellae. SBP, like compact bone, is composed of subunits called osteons consisting of concentric lamellae surrounding the central (Haversian) canal. STB and the inner surface of SBP are covered by osteoblasts and osteoclasts. Physiologically and mechanistically distinct STB is highly vascularized and it provides another route for cartilage nutrition in addition to synovial fluid (Imhof et al. 2000). The subchondral bone structure, i.e., SBP thickness and STB density vary with region in the joint (Oettmeier et al. 1992). Based on Wolffs law, both the bone density and the organization of bone trabeculae correlate with the magnitude and direction of compressive and tensile stresses of loading (Wolff 1892).
There are five different bone cell types – osteocytes, osteoprogenitors, bone lining cells, osteoblasts, and osteoclasts. Osteoblasts and osteoclasts form bone remodeling units that maintain the integrity of the bone and balance between deposited and resorbed bone (Cowin and Doty 2007b; Bartl and Frisch 2009). Bone matrix is composed of organic and inorganic components. Up to 88% of organic matrix is collagen, mainly type I collagen, which forms an organized template for the matrix mineralization by deposition of hydroxyapatite and apatite. In addition, organic matrix contains up to 12% of the dry weight of osteocalcin, osteonectin, several phosphoproteins, lipids and proteoglycans. Bone marrow of the trabecular bone maintains a heterogeneous population of various multi-potent mesenchymal stromal cells that provide progenitors for differentiation of osteochondral and other mesenchymal cell lineages as well as trophic environment for hematopoiesis.
1.3.3 Meniscus
Menisci are semi-lunar discs between tibia and femur in the knee joint protecting AC from excess shocks by distributing loads and stabilizing joints during movement (Fig. 1.4(a)) (Setton et al. 1999a; Sweigart and Athanasiou 2001). Meniscus tissue is composed of outer, dense connective tissue and inner fibrocartilage regions (Fig. 1.4(b)) (Verdonk et al. 2005). The outer region is vascularized dense fibrous connective tissue that connects to the internal knee joint capsule. Its matrix is maintained by fibroblast-like fibrochondrocytes which produce type I collagen fibrils. Elastin fibers as bridge-like connections between collagen fibrils have been suggested as contributing to the recovery after deformation (Ghosh and Taylor 1987). The inner region of the meniscus is avascular, aneural, and alymphatic tissue, which is why, similar to cartilage, its repair capacity is lower than that of the outer region. Cells in the inner region are chondrocyte-like fibrochondrocytes, and its matrix contains many similar components common to cartilage and tendon. Type II collagen forms about 60% and type I collagen about 40% of the total collagen in the inner region. Minor amounts of type III, V and VI collagens have been found in both regions (Sweigart and Athanasiou 2001).
1.4 Schematic presentation of the menisci in the knee joint. (a) Macroscopic view of the proximal tibia with menisci, and insertion sites of ligaments. (b) Vascularized outer region and avascular inner region of the meniscus. (c) Schematic organization of the collagen fibers in different layers of the meniscus. Collagen fibers at the superficial layer on both tibial and femoral sides are thin and intersect at various angles. Below that on both sides are lamellar layers where collagen fibers are arranged into lamella-like bundles that are radially arranged in the external circumference of the anterior and posterior segments of bundles, and intersect at various angles in the other regions. In the central main layer the bundles of collagen fibers are oriented in a circular manner, with a few interwoven radial tie fibers in the internal circumference. At the external circumference, also loose connective tissue from the capsule penetrates the central main layer (arrow) ((b) adapted from Verdonk et al., 2005 and (c) adapted from Petersen and Tillmann 1998).
Collagen fibers are radially and circumferentially organized to provide appropriate structure to resist tensile forces. Based on the collagen fiber orientation and thickness, three different layers can be recognized in the inner fibrocartilage region (Fig. 1.4(c)) (Petersen and Tillmann 1998; Sweigart and Athanasiou 2001).
Aggrecan is the major PG responsible for the maintenance of viscoelastic properties of the tissue, and its concentration is highest in the inner and middle parts of the meniscus, decreasing towards the periphery. It forms a spatially organized network in contrast to cartilage, where it is more diffusely distributed (Valiyaveettil et al. 2005).
Perlecan and SLRPs decorin, biglycan, fibromodulin, and keratocan, and small amounts of adhesion molecules such as fibronectin and thrombospondin are also found in the meniscus (Melrose et al. 2005, 2008). PGs residing particularly at the surface zone are thought to contribute to the smooth frictionless movement of the menisci over the articular surfaces (Melrose et al. 2005, 2008).
1.3.4 Tendon and ligaments
Tendons are specialized dense connective tissue structures connecting bones and muscles, while transmitting forces and allowing joint movements (Kannus 2000). Tenoblasts in the developing and young tendon, and tenocytes, elongated and dispersed fibroblast-like cells in the adult tendon, form about 90–95% of the cells in tendon. The remaining cells are chondrocyte-like cells at regions of pressure and insertion sites, entheses, and synoviocytes on tendon surface, and vascular cells in the endo-and epitenon regions (Benjamin et al. 2006; Cowin and Doty 2007c; Kannus 2000; Riley 2008).
The tendon matrix is maintained by tendon cells that are embedded within the long collagen fibrils running parallel to each other and arranged into bundles in a staggered fashion (Fig. 1.5). Type I collagen is the major collagen component of the ECM, but it contains also ‘minor’ collagens and elastin, glycoproteins and adhesive group molecules, e.g., fibronectin, thrombospondin, tenascin C, and undulin (Kannus 2000). Aggrecan and versican are the large PGs providing, together with HA the properties to resist compressive and tensile forces during movements. SLRPs such as fibromodulin, biglycan, and decorin are found to regulate collagen fibrillogenesis, bone morphogenic protein (BMP) activity or stem cell niche organization (Yoon and Halper 2005; Zhang et al. 2005; Bi et al. 2007).
1.5 Collagen fibril organization in (a) tendon and (b) ligament (adapted from Nordin and Frankel 1989).
Ligaments are dense connective tissue structures connecting articulating bones and giving stability to the joints (Duthon et al. 2006; Cowin and Doty 2007c; Petersen and Zantop 2007). Although they are anatomically distinct from tendon, they have an overlapping gene expression profile and matrix composition (Rumian et al. 2007). Spindle shaped fibroblasts maintain the ECM that is mainly composed of type I collagen fibers but also contains some other collagens, e.g., type II collagen in the endotendon, type III collagen in the reticular fibers, type IV collagen in the vascular basement membranes, and type VI collagen as a gliding component between functional fibrillar units (Duthon et al. 2006). Parallel organization of collagen fibrils into bundles together with PGs, glyco-conjugates and elastic components results in the formation of a unique, complex elastic network capable of withholding varying multiaxial stresses and tensile strains (Cowin and Doty 2007c). The degree of collagen fibril organization is lower than in tendon (Fig. 1.5) which also has consequences in the biomechanical properties (see below).
1.3.5 Synovial membrane and synovial fluid
Synovial membrane or synovium secretes joint lubricating components into the synovial fluid, nourishes the joints, and removes debris from the synovial space. It is composed of two layers, the intimal layer and the loose connective tissue layer (FitzGerald and Bresnihan 1995; Iwanaga et al. 2000; Sutton et al. 2009). Three basic cell types, type A synoviocytes, type B synoviocytes, and dendritic cells, are found in the intimal layer. Type A synoviocytes, or macrophage-like synoviocytes, are likely to originate from bone marrow and can be considered as resident or tissue macrophages that are mainly phagocytic with large Golgi complex and lysosomes (Iwanaga et al. 2000). Type B synoviocytes, or fibroblast-like synoviocytes, manufacture collagen, fibronectin, HA, and PG 4 (also known as lubricin, megacaryocyte stimulating factor (MSF) and superficial zone protein (SZP)) into synovial fluid to maintain joint lubrication (Rhee et al. 2005; Elsaid et al. 2007; Wann et al. 2009). They differ from other deeper, subintimal fibroblasts in that they contain characteristic lamellar bodies, and produce also surfactant protein A and VCAM-1 (FitzGerald and Bresnihan 1995; Vandenabeele et al. 2003). Dendritic cells form less than 1% of the synovial cells (FitzGerald and Bresnihan 1995). They are potent antigen presenting cells that have a pro-inflammatory role in initiation of the immune responses in rheumatoid arthritis (RA), where they are the effectors of cartilage destruction and a major source for inflammatory cytokine TNFa which indirectly induces cartilage collagenolysis (Lutzky et al. 2007).
Below the intimal layer there is a loose connective tissue layer that contains fibroblasts, macrophages, adipocytes, mast cells, nerve fibres, vascular endothelial cells, granulocytes, and lymphocytes (FitzGerald and Bresnihan 1995). It is well vascularised, innervated and supplied by lymphatic vessels (Sutton et al. 2009).
Synovial fluid is the joint lubricant and shock absorber for AC. Synovial fluid is a blood plasma dialysate, which contains HA and glycoproteins, synthesized by type B synovial lining cells (Fam et al. 2007). HA contributes to the high viscosity and lubricating properties of synovial fluid and is currently used also as a therapy for OA. Recently the synthesis and active secretion of HA were coupled to the movements and use of the joint (Ingram et al. 2008).
In addition to substances secreted by the lining cells, synovial fluid contains plasma proteins that originate from the blood vessels vascularizing synovium (Fam et al. 2007). Cellular components are present in small amounts in normal synovial fluid, including different leukocytes: lymphocytes, monocytes, synovial lining cells, and polymorphonuclear cells (Fam et al. 2007).
The rate and mechanism of passage of substances going through synovium depend on the size of the molecules. Gases and crystalloids diffuse rapidly in both directions. Larger proteins are taken out of the synovial fluid by way of lymphatics. Macrophages phagocytose cellular debris and particles that are too large to be removed otherwise (Iwanaga et al. 2000). The turnover time for synovial fluid volume is estimated to be about one hour in rabbit and normal human knees, while that for HA is much longer, 17–30 hours in rabbit knee joints (Levick 1987; Ingram et al. 2008).
It is now commonly believed that synovial macrophages are responsible for producing the proinflammatory cytokines into the joint space and drive the inflammatory responses with stimulation of cartilage cytokines and matrix degrading proteases under pathological conditions such as OA or RA (Blom et al. 2007; Sutton et al. 2009).
1.3.6 Biomechanical properties of joint tissues
The mechanical properties of tissues can be determined from the load–deformation behavior in compression, tension, bending, or shear geometry. The 3- or 4-point bending tests have actively been used for bone samples and tension tests for tendons and ligaments. For mechanical testing of AC, compression, tension, and shear techniques have traditionally been applied. In compression, unconfined compression, confined compression, and indentation geometries (Fig. 1.6) and stress–relaxation or creep (Fig. 1.7) test protocols are generally in use.
1.6 Schematic presentation of the typical measurement configurations in use for mechanical testing of the articular cartilage, (a) Unconfined compression: the tissue is compressed between two smooth metallic plates allowing fluid flow in the lateral direction, (b) Confined compression: the tissue is placed in a metallic chamber and compressed with a porous filter allowing fluid flow axially through the filter, (c) Indentation: the tissue is compressed with a cylindrical plane-ended or spherical-ended indenter allowing fluid flow in both lateral and axial directions (from Saarakkala 2007).
1.7 (a) In a creep measurement, cartilage tissue deformation (strain) is recorded under a constant load (stress) applied at t0. (b) In a stress-relaxation measurement, the cartilage tissue load (stress) is recorded under a constant deformation (strain) applied at t0 (from Saarakkala 2007).
To calculate the true material properties of joint tissues, theoretical analysis of the measurements is needed. In most classical models, soft tissues are simplified as homogeneous isotropic linearly elastic materials. The relationship between the stress and strain is described as linear and two independent elastic constants are needed to describe the material, i.e., the elastic (Young’s) modulus (E) and Poisson’s ratio (υ) (Table 1.1). Consequently, this model is inadequate for characterizing time-dependent mechanical behavior of soft tissues, especially that of AC. However, it has been used actively to calculate the instantaneous (dynamic) or equilibrium (static) modulus for AC (Hayes et al. 1972; Jurvelin et al. 1990).
Table 1.1
Basic equations for the determination of isotropic elastic parameters of cartilage
F reaction force
A area of the surface in which the force is acting
L initial thickness
L′ thickness after compression/tension
σa and ∈a axial stress and strain
∈l lateral strain
a indenter radius
h cartilage thickness
κ,(a/h, υ) theoretical scaling factor due to finite and variable cartilage thickness (Hayes et al. 1972; Jurvelin et al. 1990)
The joint tissues are all viscoelastic in their mechanical behavior, i.e., the mechanical response, depends significantly on the rate of loading. Depending on the tissue type, the viscoelasticity may originate from the intrinsic property of the solid tissue, or from the interstitial fluid flow within the tissue under load. Under loads with volumetric dilatation, these two viscoelastic mechanisms are difficult to separate. The latter, called poroelasticity, is especially well recognized in AC. A traditional model for AC, taking the interstitial fluid movement into account, is the linear isotropic biphasic model (Mow et al. 1980). In addition to elastic parameters of the solid matrix, knowledge of the tissue permeability is needed for characterizing the time-dependent behavior of the tissue. To extract the model parameters, experimental mechanical measurements are conducted and, subsequently, the theoretical model is fitted to the experimental data.
As an extension of the biphasic model, fibril reinforced models have been introduced (Soulhat et al. 1999; Korhonen et al. 2003; Wilson et al. 2004). In these models, the compression-tension nonlinearity is taken into account by inclusion of the collagen fibril network. The material parameters of the fibril-reinforced model are Young’s modulus and Poisson’s ratio of the drained porous matrix, permeability, and Young’s modulus of the fibril network. Triphasic theory is an extension of the biphasic model incorporating three phases: an incompressible solid, an incompressible fluid, and a monovalent ionic phase (Lai et al. 1991). The model assumes that the total stress of the tissue is composed of the fluid stress, solid stress and chemical potentials. This model can be used to accurately include the effect of cartilage swelling. Owing to tissue heterogeneity and anisotropy, as well as to mimic realistic loading geometries, the model implementations for nonlinear behavior of AC are most often conducted numerically using finite-element analysis.
The structure, composition, and properties of all joint components have evolved on the grounds of their biological and mechanical function. In a healthy joint, uncalcified cartilage and meniscus (elastic modulus in compression of 0.1–1 MPa), calcified cartilage (elastic modulus ~ 0.3 GPa), and subchondral bone (elastic modulus > 1 GPa) establish a structural and functional continuum with optimal mechanical properties (Mente and Lewis 1994; Setton et al. 1999a; Arokoski et al. 2000; Helminen et al. 2000). The tibio-femoral contact stresses may be very high (> 10 MPa), compared with typical Young’s modulus of 1 MPa for solid matrix of normal cartilage, indicating that hydrostatic pressure within cartilage must serve as a primary mechanism for successful load bearing. Further, intrinsic fluid pressurization contributes significantly to low friction between articulating surfaces (Ateshian 2009). In OA, critical loss of fluid pressurization mechanism of load support takes place.
Tendons, with densely packed collagen fibers, show typically very high tensile modulus (> 1 GPa) and strength (> 100 MPa). Tendons are highly elastic with minor viscoelastic effects and their nonlinear tensile behavior is related to gradual alignment and stretching of the fibers (Ker 2007). Ligaments, owing to lower collagen content and highly woven collagen structure, are less stiff and strong than tendons.
Articular cartilage exhibits significant compression–tension nonlinearity. Compressive equilibrium modulus of healthy cartilage in unconfined compression is ~ 1 MPa; however, under highly dynamic loads hydraulic stiffening produces a modulus that is much higher, and comparable to that of tensile modulus (5–25 MPa, Setton et al. 1999b). Owing to inhomogeneous structure, e.g., depth-dependent increase of PG content, the compressive modulus and permeability increase and decrease, respectively, along cartilage depth (Schinagl et al. 1997; Boschetti et al. 2004). The tensile modulus is highest in the superficial cartilage zone, where the direction of the collagen fibers is parallel to articular surface, and the collagen content is highest. Deeper in the tissue, the more random orientation produces lower tensile stiffness (Kempson et al. 1973).
In joint tissues, each structural component and their interactions contribute to overall mechanical characteristics of the tissue. In cartilage, PGs, owing to the swelling stress they produce, and their effect to tissue permeability, are considered important for mechanical characteristics in compression, while collagen is the primary structure resisting tension (Huang et al. 2001). However, joint tissues can be considered to be biological composites, and the structural interactions critically control the mechanical behavior as well. Therefore, these sophisticated structures have remained difficult to replicate using tissue engineering methods, making tissue repair of, for example, AC, challenging. It is well shown that proper collagen cross-linking is essential for a functional matrix in both native and engineered cartilage (Broom 1984; Bastiaansen-Jenniskens et al. 2008). Further, mechanical properties of AC are sensitively modulated by the changes in structural integrity of the tissue (Fig. 1.8).
1.8 Minor degenerative changes in matrix, based on the scoring the histological integrity of the cartilage using Mankin score, can lead to inferior mechanical properties (e.g. dynamic modulus) of AC (from Laasanen et al. 2003).
1.4 Resident mesenchymal progenitor cells in synovial joint tissues
Remarkable progress during the last ten years of stem cell research has increased our understanding of how stem cells can be induced and manipulated to form repair tissue. Adult stem cells, especially bone marrow MSCs, which are a highly variable population of multi-potent mesenchymal stem cells and progenitors, have been actively characterized due to their great potential in regenerative medicine (reviewed by, for example, Barry and Murphy 2004; Keating 2006; Caplan 2007; Phinney and Prockop 2007; Abdallah and Kassem 2008, 2009; Chen and Tuan 2008; Jones and McGonagle 2008; Nöth et al. 2008; Arthur et al. 2009). Currently, MSCs and progenitors have been found residing virtually in all organs and tissues (Sakaguchi et al. 2005; da Silva Meirelles et al. 2006; Chamberlain et al. 2007; El Tamer and Reis 2009).
While the aim of this whole book is to gather together current understanding of the normal biology, disease pathogeneses, and different therapeutic approaches of connective tissue disorders, especially those related to joint and associated tissues, it is important also to be aware of the endogenous stem cells and progenitors residing in these tissues. Therefore, inventory of resident stem cells and progenitors in human synovial and associated tissue joints (Table 1.2), and some of their properties will be briefly discussed in this chapter.
Table 1.2
Studies demonstrating presence of multipotent progenitor cells in human synovial joint and associated tissues
¹Tested differentiation capacity to adipogenic (A), chondrogenic (C), osteogenic (O), ormyogenic (M) lineages.
²Cell source(s) used for MSC isolation are in parentheses: articular cartilage (AC), synovial membrane (SM), synovial fluid (SF), adipose synovium (AS), adipose tissue (AD), anterior cruciate ligament (ACL), posterior cruciate ligament (PCL),medial collateral ligament (MCL), tendon (TE),meniscus (ME), skeletalmuscle (MU), periosteum(PE).
1.4.1 Mesenchymal stromal cells
Stem cells have a potential to self-renew, proliferate, and differentiate into multiple cell types. Adult multi-potent stem cells have a more limited differentiation potential in comparison to embryonic stem cells. Early embryonic stem cells (derived from the inner layer of blastocyst) are totipotent and can give rise to all germ layers. Pluripotent embryonic stem cells lack differentiation potential to placental cells, but can differentiate to form other tissues. Two major classes of multi-potent stem cells are found in adult bone marrow, hematopoietic stem cells, and nonhematopoietic stromal cells. MSCs are multi-potent nonhematopoietic cells that can differentiate into mesodermal lineages (Fig. 1.9(a)). They represent a small percentage of the total population of nucleated cells in the bone marrow, where the majority of the cells consist of hematopoietic stem cells and hematopoiesis supporting stromal cells (Pittenger et al. 1999).
1.9 Definition of adult mesenchymal stem/stromal cell (MSC). (a) Mesenchymal tissues contain MSCs and progenitor cells that under defined conditions have a capacity to differentiate into multiple connective tissue cell types. (b) The minimal criteria for defining the term ‘multipotent mesenchymal stromal cell’ as suggested by the International Society for Cellular Therapy: (1) they must be plastic adherent, (2) express certain cell surface antigens, and (3) have a capacity to differentiate to at least chondrocytic, osteogenic and adipogenic lineages (Horwitz et al. 2005; Dominici et al. 2006) (adapted from Säämänen et al. 2008).
MSCs are defined as highly clonogenic cells having potential for self-renewal and differentiation into multiple mesenchymal tissues (Pittenger et al. 1999). Johnstone was the first to induce chondrogenic differentiation of mesenchymal progenitor cells isolated from rabbit bone marrow (Johnstone et al. 1998). In contrast to the hematopoietic stem cells, no single mesenchymal stem cell specific marker has been found so far. They appear to be a rather heterogeneous population and most of the cells seem to be progenitors rather than true stem cells. To clarify the confusion in the nomenclature and to attempt to standardize the research in this field, the International Society for Cellular Therapy has made two statements (Fig. 1.9(b)). First, they recommend the term ‘multi-potent mesenchymal stromal cell’ instead of the mesenchymal stem cell, the acronym still remaining the same ‘MSC’ for both (Horwitz et al. 2005). Second, multipotent MSCs should fulfill the minimal criteria of expressing certain surface antigens characteristic for mesenchymal cells. In addition, MSCs should not express some hematopoietic and epithelial cell surface antigens, and they should also have a capacity to differentiate under appropriate conditions to chondrogenic, osteogenic, and adipogenic lineages (Dominici et al. 2006). Several controversial issues prevailing in adult stem cell research including nomenclature, and other MSC characteristics, were critically discussed in a recent review by Darwin Prockop (2009).
1.4.2 Role of mesenchymal progenitor cells in joint homeostasis
Being resident in subchondral bone, bone marrow stromal cells may give rise to a spontaneous AC repair that is seen when cartilage lesions extend to the underlying bone, resulting in a formation of cartilage repair tissue. Already in the 1940s, before the era of the modern arthroplasty surgery, abrasion of the osteoarthritic joint surfaces and drilling several holes 6 mm in diameter to the subchondral bone were used as a therapy for OA (Magnuson 1941; Pridie 1959). However, functionally impaired fibrocartilagenous repair tissue did not give satisfactory results. Currently microfracture is a frequently used technique for the repair of AC lesions of the knee. In this ‘marrow stimulating’ technique an awl is used to penetrate the subchondral bone to produce small holes in cartilage defects, allowing marrow cells to migrate to the cartilage lesion site (Steadman et al. 2002). Recent studies have shown that microfracture provides effective short-term functional improvement of knee function, but often results in formation of suboptimal fibrocartilage (Knutsen et al. 2007; Mithoefer et al. 2009).
The principal role of adult MSCs and progenitors has been considered to maintain physiological balance in the organism by serving a cellular reserve for tissue remodeling and rejuvenation, but they can do more than just respond to stimuli and differentiate. Newly committed progenitor cells have been shown to secrete several growth factors and cytokines (Haynesworth et al. 1996), and immunosuppressive factors, e.g., HLA-G that interfere with the immune recognition system (Selmani et al. 2009; Siegel et al. 2009). Caplan and Dennis (2006) introduced a term ‘trophic mediator’ to MSCs, and defined the trophic effects as ‘those chemotactic, mitotic, and differentiation-modulating effects, which emanate from cells as bioactive factors that exert their effects primarily on neighboring cells and whose effects never result in differentiation of the producer cell’. Stem cells are maintained in so-called stem cell niches at specific sites in the tissues (Gregory et al. 2005; Jones and Wagers 2008; Walker et al. 2009). Stem cell niches have been characterized in tendon, AC, and zone of Ranvier, where PGs or their GAG sulfation patterns have been suggested as having important roles in maintaining and organizing the niches, and regulating the local BMP activity (Bi et al. 2007; Hayes et al. 2008; Karlsson et al. 2009). Hence, there seems to be a complex and bidirectional regulation system of the stem cell response to stimuli for differentiation and secretion of bioactive factors, thereby influencing tissue homeostasis (Fig. 1.10) (Caplan and Dennis 2006; Caplan 2009).
1.10 Mesenchymal stem cells are maintained in stem cell niches and function as trophic mediators and reservoir for tissue remodeling and repair. Proteoglycans, particularly glycosaminoglycan sulfation patterns of aggrecan and perlecan in cartilage (Hayes et al. 2008), and small leucine rich repeat proteoglycans (SLRPs) fibromodulin (Fmn) and biglycan (Bgn) in tendon (Bi et al. 2007), have been suggested to have important functions in regulating the organization and/or growth factor presentation in the niches (principle of the graph adapted from Caplan and Dennis 2006).
1.4.3 Articular cartilage
Articular cartilage was earlier thought to lack stem cells or progenitors but several recent studies have demonstrated their existence in the tissue (Table 1.2). A few years ago it was first shown that young bovine AC contains a multi-potent progenitor cell population in the superficial zone with differentiation plasticity into various connective tissues, including bone, tendon, and perimysium (Dowthwaite et al. 2004). Several studies have shown the presence of multipotent progenitors with limited expandability in AC of healthy young individuals (reviewed by Tallheden et al. 2006). In OA cartilage, increased number of cells with MSC phenotype has been found (Alsalameh et al. 2004; Fickert et al. 2004; Hiraoka et al. 2006). Adult human AC contained a small population of cells that coexpressed surface antigens CD105 and CD166 (ALCAM, activated leukocyte adhesion molecule) (Alsalameh et al. 2004). These markers have been proposed to define a population of MSCs in bone marrow stroma and have properties similar to mesenchymal progenitor cells (Majumdar et al. 1998). As the presence of CD105 +/CD166 + progenitor cells was significantly increased in OA cartilage, they were speculated to have a role in pathogenesis of OA (Alsalameh et al. 2004). As CD105 is endoglin, a TGFβ receptor III, its expression in MSC population is likely to enhance the chondro-genic potential due to responsiveness to exogenous TGFβ, which is used for induction of chondrogenesis.
Fate and differentiation regulating factor Notch1 has been regarded as a progenitor marker. Surprisingly high numbers of Notch1 positive cells have been found both in healthy and OA cartilage. Over 70% of the cells in primary culture of cells isolated from AC with induction in OA were Notch1 positive (Hiraoka et al. 2006). In normal human AC, taking all cartilage zones together, over 45% of the cells expressed progenitor markers Notch1, a stem cell marker Stro-1 and vascular endothelial molecule VCAM-1, with the highest expression in the superficial zone (Grogan et al. 2009). Most of the cells in chondrocyte clusters in OA cartilage also expressed all these progenitor markers, suggesting responses to OA pathogenesis. In another study with adult bovine knee AC, Notch1 expression did not correlate with multi-potent properties of the progenitors, thus questioning the value of Notch1 as an early progenitor marker, and suggesting that the actual progenitor cell population is much smaller in adult AC (Karlsson et al. 2008). Supporting this, normal and OA cartilage contained 0.14% so-called side-population (SP) cells identified by their negative staining for Hoechst 33342 dye, that differentiated into chondrocytes and osteocytes, but not adipocytes, thus likely representing a more primitive osteoprogenitor population than Notchl, Stro-1, and VCAM-1 expressing cells (Grogan et al.
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