Tissue Engineering

of

Cartilage
Evan Witmer
Gregory Lynn

Overview
Cartilage Basics
Ailments, Traditional Treatments, &
Tissue Engineering
Six Current Publications
Objective & Justification
Methods & Materials
Results
Summary
Acknowledgements

What is Cartilage?
Gel-like connective tissue made
up of chondrocytes, collagen,
proteoglycans, and other ECM
proteins
70-80% water
Avascular
no nutrients, no regeneration
3 types:

Hyaline (low-friction)
Elastic (epiglottis)
Fibrocartilage (shock absorbing)

Ailments & Traditional Treatment

Causes: Trauma, chronic wear,
developmental disorders, immobility
Little-to-no natural regeneration
Autografts (self) / Allografts
transplantation of donor cartilage
Debridement (smoothing)
Marrow stimulation
Problems: site morbidity, limited
availability, immune responses
Primary Source: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3146065/

Advent of Tissue Engineering

Creates an embryonic-like environment that stimulates growth

Custom-made scaffolds for strength, structure, and function
Resulting structure is true self-regenerated tissue
Single surgery required

IF: 5.68 - (2006)

Objective: To better assess the applicability of the electrospinning
technology for scaffold fabrication, through electrospinning six common poly
(-hydroxy esters) and testing their characteristics

Electrospinning
high surface area to volume ratio
similar structural morphology to the fibrillar ECM
Poly(-hydroxy esters)
biodegradable & FDA approved

Methods & Materials

6 Poly(-hydroxy esters)
spun into fibrous scaffolds
A.) PGA
B.) PLGA5050
C.) PDLLA
D.)PLGA8515
E.) PLLA
F.) PCL

Analysis
Coat in gold & SEM
Apply load

Results

SEM
uniform, randomly oriented fibers
Tensile testing
PLGA5050 & PGA highest modulus & yield stress

Results

Cell-Matrix Interaction (After 7 days)

Remained on surface of PGA, PDLLA, PLGA5050, and PLGA8515
Chondrocytes integrated into PLLA & PCL
Cellular proliferation
Chondrocytes

Mesenchymal Stem Cells

(2012) - IF: 5.68

Objective: To describe the developmental physicochemical properties of
silk fibroin scaffolds derived from high-concentration aqueous silk fibroin
solutions.

Silk fibroin in vivo

degrades
easily tailored & processed
More than 10% aqueous silk fibroin solution

Methods & Materials

Silk fibroin scaffolds with

different initial
concentrations (in wt.%)
A.) 8%
C.) 10%
E.) 12%
G.) 16%,

Combined methodology
Salt-leaching & freezedrying
Create different
structures

Results

XRD
no significant differences between crystallinity of four scaffolds
low crystallinity and an uncertain amount of random coil
SEM
Silk-8 & Silk-10 had microspheres (100nm-10m)
Silk-12 and Silk-16 had pores (<10m)
Morphology varies by initial concentration

Silk-8

Silk-10

Silk-12

Silk-14

Results

All above 90% interconnectivity and

79% porosity
~15% of pores larger than 300m
Water up-take (30 days immersion)
Original weights
No significant difference in
morphology
Mechanical Properties
Silk-16 & Silk-12 values suitable
for meniscus and cartilage
engineering

Compressive Strength

European Cells and Materials

(2008) - IF: 4.89
Objective: To evaluate in vivo cartilaginous tissue formation by chondrocyteseeded fibrin/PLGA hybrid scaffolds.

Joint replacement
PGLA & fibrin scaffolds
in vitro
promoted cartilage constructs

Methods & Materials

fibrin/PLGA hybrid (A)

PLGA scaffolds soaked in
chondrocyte-fibrin solution

Control: Chondrocytes seeded

into just PLGA (B)

Implanted into dorsum of nude

mice
4 weeks

Results

Macroscopic observation
Original rounded cylindrical shapes
Smooth and glistening cartilage properties
No malignant invasion
1 Week in Vivo

2 Weeks in Vivo

4 Weeks in Vivo

PLGA

Results

Fibrin/PLGA hybrid superior

Higher cell viability and
proliferation
Superior cell distribution, cellmatrix organization, overall
ECM production

Fibrin/PLGA

Biomacromolecules
(2005) - IF: 5.78
Objective: To combine the benefits of a photocrosslinkable network with
the desirable material HA for cartilage tissue engineering.

Hyaluronic Acid (HA) forms aggrecans (proteoglycan CSPCP) & can aid in
wound healing
Hydrogels photoencapsulate chondrocytes
Easier to fill tight spaces and irregularly shaped defects

Materials & Methods

Materials:
HA and methacrylic anhydride
form MeHA
Polyethylene glycol
dimethacrylate (PEGDM)
Swine chondrocytes
Nude mice

Methods:
Fabrication of MeHA & PEGDM
Encapsulation by polymerizing with UV light
Culture in nude mice
4, 6, and 8 week dissection

Results

Mechanical properties
linear slope at low strains
(<20%), modulus correlates
with cross-linking density

Degradation over time

Strong correlation between
time and hydrogel crosslinking
density

Results

Cell viability
Decrease in viability with increase in
monomer concentration
Effect of molecular weight was
negligible
1 day (black) 1 week (white)

immediate (black), 4 weeks (gray), 8 weeks (white)

Neocartilage formation
After 4 weeks, all constructs
became opaque. Stains for
GAG formation
As effective as the PEGbased hydrogel

(2009)
IF: 8.31

Objective: Study aims to form non-toxic composite and study mechanical

properties and degradation of chitosan-HA in vitro.

New Material: water-soluble chitosan and oxidized hyaluronic acid (HA)

without toxic cross-linking
Crosslinking action with amino functions

No photopolymerization to prevent prolonged irradiation, no

photosensitizer (toxic)

Methods & Materials

Materials:
Chitosan
HA sodium
Succinic anhydride
Bovine chondrocytes
Methods
Succinyl-CS and Aldehyde-HA
synthesized and lyophilized
Hydrogels formed by mixing ratios of
1:9, 3:7, 7:3, 9:1
Schiffs base crosslinking reaction
Encapsulation:
Mix cells with S-CS,
Add A-HA to form gel

Results

Degradation rate
strong correlation to ratio of SCS to A-HA
more S-CS = slower weight loss

Cell adhesion
5:5 & 7:3 significantly
greater than 3:7 & 9:1

Results

Cell proliferation and viability

successful encapsulation
in a normal spherical
morphology
promoted cell survival

Successful crosslinking
possible with mild, non-toxic
reagents

(2004)
IF: 8.31

Objective: To study the response of bovine chondrocytes on native and

chemically modified BC (Phosphorylation & Sulfation)

Bacterial cellulose (BC) has unusual material properties & degradability

high water retention, fiber network, high wet tensile strength.
cost-effective mass producible BC mold

Lack of literature describing native mechanical properties for tissue

constructs

Methods & Materials

Materials
G. xylinus
H3PO4
NH2SO3H
Bovine chondrocytes
Methods
G. xylinus grown & BC purified
Phosphorylation
BC-P1 (30 min), BC-P2 (2 hr)
Sulfation
BC-S
Cultured human arterial cartilage for 8
days
collagen type II, plant-derived
cellulose, calcium alginate, and
tissue culture plastic

Results

Modifications changed
surface morphology to
adhere more strongly

Strong immune response

to native BC

Modified BC shows
higher growth at similar
immune response to
alginate & tissue culture
plastic

Results

Presence of cell
ingrowth promising
for scaffold
material
engineering

BC does not
prematurely
differentiate cells
to form fibroblasts

Summary
Scaffold Materials:

Synthetics
PGA, PLLA, PDLLA,
PLGA5050, PLGA8515, PCL
Natural
Silk, Bacterial Cellulose, HA,
Chitosan
Huge variety of materials

Techniques:

Electrospinning,
Lyophilization,
Injectable Hydrogels,
Photopolymerization

Cross-linking
generally determines
degradation rate

Future of Cartilage TE

Hydrogels mimicking ECM-like matrices

Self-assembling nanoscaffolds
Thermosensitive injectable materials
Biomimetic novel materials (coral, silk, etc.)

Acknowledgements
Dr. Abidian
Evan Witmer
Greg Lynn