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Articular Cartilage
Structure, Composition, Function
Composition
Sparse population of cells- chondrocytes
Large extracellular Matrix-water-proteoglycan-collagen
Comparing Skeletal Tissue Composition
StructureLamina Splendens
Superficialtangential zone
Zonal Topography
• STZ– Parallel collagen fibrils– Flattened cells– High water
• Middle Zone– Less organized, larger diameter collagen fibrils– Rounded cells
• Deep Zone– Perpendicular collagen fibrils– Highest proteoglycan content– Rounded cells arranged in columns
• Calcified Zone• Subchondral Bone
Collagen
Collagen Fiber Architecture
Collagen Fibril Organization
Summary of Collagen Synthesis
Proteoglycan
Proteoglycans are complex macromolecules
• Protein core• Polysaccharide
chains
Macromolecular ProteoglycanStructure
SEM View of the Proteoglycan Superstructure
How Collagen and Proteoglycan Interact
Nutrition & Articular Cartilage
• Main source originates from vascularity in the synovium
• Factors, vitamins, minerals, carbohydrates, metabolites rapidly diffuse through the synovial fluid
• Diffusion through the cartilage matrix is significantly slower
Proteoglycan Synthesis
Proteoglycan Degradation
Important Growth Factors
• PDGF– Stimulator of mitogenesis– Only important in OA and lacerative injury
• bFGF– Powerful mitogen– Works most effectively with other factors
• IGF-I and II– Mitogenic and anabolic (matrix inducer)– Maintains steady proteoglycan synthesis
• TGF-– Complex constellation of actions– Alterations in signaling correlate with OA
Degradative Enzymes Important in Cartilage
– Metalloproteinases• Collagenase, gelatinase, stromelysin• Depend on zinc binding• Collagenase targets triple helical collagen• Gelatinase targets individual collagen a chains• Stromelysin targets col2 and 9 and possibly aggrecan
– Cathepsins/Aggrecanases• Common forms include cathepsin D and B and
aggrecanase 1 and 2 (ADAMTS 4 and 5)• Exclusively targets aggrecan
Articular Cartilage:Development and Aging
immature maturing adult
Most Important Biomechanical Consideration
Donnan osmotic pressure Na+, Ca2+
H2O
Effects of Joint Loading and Motion
• Reduced loading (immobilization) = atrophy• Continuous static compression induced lesion
and chondrocyte apoptosis• Single high impact or repetitive trauma
induces catabolism• Repetitive moderate loading (e.g. running)
thought to be anabolic for proteoglycan• Failure of structural mechanisms induces
catabolism• How loading influences chondrocyte function
is unknown
Growth Plate Cartilage
& Endochondral Ossification
The Human Growth Plate
Endochondral Bone Formation Growth plate chondrocyte
differentiation
Proliferating
Hypertrophic
Resting
Bone
• Complex interplay of intercellular signals that co-ordinate
proliferation hypertrophyossification
• TGF-• BMPs• Retinoic Acid• PTHrP• Ihh• Wnts• Cytokines
Stages of Chondrocyte Maturation
Undiffere
ntiated
Prolife
rativ
e -
Prehypertr
ophic
Hypertrophic
Terminal
Matura
tion
Growth PlateChondrocytes
TGF- BMP
Type X CollagenMMP13
Alkaline PhosphataseOsteocalcin
VEGFApoptosis
Type II Collagen
Type X
28s rRNA
BMP-2 (ng/ml) 50 10025100
BMP-2 Stimulates Chondrocyte Maturation
Chick Caudal Sternal Chondrocytes treated for 8 days
Li, et al., Endocrinology 144: 2514-23, 2003
24h 48h 72h 96h 24h 48h 72h 96h TGF- - - - - + + + +
colX
18s RNA
TGF- Inhibits Chondrocyte Maturation
Chick Cephalic Sternal Chondrocytes
Ionescu, et al., Exp. Cell Res. 288:198-207, 2003
Smad1,5
P P
Smad4Smad4
Smad2,3
Smad2,3
BMP-2 responsive genes
Smad1,5
P
Smad4Smad
1,5
TGF- receptor BMP receptor
Smad4P Smad
2,3
TGF- responsive genes
TGF- and BMP Activate Smad Pathways
Smad3
Control TGF- BMP-2
Smad2
TGF- Induces Nuclear Localization of Smad2 and 3
What in vivo evidence is there that these signaling pathways are important in
regulating maturation of chondrocytes?
Smad3 deficient mice have accelerated chondrocyte maturation and OA.
How is TGF- signaling effected in chondrocytes isolated from the
neonatal sternum of wild type and Smad3-/- mice?
Assessment of signaling using a TGF--responsive promoter/reporter
4xSBE luciferase
P3TP-luc
1) Transfect
2) Treat with TGF-
3) Measure luciferase luminescence
Measuring activation of TGF-/Smad signaling induced by TGF-
SB
E L
uci
fera
se
0
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
WT KO
TGF- – + – +
Activation of the SBE-Luc Reporter in Smad3-/- Chondrocytes is Completely
Blocked
What is the phenotype of chondrocytes isolated from the
neonatal sternum of wild type and Smad3-/- mice?
Assessment of phenotypic gene expression
colX Expression is Elevated in Smad3-/- Chondrocytes
0
0.1
0.2
0.3
0.4
0.5
0.6
2 Days 4 Days 8 Days
co
lX E
xp
res
sio
nWT
KO
28S RNA
colX
0
0.5
1
1.5
2
2.5
3
3.5
AP
MM
P-9
MM
P-13
VEGF-A
Osteo
calc
in
WT
KO
Other markers of maturation are up-regulated in Smad3-/- Chondrocytes
Rel
ativ
e E
xpre
ssio
n b
y R
T-P
CR
(co
mp
ared
to
b-a
ctin
co
ntr
ol)
What other in vivo evidence is there that the BMP/TGF- signaling pathways are
important in regulating maturation of chondrocytes?
BMP signaling induction of the transcription factor Runx2 is critical for
terminal hypertrophy of chondrocytes and skeletal mineralization
WT Runx2 KO
Inactivating mutations of the Runx2 gene are linked to the development of cleidocranial dysplasia
Osteoarthritis
Osteoarthritis
Undiffere
ntiated
Prolife
rativ
e -
Prehypertr
ophic
Hypertrophic
Terminal
Matura
tion
Growth PlateChondrocytes
ArticularChondrocytes
Ihh colxalk phosBMP-6
MMP9, 13
Sox9col2
aggrecan/proteoglycans
VEGF OCapoptosismatrix calcification
osteoarthritis
TGF- BMP
Chondrocytes Express Hypertrophic Markers During Osteoarthritis
BMP-6 Immunostain
During OA, articular chondrocytes exhibit:
- Increased proliferation (cloning)
- Expression of MMPs, colX, BMP-6 and other hypertrophic markers
- Terminal hypertrophy and apoptosis
Cloning, Fibrillation and Ulceration
normal
OA
Definition and Pathology
• Progressive loss of articular cartilage without a major inflammatory component– Focal fibrillation and ulceration– Cartilage swelling due to ‘loosening’ of the
collagen matrix leading to increased Donnan osmosis
– Cartilage loss and destruction– Subchondral sclerosis– Cyst and osteophyte formation
Cartilage Loss and Destruction
Etiology
• Aging
• Alterations in matrix
• Alterations in cell activity/function
• Alterations in cell mediators
• Altered joint mechanics
• Immune responses
Loss of TGF- signaling is a candidate pathogenic mechanism for OA
Undiffere
ntiated
Prolife
rativ
e -
Prehypertr
ophic
Hypertrophic
Terminal
Matura
tion
ArticularChondrocytes
TGF-
WT
4 Month
4 Month KO
7 Month
KO
7 Month KO
Smad3-/- mice display an OA-like cartilage degeneration
Yang, et al., J Cell Biol. 153:35-46, 2001
1 Month