Articular Cartilage

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Articular Cartilage

• Structure, Composition, Function

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Composition
Sparse population of cells - chondrocytes Large
extracellular Matrix -water -proteoglycan -coll
agen
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Comparing Skeletal Tissue Composition
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Structure
Lamina Splendens
Superficial tangential zone
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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

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Collagen
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Collagen Fiber Architecture
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Collagen Fibril Organization
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Summary of Collagen Synthesis
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Proteoglycan
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Proteoglycans are complex macromolecules

• Protein core
• Polysaccharide chains

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Macromolecular Proteoglycan Structure
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SEM View of the Proteoglycan Superstructure
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How Collagen and Proteoglycan Interact
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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

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Proteoglycan Synthesis
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Proteoglycan Degradation
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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-b
• Complex constellation of actions
• Alterations in signaling correlate with OA

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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

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Articular CartilageDevelopment and Aging
immature
maturing
adult
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Most Important Biomechanical Consideration
Na, Ca2
Donnan osmotic pressure
H2O
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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

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Growth Plate Cartilage

• Endochondral Ossification

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The Human Growth Plate
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Endochondral Bone Formation
Growth plate chondrocyte differentiation

• Complex interplay of intercellular
• signals that co-ordinate
• proliferation
• hypertrophy
• ossification

Resting
Proliferating
Hypertrophic

• TGF-b
• BMPs
• Retinoic Acid
• PTHrP
• Ihh
• Wnts
• Cytokines

Bone
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Stages of Chondrocyte Maturation
Proliferative - Prehypertrophic
Terminal Maturation
Undifferentiated
Hypertrophic
Growth Plate Chondrocytes
TGF-b
BMP
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BMP-2 Stimulates Chondrocyte Maturation
Chick Caudal Sternal Chondrocytes treated for 8
days
100
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10
0
BMP-2 (ng/ml)
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Type X
28s rRNA
Li, et al., Endocrinology 144 2514-23, 2003
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TGF-b Inhibits Chondrocyte Maturation
Chick Cephalic Sternal Chondrocytes
24h 48h 72h 96h
24h 48h 72h 96h TGF-b -
- - -

colX
18s RNA
Ionescu, et al., Exp. Cell Res. 288198-207, 2003
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TGF-b and BMP Activate Smad Pathways
TGF-b receptor
BMP receptor
Smad 1,5
Smad 2,3
P
P
Smad 1,5
Smad 2,3
Smad4
Smad4
P
Smad 1,5
Smad4
BMP-2 responsive genes
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TGF-b Induces Nuclear Localization of Smad2 and 3
Control TGF-b BMP-2
Smad2
Smad3
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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.
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How is TGF-b signaling effected in chondrocytes
isolated from the neonatal sternum of wild type
and Smad3-/- mice?
Assessment of signaling using a TGF-b-responsive
promoter/reporter
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Measuring activation of TGF-b/Smad signaling
induced by TGF-b
4xSBE
luciferase
P3TP-luc
1) Transfect
2) Treat with TGF-b
3) Measure luciferase luminescence
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Activation of the SBE-Luc Reporter in Smad3-/-
Chondrocytes is Completely Blocked
2000000
1800000
1600000
1400000
SBE Luciferase
1200000
1000000
800000
600000
400000
200000
0
WT
KO

TGF-?
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What is the phenotype of chondrocytes isolated
from the neonatal sternum of wild type and
Smad3-/- mice?
Assessment of phenotypic gene expression
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colX Expression is Elevated in Smad3-/-
Chondrocytes
0.6
0.5
0.4
WT
colX Expression
0.3
KO
0.2
0.1
0
2 Days
4 Days
8 Days
colX
28S RNA
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Other markers of maturation are up-regulated in
Smad3-/- Chondrocytes
3.5
3
2.5
Relative Expression by RT-PCR (compared to
b-actin control)
2
WT
KO
1.5
1
0.5
0
AP
MMP-9
VEGF-A
MMP-13
Osteocalcin
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What other in vivo evidence is there that the
BMP/TGF-b signaling pathways are important in
regulating maturation of chondrocytes?
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BMP signaling induction of the transcription
factor Runx2 is critical for terminal hypertrophy
of chondrocytes and skeletal mineralization
Inactivating mutations of the Runx2 gene are
linked to the development of cleidocranial
dysplasia
WT
Runx2 KO
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Osteoarthritis
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Osteoarthritis
Proliferative - Prehypertrophic
Terminal Maturation
Undifferentiated
Hypertrophic
Growth Plate Chondrocytes
TGF-b
BMP
Articular Chondrocytes
osteoarthritis
Ihh
colx alk phos BMP-6 MMP9, 13
Sox9 col2 aggrecan/ proteoglycans
VEGF OC apoptosis matrix calcification
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Chondrocytes Express Hypertrophic Markers During
Osteoarthritis

• During OA, articular chondrocytes exhibit
• - Increased proliferation (cloning)
• Expression of MMPs, colX, BMP-6 and other
  hypertrophic markers
• - Terminal hypertrophy and apoptosis

BMP-6 Immunostain
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Cloning, Fibrillation and Ulceration
OA
normal
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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

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Cartilage Loss and Destruction
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Etiology

• Aging
• Alterations in matrix
• Alterations in cell activity/function
• Alterations in cell mediators
• Altered joint mechanics
• Immune responses

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Loss of TGF-b signaling is a candidate pathogenic
mechanism for OA
Proliferative - Prehypertrophic
Terminal Maturation
Undifferentiated
Hypertrophic
Articular Chondrocytes
TGF-b
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Smad3-/- mice display an OA-like cartilage
degeneration
WT
KO
1 Month
4 Month KO
4 Month
7 Month KO
7 Month
Yang, et al., J Cell Biol. 15335-46, 2001