1. Structure, Composition, Function
Articular Cartilage
Structure, Composition, Function

2. Composition Sparse population of cells - chondrocytes
Large extracellular Matrix
-water
-proteoglycan
-collagen

4. Comparing Skeletal Tissue Composition

5. Structure
Lamina Splendens
Superficial
tangential zone

6. Zonal Topography STZ Middle Zone Deep Zone Calcified Zone
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

7. Collagen

8. Collagen Fiber Architecture

10. Collagen Fibril Organization

11. Summary of Collagen Synthesis

12. Proteoglycan

13. Proteoglycans are complex macromolecules
Protein core
Polysaccharide chains

14. Macromolecular Proteoglycan
Structure

15. SEM View of the Proteoglycan Superstructure

16. How Collagen and Proteoglycan Interact

17. 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

18. Proteoglycan Synthesis

19. Proteoglycan Degradation

21. 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

22. 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

23. Articular Cartilage: Development and Aging
immature
maturing
adult

24. Most Important Biomechanical Consideration
Donnan osmotic pressure
Na+, Ca2+
H2O

25. 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

26. Growth Plate Cartilage
& Endochondral Ossification

27. The Human Growth Plate

28. Growth plate chondrocyte differentiation
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

29. Stages of Chondrocyte Maturation
Proliferative -
Prehypertrophic
Undifferentiated
Terminal
Maturation
Hypertrophic
Growth Plate
Chondrocytes
TGF-b
BMP
Type X Collagen
MMP13
Alkaline Phosphatase
Osteocalcin
VEGF
Apoptosis
Type II Collagen

30. Li, et al., Endocrinology 144: 2514-23, 2003
BMP-2 Stimulates Chondrocyte Maturation
Chick Caudal Sternal Chondrocytes treated for 8 days
BMP-2 (ng/ml) 10 25 50
100
Type X
28s rRNA
Li, et al., Endocrinology 144: , 2003

31. Ionescu, et al., Exp. Cell Res. 288:198-207, 2003
TGF-b Inhibits Chondrocyte Maturation
Chick Cephalic Sternal Chondrocytes
24h h h h h h h h
TGF-b
colX
18s RNA
Ionescu, et al., Exp. Cell Res. 288: , 2003

32. TGF-b and BMP Activate Smad Pathways
TGF-b receptor
BMP receptor
Smad
2,3
Smad
1,5 P P
Smad
2,3
Smad
1,5
Smad4
Smad4
Smad4 P
Smad
2,3
TGF-b responsive genes P
Smad
1,5
Smad4
BMP-2 responsive genes

33. TGF-b Induces Nuclear Localization of Smad2 and 3
Control TGF-b BMP-2
Smad2
Smad3

34. Smad3 deficient mice have accelerated chondrocyte maturation and OA.
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.

35. Assessment of signaling using a TGF-b-responsive promoter/reporter
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

36. 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

37. Activation of the SBE-Luc Reporter in Smad3-/- Chondrocytes is Completely Blocked
SBE Luciferase
800000
600000
400000
200000 WT KO
– –
TGF-

38. Assessment of phenotypic gene expression
What is the phenotype of chondrocytes isolated from the neonatal sternum of wild type and Smad3-/- mice?
Assessment of phenotypic gene expression

39. colX Expression is Elevated in Smad3-/- Chondrocytes
0.6
0.5
0.4
colX Expression WT
0.3 KO
0.2
0.1
2 Days
4 Days
8 Days
colX
28S RNA

40. 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 AP
MMP-9
MMP-13
VEGF-A
Osteocalcin

41. What other in vivo evidence is there that the BMP/TGF-b signaling pathways are important in regulating maturation of chondrocytes?

42. 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

43. Osteoarthritis

44. Osteoarthritis osteoarthritis Growth Plate Chondrocytes TGF-b BMP
Proliferative -
Prehypertrophic
Undifferentiated
Terminal
Maturation
Hypertrophic
Growth Plate
Chondrocytes
TGF-b
BMP
Articular
Chondrocytes
osteoarthritis
Sox9
col2
aggrecan/
proteoglycans
Ihh
colx
alk phos
BMP-6
MMP9, 13
VEGF OC
apoptosis
matrix calcification

45. 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

46. Cloning, Fibrillation and UlcerationOA
normal

47. 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

48. Cartilage Loss and Destruction

50. Etiology
Aging
Alterations in matrix
Alterations in cell activity/function
Alterations in cell mediators
Altered joint mechanics
Immune responses

51. Loss of TGF-b signaling is a candidate pathogenic mechanism for OA
Proliferative -
Prehypertrophic
Undifferentiated
Terminal
Maturation
Hypertrophic
Articular
Chondrocytes
TGF-b

52. Smad3-/- mice display an OA-like cartilage degenerationWT KO
1 Month
4 Month KO
4 Month
7 Month KO
7 Month
Yang, et al., J Cell Biol. 153:35-46, 2001