Structure, Composition, Function Articular Cartilage Structure, Composition, Function

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

Comparing Skeletal Tissue Composition

Structure Lamina Splendens Superficial tangential zone

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

Collagen

Collagen Fiber Architecture

Collagen Fibril Organization

Summary of Collagen Synthesis

Proteoglycan

Proteoglycans are complex macromolecules Protein core Polysaccharide chains

Macromolecular Proteoglycan Structure

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-b 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

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

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

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: 2514-23, 2003

Ionescu, et al., Exp. Cell Res. 288:198-207, 2003 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. 288:198-207, 2003

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

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

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.

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

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

Activation of the SBE-Luc Reporter in Smad3-/- Chondrocytes is Completely Blocked 2000000 1800000 1600000 1400000 SBE Luciferase 1200000 1000000 800000 600000 400000 200000 WT KO – + – + TGF-

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

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

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

What other in vivo evidence is there that the BMP/TGF-b 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 Inactivating mutations of the Runx2 gene are linked to the development of cleidocranial dysplasia WT Runx2 KO

Osteoarthritis

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

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

Cloning, Fibrillation and Ulceration OA normal

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

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. 153:35-46, 2001