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