ECM

Structure, Function and Pathology

Margaret Smith mobsmith@sydney.edu.au

Today

Extracellular matrix

• Structure

• Function

• Pathology

• Current research at the Institute of Bone

and Joint Research at Royal North Shore

Hospital

Extracellular matrix

• All tissues have ECM but some have more

than others:

bone

articular cartilage

intervertebral disc

ligament

tendon

skin

Components of ECM

• Water

• Collagen (more than 25 types)

• Proteoglycans (lots of these too)

• Non-collagenous proteins (elastin,

enzymes, inhibitors, cytokines, signaling

molecules, growth factors etc)

Collagen

• More than 25 types

• Type I - main type in tensile tissue

skin

tendon

ligament

• Type II - main type in compressed tissue

cartilage

compressed tendon

Collagen fibres in compressed sheep tendon

• Toluidine Blue stains

collagen fibres pale

bluegreen,

proteoglycans purple

and nuclear material

(eg DNA) inside cells

dark blue.

Proteoglycans

• Large (space filling)

Aggrecan

Versican

• Small (leucine rich repeat) = SLRPs

Decorin

Biglycan

Fibromodulin

Lumican

Keratocan

Non-collagenous proteins

• Elastin

• COMP and other mediators

• Collagenases (MMPs)

• Aggrecanases (ADAMTSs)

• Other enzymes

• TIMPS and other inhibitors

Composition of Articular Cartilage

(by wet weight)

Solid Phase

Proteoglycans - Aggrecan (5-10%)

Collagens - Type II (15-20%) (also types III, VI, IX, X, XI, XII, XIV) Fluid Phase

Water (70-80%)

Normal sheep

articular cartilage

Osteoarthritic

sheep articular

cartilage

Structure of articular cartilage

Cartilage degeneration in osteoarthritis

First the proteoglycans

are lost, and then the

collagen network

degrades and cells are

lost.

FUNCTION: Flexibility vs Mechanical Stability

Co-ordinated interplay between:-

• Muscle

• Ligament

• articulating facet joints

• intervertebral disc

IVD provides mechanical Stability

during axial/torsional spinal loading

FLEXION: forward bending

EXTENSION: straightening of spinal curves

TORSION: bending/twisting movement

THE SPINAL COLUMN

The Intervertebral Disc

1. AF - tough collagenous lamellae-tensile strength

2. NP - proteoglycan rich, hydrated gel- weight bearing cushion

3. CEP hyaline like cartilage at IVD-VB interface

4. Vertebral growth plate contiguous with CEP in human IVD

1

1 2

3

3

4

4

Several morphologically distinct cell populations in IVD

AF cells have a flattened fibroblastic morphology

NP cells have a rounded chondrocytic morphology

Notochordal cells

Chondrocyte clone

Cells in the CEP have a rounded chondrocyte like

morphology

Bone

BV

Bone

CEP

Tendon Function

Attach muscle to bone

allowing movement of

joints

Allow muscles to act at a distance

from joint

Focus action of multiple muscles on

single bone (Achilles)

Focus action of single muscle on

multiple bones (Tib. Post.)

Limited ability to stretch - improve efficiency of movement

Tendon Function

fibrochondrocyte

proteoglycan

tenocyte

endotenon

Tendon Composition - Collagen

• Highly ordered collagen fibrils

• Tensile tendon Collagen type I (95%) III and V

• Compressed tendon Collagen types I and II

IX, X and XI

Tendon composition -

Proteoglycans (PGs)

• Aggrecan & Versican

• SLRPS - decorin, biglycan, fibromodulin, lumican, keratocan

• Decorin and versican in tensile tendon

• Biglycan and aggrecan in compressed tendon and near bone insertion sites

Tendon composition -

Proteoglycans (PGs)

• Aggrecan & Versican

• SLRPS - decorin, biglycan, fibromodulin, lumican, keratocan

• Decorin and versican in tensile tendon

• Biglycan and aggrecan in compressed tendon and near bone insertion sites

Normal tensile tendon Normal compressed tendon

Tensile tendon - stress

deprived for 2 weeks

Tensile tendon - stress

deprived for 52 weeks

Fibroblastic v chondrocytic

Tension Compression

Stress-deprived tendon

ex vivo

0.01

1

100

10000

collagen-1 collagen-2 MMP 1 MMP 13 fold

change f

rom

snap f

rozen

* * *

* *

#

Cells respond quickly to changes in their environment

In vitro tensile loading

Can we model the effects of tensile load in vitro? Could we use this system to evaluate KO and KI mice?

Developed apparatus to enable variable cyclic tensile loads to be applied to mouse tendon

Tail tendon (± flexor digitorum longus) tensile and not attachment region?

0, 10, 20 & 40g load 16 hours 3 cycles/minute with ~ 50% of each cycle loaded v unloaded DMEM 10% FCS

Cartilage Tissue Engineering

HEALTH REPAIR

DISEASE

Catabolism Anabolism

Orthopaedic Disease & Research

HEALTH

Tissue which is

biochemically and

biomechanically suited

to its role

Altered protein synthesis

Increased proteolysis

Cell death

DISEASE

Degenerate Tissue

Loss of biomechanical

properties

Biologics

Cells - stem cells

Matricies

REPAIR / REGENERATION

Tissue which is

biomechanically functional

Cartilage Tissue Engineering

Isolated Chondral/Osteochondral lesions

Acute injury

Focal

No underlying biomechanical,

degenerative or inflammatory

issues?

Osteoarthritis Chronic disease

Focal?

Numerous complicating factors:

- instability/deformity

- additional injuries (ACL, Men)

- subchondral bone changes

- synovial inflammation & fibrosis

- degeneration

Defining Therapeutic Targets

Growth factors

(TGF-b, IGF)

Biomechanics Autoimmunity Infection Hormones

Bone remodelling

Ligament/meniscus injury

Inflammatory cells

cytokines

(IL-1, TNF, IL-17, IL-6)

Common Endpoint

OA - a focal disease? Meniscectomy model of OA in sheep

Sham 3 mth post Mx 6 mth post Mx

Sham Meniscectomy

..EGE

..PEN

Aggrecan & Collagen Proteolysis Focal cleavage of aggrecan by ADAMTS (..EGE neoepitope)

No increase in aggrecan cleavage by MMPs (..PEN neoepitope)

Increased collagen proteolysis by MMPs (9A4 neopeitope)

Meniscectomy Sham

Mab 9A4

(collagenase-cleaved neoepitope)

Diffuse & prolonged

aggrecanolysis in

human knee

cartilage after

ligament injury

Dr. AY

Healthy control for dGEMRIC study

Acute PCL rupture

1 mo - effusion and pain

3 mo follow up - no effusion and

completely asymptomatic

Controversies: Next Steps Towards Success

• Targeting catabolism is a valid approach to

enhance repair/regeneration

• Cartilage Tissue Engineering:

- cells, scaffolds, growth “factors”

- inhibitors of aggrecan ± collagen proteolysis

HEALTH REPAIR

DISEASE

Catabolism Anabolism

bone muscle 1

Underload

pathology

4

High aggrecan

Low ADAMTS

Low aggrecan

High ADAMTS

Overload

Pathology

in tendon

“A tale of two tissues” Health Disease

High aggrecan

Low ADAMTS

Low aggrecan

High ADAMTS

Overload

Underload

OA

in

cartilage