ecm - university of sydneyweb.aeromech.usyd.edu.au/amme5971/course_notes/ecm-2010-smith.pdf ·...
TRANSCRIPT
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