bsmb presentation 2013
TRANSCRIPT
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Mechanical load
Mechanical loading
Up-regulation of matrix
macromolecules
Matrix synthesis
Maintenance of tissue integrity
Abnormal mechanical
loading
Predominantly catabolic activity
Limited matrix synthesis Tissue atrophy
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Canonical WNT signalling
WNT
Casein kinase
Axin APC GSK-3β
Nucleus
Β-catenin
Β-catenin
Β-cateninΒ-catenin
Β-catenin
Dishevelled Key:APC= Adenomatous polyposis coliGSK-3β= Glycogen synthase kinase 3β
MMP 3WISP1WIF1
SFRP1FRZB
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Why look at WNT signalling?
ChondrogenesisChondrocyte
differentiation
Cartilagehomeostasis
↑WNTcomponents
Osteoarthritis
END STAGE DISEASE
WNT signalling
Mechanical loading
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Hypothesis
Wnt signalling is mechano-regulated in cartilage
Aims
1. Examine the effect of physiological load on WNT signalling
components (activators/inhibitors) in chondrocytes
2. Determine the influence of physiological and non-physiological
load on WNT signalling components in explants.
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Methods: Mechanical loading
Chondrocytes Explants
Primary chondrocytes released from cartilage using pronase (1mg/ml) and
collagenase type II (0.4mg/ml) in DMEM F-12 media.
- Seeded 4×106/well- 48 hours stabilisationperiod
7-day old bovine Metacarpophalangeal
joint
4mm explants
Full-depth cartilage explant
- Multiple washes in Hank’s balanced salt solution (HBSS) with 4× strength antibiotics- 48 hours stabilisation period
Bose Electroforce 3200
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Methods: Post-load analysis
Chondrocytes
o 7.5% strain 1Hz (1hr, 2, 4 hrs)
• Physiological regime
o 7.5%, 10%, 14% 1Hz (2hrs)
• Physiological injurious
Explants
o Physiological load 2.5MPa 1Hz 10minso Injurious load 8MPa 1 Hz 10 cycles
o Gene level analysis
• Quantitative PCR
o Protein level analysis
• Immunofluorescence in conjunction with confocal microscopy
Evaluation of WNT components
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Results: Effect of loading duration (7.5% strain)β-catenin nuclear translocation in chondrocytes subjected to tensile strain
o Loading regime: 7.5% strain 1Hz (physiological)
FITC green= β-catenin; DAPI blue
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Differential expression of WNT signalling components exposed to strain
Most WNT activity is
detected at 2hrs
Results: Effect of loading duration (7.5% strain)
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Results: Effect of strain level (1 Hz, 2hrs)
β-catenin nuclear translocation in chondrocytes subjected to tensile strain
FITC green= β-catenin; DAPI blue
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Results: Explants 2.5MPa 10mins 1 Hz
β-catenin nuclear translocation in chondrocytes subjected to physiological compressive loading.
FITC green= β-catenin; DAPI blue
Canonical WNT
signalling activation
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Results: Explants 8 MPa 10mins 1 Hz
FITC green= β-catenin/ WIF-1; DAPI blue
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Conclusions
Mechanical loading
Activates canonical WNT
signalling
Up-regulation of WNT components
(gene/protein levels)
Downstream effects?
Mechanism to maintain
tissue integrity
Future aim of project
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Dr. Emma Blain
Prof. Vic Duance
Dr.Yadan Zhang
Paulina Stadnik
Kuwait Cultural Office (UK)
Arthritis Research UK Biomechanics and
Bioengineering Centre