1) The document examines the effect of mechanical loading on WNT signaling components in cartilage. 2) It finds that physiological loading activates canonical WNT signaling in chondrocytes, as shown by β-catenin nuclear translocation. WNT activity peaks at 2 hours of loading. 3) Higher strain levels and non-physiological loading decrease WNT activity, as seen through reduced β-catenin translocation and upregulation of WNT inhibitors. 4) The results suggest mechanical loading regulates WNT signaling to maintain cartilage tissue integrity, and abnormal loading disrupts this process. Further study is needed to understand the downstream effects and mechanisms involved.

2. 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

3. CanonicalWNT signalling
WNT
Casein kinase
Axin APC GSK-3β
Nucleus
Β-catenin
Β-catenin
Β-catenin
Β-catenin
Β-catenin
Dishevelled Key:
APC= Adenomatous polyposis
coli
GSK-3β=Glycogen synthase
kinase 3β
MMP 3
WISP1
WIF1
SFRP1
FRZB

4. Why look atWNT signalling?
Chondrogenesis
Chondrocyte
differentiation
Cartilage
homeostasis
↑WNT
components
Osteoarthritis
END STAGE
DISEASE
WNT
signalling
Mechanical
loading

5. Hypothesis
Wnt signalling is mechano-regulated in cartilage
Aims
1. Examine the effect of physiological load onWNT signalling
components (activators/inhibitors) in chondrocytes
2. Determine the influence of physiological and non-physiological
load onWNT signalling components in explants.

6. 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 stabilisation
period
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

7. 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 10mins
o Injurious load 8MPa 1 Hz 10 cycles
o Gene level analysis
• Quantitative PCR
o Protein level analysis
• Immunofluorescence in conjunction with confocal microscopy
Evaluation ofWNT components

8. 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

9. Differential expression of WNT signalling components exposed to strain
Most WNT
activity is
detected at
2hrs
Results: Effect of loading duration (7.5% strain)

10. Results: Effect of strain level (1 Hz, 2hrs)
β-catenin nuclear translocation in chondrocytes subjected to tensile strain
FITC green= β-catenin; DAPI blue

11. 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

12. Results: Explants 8 MPa 10mins 1 Hz
FITC green= β-catenin/ WIF-1; DAPI blue

13. Conclusions
Mechanical
loading
Activates
canonical WNT
signalling
Up-regulation ofWNT
components
(gene/protein levels)
Downstream
effects?
Mechanism to
maintain
tissue integrity
Future aim of
project

14. Dr. Emma Blain
Prof.Vic Duance
Dr.YadanZhang
Paulina Stadnik
Kuwait Cultural Office (UK)
Arthritis Research UK
Biomechanics and
BioengineeringCentre