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

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

Why look at WNT signalling?

ChondrogenesisChondrocyte

differentiation

Cartilagehomeostasis

↑WNTcomponents

Osteoarthritis

END STAGE DISEASE

WNT signalling

Mechanical loading

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.

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

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

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

Differential expression of WNT signalling components exposed to strain

Most WNT activity is

detected at 2hrs

Results: Effect of loading duration (7.5% strain)

Results: Effect of strain level (1 Hz, 2hrs)

β-catenin nuclear translocation in chondrocytes subjected to tensile strain

FITC green= β-catenin; DAPI blue

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

Results: Explants 8 MPa 10mins 1 Hz

FITC green= β-catenin/ WIF-1; DAPI blue

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

Dr. Emma Blain

Prof. Vic Duance

Dr.Yadan Zhang

Paulina Stadnik

Kuwait Cultural Office (UK)

Arthritis Research UK Biomechanics and

Bioengineering Centre