Ascb Comp

Regulation of the membrane bound matrix metalloprotease (MT1-MMP) in mechanically stimulated bioengineered articular cartilage. 

1CIHR BioEngineering of Skeletal Tissues Team, Mount Sinai Hospital2Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Canada.

1De Croos, J. N. A., 1Séguin, C. A., 1,2Grynpas, M. D., 1,2Pilliar R. M., and 1,2,Kandel, R. A.

• Cartilage– Designed to transmit load to the

underlying bone – Lowers the friction between articulating

ends of bone– Avascular

• Chondrocyte– Comprises about 10% of the tissue

Background

• Damage can result from injury or disease (arthritis)

• Repair potential is limited given the avascular nature of cartilage

Background

Formation of Articular Cartilage in vitro

Cell cultureChondrocytes

Bone Substitute

Cartilage Tissue Engineering

• In vitro-formed tissue does not attain the 3:1 ratio of collagen-to-proteoglycan of native cartilage– in part due to the decreased

collagen content

• In vitro-formed tissue also displays only a fraction of the mechanical properties of native cartilage

In vitro

Native

StrainS

tre

ss

(k

Pa

)

Implantation of a biphasic construct

Sheep Knee, 9 months post-implantation

Toluidine blue/trichrome, X25 magnification

Mechanical Stimulation• Previous studies have

demonstrated that long-term exposure to dynamic mechanical forces:– increases matrix accumulation

(both collagen and proteoglycans)

– improves mechanical properties of the developed tissue

• The mechanism(s) by which mechanical stimulation improves in vitro grown cartilage tissue formation is not known

Mechanical Stimulation

Chondrocytes were cyclically compressed within three days of initial seeding for 30 minutes (1kPa, 1Hz)

MACH-1 mechanical tester

Tissue

CPP

Tygon Tubing

Agarose Plug

MACH-1Mechanical Stimulator

Background

Waldman et al. 2006. Osteoarthritis.Cartilage.De Croos et al. 2006. Matrix Biol. De Croos et al. 2007. Osteoarthritis.Cartilage.

24 h0 h

mmp-13aggrecan type II collagenMMP-13

Proteoglycan CollagenEgr-1

pERK1/2,

egr-1

2h0h 6h 12h 24h0.5hDuring cyclic compression(30 minutes, 1kPa, 1Hz).

Stimulation ends

Identification of Egr-1

• Egr-1 initially identified in a TF array as being upregulated after mechanical stimulation.

• Egr-1 (early response gene)– Binds to promoter of

MT1-MMP Unstimulated

Stimulated

Egr-1

Egr-1

MT1-MMP

MT1-MMPs

ECM

Sp-1Egr-1

Egr-1 binds to MT1-MMP promoter

Supershift

DNA/Protein

DNA/Protein

US SUS SUS S

0 min 15 min 60 min

US S US S S S S

MT1-MMP + + - - + + +

Mutant MT1-MMP - - + + - - -

Egr-1 competitor (10X) - - - - + - -

Sp-1 competitor (10X) - - - - - + -

Egr-1 Antibody - - - - - - +

A)

B)

Transfection of chondrocytes(3D tissue)

Cell Culture

SeedingCyclic

Compression Luciferase

Luciferase Assay –MT1-MMP

• FuGene6 (6:2)• Transfected with pLuc-MT1-MMP prior to

seeding0

0.1

0.2

0.3

0.4

0.5

Unstimulated Stimulated MT1-MMP mutant

Re

lati

ve

Lu

cif

era

se

Un

its

*

*significant difference compared to corresponding controls, p<0.05, n=4.

MT1-MMP expression patterns

0

0.5

1

1.5

2

2.5

3

3.5

4

-15min 0min 15min 30min 1hour 2h 12h 24h

Time

Fo

ld C

han

ge

Sti

mu

late

d:U

nsti

mu

late

d

Post-Stimulation

DuringStimulation

*

* *

*

* p<0.05, n=4

*significant difference compared to corresponding controls, p<0.05, n=4.

0

1

2

3

4

5

0.5 hour 1 hour 2 hours 6 hours

Time

*

*

Blocking MT1-MMP

• Double stranded oligonucleotide containing a transcription factor binding site for Egr-1.

• Direct incubation of in vitro grown tissue with modified MT1-MMP decoy oligodeoxynucleotides (ODN)

• Endogenous transcription factor binding is prevented and the effects can be analyzed.

Egr-1

ODN

Egr-1

, MT1-MMP

Blocking Egr-1 binding

0

0.5

1

1.5

2

2.5

3

3.5

4

Vehicle 1.0 uMODN

Fo

ld C

han

ge

(Sti

mu

late

d:U

nst

imu

late

d)

*

US S

Vehicle

US S

ODN (1M)

DNA/Protein

• Decreased Egr-1 binding with ODN (1 M).

• RT-PCR of MT1-MMP is significantly lower by 15 minutes post-stimulation.

*significant difference compared to corresponding controls, p<0.05, n=4.

*significant difference compared to corresponding controls, p<0.05, n=4.

1000

1500

2000

2500

3000

3500

4000

0 uM 0.1 uM 1.0 uM

ODN concentration

cpm

/ug

DN

A

Unstimulated

Stimulated

1000

2000

3000

4000

5000

6000

7000

0 uM 0.1 uM 1.0 uM

ODN concentration

cpm

/ug

DN

A

Unstimulated

Stimulated

Blocking MT1-MMP – 24 hours• MT1-MMP

promoter ODN– Effect of

mechanical stimulation decreases by 1.0uM of ODN

– Suggests expression of MT1-MMP is necessary for increase in ECM.

Pro

teog

lyca

nC

olla

gen

*

*

*significant difference compared to corresponding controls, p<0.05, n=6.

Collagen and Proteoglycan accumulation (at 4 weeks)

0

20

40

60

80

100

120

140

C S C S C S

Vehicle Inhibitor Mutantug

GAG

/ ugD

NA

0

2

4

6

8

10

12

C S C S C S

Vehicle Inhibitor Mutant

ug O

H-pr

o/ u

gDNA

*significant difference compared to corresponding controls, p<0.05, n=6.

*

*

**

Conclusions

• Cyclic compression can improve extracellular matrix synthesis

• Improved tissue formation is dependant on early MT1-MMP regulation

MT1-MMP

MT1-MMPs

ECM

Sp-1Egr-1

• Dr. Rita Kandel • Dr. Marc Grynpas• Dr. Robert Pilliar

• Dr. Cheryle Séguin • Dr. Jian Wang• Dr. Stephen Waldman• Dr. Jason Hong• Dr. Suzanne Bernier

• Jennifer Lee

• Shu Qui Li

• Sandeep Dhaliwhal

• Ben Jang

Acknowledgements