UNIXAXIAL TENSILE STRAIN AND COLLAGEN

STRUCTURE AFFECT VASCULAR CELL

ORIENTATION AND PROLIFERATION Mathieu, P.S.

1,2, Fitzpatrick, E.

1,2, Cahill, P.A.

3, Lally, C.

1,2

1. Trinity Centre for Bioengineering, Trinity Biomedical Sciences Institute, Trinity College Dublin,

Dublin, Ireland.

2. Department of Mechanical & Manufacturing Engineering, School of Engineering, Trinity College

Dublin, Ireland.

3. School of Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland.

In-stent restenosis, which occurs in 5-10% of all

stents[1], is characterised by excessive cell

proliferation which may be driven by the proliferation

of cells found in the arterial media: vascular smooth

muscle cells (VSMC)[2] and multipotent vascular

stem cells (MVSC)[3]. In vitro, VSMC have been

shown to reorient perpendicular to uniaxial strain[4].

VSMC in vivo are constrained by collagen fibres and

align parallel to the primarily circumferential strain in

the vessel as shown in Figure 1.

Stenting is known to alter

the cyclic strain within a

stented vessel[5], and

may alter the underlying

collagen structure[6]. In

order to explore the role

of uniaxial tensile strain

and collagen structure in

the growth response of

vascular cells, prolifer-

ation and alignment were

determined in both rat

aortic VSMC (RASMC)

and rat MVSC (rMVSC) exposed to uniaxial cyclic

tensile strain with no structure. Then, the proliferation

and alignment response of rMVSC were determined,

following being seeded on decellularized arteries,

with the collagen aligned parallel or perpendicular to

the direction of strain.

rMVSC RASMC

24hr 72hr 24hr 72hr

Figure 4: rMVSC and RASMC strained for 24 or 72 hours at 0-10%, 1Hz uniaxial tensile strain. Red: F-actin Green: Ki67 Blue: Nuclei

Figure 5: Change in cell number and Ki67+ nuclei for rMVSC and RASMC after 24 and 72 hours of 0-10%, 1Hz uniaxial tensile strain. Strain decreased cell number vs unstrained, but did not significantly affect percentage of proliferating, Ki67+ cells.

Figure 6: rMVSC and RASMC both realigned perpendicular to the strain direction by 24 hours after onset of strain. rMVSC showed greater perpendicular alignment than RASMC at 24 hours, while RASMC showed greater perpendicular alignment at 72 hours.

Funding from Science Foundation

Ireland (13/CDA/2145).

Introduction

Acknowledgements

1. Byrne (et al.), Eur. Heart J. 36:3320-3331, 2015 2. Newby (et al.), Toxicoll Lett 112-113:519-529, 2000 3. Tang (et al.), Nat. Commun. 3:875, 2012 4. Rodriguez (et al), Arterioscler Thromb Vasc Biol 35:430-438,

2015 5. Colombo (et al.), Biomech Model Mechanobiol 12:671-683,

2013 6. Khilji, In-Vitro Effects of Intravascular Stenting on Collagen Fiber

Reorientation and Tissue Remodeling, MAI, University of Dublin, Trinity College, 2017

References

Conclusions

First experiments to study MVSC response to

tensile strain and collagen structure.

Both rMVSC and RASMC exhibit strain-avoidant

behaviour.

Underlying ECM alignment is critical to rMVSC

strain response.

Cells aligned parallel to strain direction increased

proliferation.

Cells aligned perpendicular to the strain direction

showed no difference from unstrained cells.

These results demonstrate that any intervention

that alters collagen fibre alignment, such as

stenting, would influence the strain sensed by cells

and their subsequent growth profile.

Figure 7: rMVSC on decellularized arteries

strained for 10 days at 0-10%, 1Hz uniaxial tensile

strain parallel or perpendicular to fibre direction.

Red: F-actin Green: Ki67 Blue: Nuclei

Figure 8: rMVSC remain aligned with collagen

fibre direction even after 10 days of 0-10%, 1Hz

uniaxial tensile strain. While cells exposed to strain

parallel to fibre direction show an increase in

proliferative, Ki67+ cells after 10 days of strain, cells

exposed to strain perpendicular to fibre direction

show no such strain-induced Ki67 increase.

Parallel Perpendicular0

20

40

60

80

%K

i67+

Nu

cle

i

Day 0

Unstrained

Strained

✱

✱✱✱

Results

24h

Fo

ld C

han

ge in

Cell N

um

ber

vs D

ay 0

rMVSC RASMC0

1

2

3

4

Unstrained

Strained✱ ✱ ✱

72h

Fo

ld C

han

ge in

Cell N

um

ber

vs D

ay 0

rMVSC RASMC0

1

2

3

4

Unstrained

Strained✱ ✱ ✱ ✱

✱ ✱ ✱ ✱

24h

%K

i67+

Nu

cle

i

rMVSC RASMC0

20

40

60

80

100Day0

Unstrained

Strained

72h

%K

i67+

Nu

cle

i

rMVSC RASMC0

20

40

60

80

100Day0

Unstrained

Strained

✱ ✱ ✱ ✱

✱ ✱

✱ ✱

24h

Orientation Relative to Strain Direction (Degrees)

Fre

qu

en

cy

(%

To

tal

Nu

cle

i)

0-10 10-20 20-30 30-40 40-50 50-60 60-70 70-80 80-900

10

20

30 rMVSC

RASMC

✱

✱

✱ ✱ ✱ ✱

72h

Orientation Relative to Strain Direction (Degrees)

Fre

qu

en

cy

(%

To

tal

Nu

cle

i)

0-10 10-20 20-30 30-40 40-50 50-60 60-70 70-80 80-900

10

20

30 rMVSC

RASMC✱

✱ ✱ ✱ ✱

0-10 10-20 20-30 30-40 40-50 50-60 60-70 70-80 80-90

0

10

20

30

40

Orientation Relative to Strain Direction (Degrees)

Fre

quen

cy (%

Tot

al N

ucle

i) Parallel

Perpendicular

RASMC and rMVSC were cultured on pronectin-

coated PDMS strips and strained at 0-10%, 1Hz,

uniaxial strain for 24 or 72 hr in a Bose Biodynamic

5200 as shown in Figure 2.

Rat MVSC were

seeded on the medial

layer of decellularized

porcine carotid

arteries and strained

at 0-10%, 1Hz,

uniaxial strain parallel

or perpendicular to

collagen fibre

direction for 10 days,

as shown in Figure 3.

Cell nucleus number

and alignment was

assessed using

ImageJ. Cells were

immunostained for

Ki67, and the

percentage of Ki67

positive nuclei was

determined using

MatLab and ImageJ.

Methods

Figure 2: Bose Biodynamic

5200 and chamber setup for

PDMS experiments

Figure 3: Chamber setup

showing parallel and

perpendicular fibre orientation

Figure 1: In Vivo both

VSMC and collagen fibres

align circumferentially