unixaxial tensile strain and collagen structure …
TRANSCRIPT
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