Download Research Day - Andrew Whitton Poster

Cell Invasion in Vascular Disease
Introduction
Cell Attachment Strength
However, occasionally it has an undesirable effect; for example
in cancer growth and vascular (blood vessel) disease.
Many surgical interventions used to treat vascular disease, such
as the stent in the centre picture, are disrupted by the invasion of
the surrounding tissue onto the implanted device.
Proteins present in the blood can adsorb onto the surface of
implanted materials and effect the way the tissue interacts with it.
The aim of this work is to study how the interaction between
human cells and implantable materials is affected by the nature
of the material and the amount of protein adsorbed on it.
Medical grade polyurethane of two different stiffnesses was used
as the substrata.
The protein investigated was fibronectin, an abundant protein in
the blood and one which has a substantial effect on cell
attachment.
The adhesion of a cell to its substrate is central to its migration.
A centrifugal detachment force was applied to the cells to
determine the strength of attachment to the fibronectin-coated
substrates.
Median Detachment Force (nN)
Cell migration is critical in many biological processes including
foetal development, wound healing and the immune response.
6.5
6
Figure 4: Graph
showing median
attachment
strength of
vascular cells on
polymer surfaces
of differing
stiffnesses at
various protein
surface densities.
3.1MPa
5.5
10.6MPa
5
4.5
4
3.5
3
2.5
2
0
20
40
60
80
Fibronectin Adsorption (% Saturation)
The attachment strength was found to increase with increasing
fibronectin adsorption and was greater on the stiffer substrate.
Cell Structure
Cell Migration Rate
A barrier migration assay (modified from the Teflon fence
migration assay1) was used:
Vascular cells were grown on fibronectin-coated polymer
membranes, restricted by a barrier.
The barrier was then removed and the cells microscopically
imaged as they populated the polymer surface.
The mechanism by which a cell propels itself across a surface
involves an internal framework, called the cytoskeleton, which
attaches at numerous points to the substratum via the cell
membrane.
The cytoskeleton can provide traction forces to pull the cell along.
Cells attached to the different stiffness polymers with various
fibronectin coating densities were imaged using fluorescent
microscopy to study the effect of the substrate on the cytoskeleton.
A
B
Figure 1: Illustration of barrier migration assay.
Figure 5: Example fluorescent images of vascular cells attached to; A)
soft substratum at low fibronectin densities and B) hard substratum at
high fibronectin densities. Cell cytoskeleton is green, nuclei in blue.
An increase in the substratum stiffness or adsorbed fibronectin
density lead to a more pronounced cytoskeleton and an increase
in the spread area of the attached cells.
44hr
26hr
0hr
72hr
Figure 2: Representative phase-contrast images of the migration of a
cell population after the removal of the constraint at t=0 hours.
The migration rate was maximal at intermediate protein coating
densities on both polymer grades.
The maxima occurred at lower densities on the stiffer substrate.
Average Migration Rate (um/hr)
The maximum migration rate was the same magnitude for both
polymers.
22
3.1MPa
20
10.6MPa
Conclusions
Many cell types have been found to have maximal migration rates
on surfaces to which they adhere at intermediate adhesion
strengths2. Furthermore, both substrate stiffness and the density of
surface-bound cell-adhesion sites have an effect with an increase
in either producing an increase in the strength of cell attachment3.
An explanation for this maximal migration rate is that at lower cellattachment strengths there is not enough traction to provide
adequate propulsion at a cell’s leading end while with stronger
adhesions a cell takes longer to detach from the substratum at its
trailing end. The maximum migration rate occurs between the two.
18
This study is the first to demonstrate that vascular cells have a
maximal migration rate across medical grade polymers of various
stiffnesses with a range of adsorbed fibronectin densities.
16
14
These results have important implications for the design of
implanted devices irrespective of whether or not migration of cells
from the surrounding tissue is the desired outcome.
12
10
0
20
40
60
80
Fibronectin Adsorption (% Saturation)
Figure 3: Graph showing migration rate of vascular cells over
polymer surfaces of differing stiffnesses at various protein surface
densities.
Andrew Whitton
David J. Flint
Richard A. Black
References
1 Pratt, B. M.; Harris, A. S.; Morrow, J. S. Am. J. Pathol., 1984, 117 (3), 349-354.
2 DiMilla, P, A, et al., J. Cell Biol., 1993, 122, 3, 729-737.
3 Peyton, S. R.; Putnam, A. J. J. Cell. Physiol., 2005, 204, 198–209
Bioengineering Unit, University of Strathclyde
Strathclyde Institute of Pharmacy and
Biomedical Sciences