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An In vitro Shear Stress System for the Study of Cell-seeded Scaffolds:
A Molecular Biology & Computational Analysis
Meaney, C. L., Piterina A. V., Carroll, G. T., McGloughlin, T. M
Centre for Applied Biomedical Engineering Research (CABER),
Department of Mechanical Aeronautical & Biomedical Engineering,
Materials Surface Science Institute (MSSi), University of Limerick, Ireland
E-mail: claire.meaney@ul.ie
Abstract
Novel materials for vascular graft applications are
continually emerging from research. To attain
comprehensive pre-clinical evaluation, cell-seeded
scaffold materials require exposure to physiological
shear stresses mimetic of those produced in vivo. This
field of testing allows the shear-resistance of the
endothelial layer to be assessed while also providing
indication of anticipated host response to shear through
gene expression analysis. An In vitro methodology was
developed to assess 3D scaffolds in terms of cellular
compatibility under physiological flow conditions. To
ascertain the feasibility of this system computational
and cellular studies were conducted.
1. Introduction
Cellular mechanotransductory pathways are known
to be highly regulated by the mechanical and chemical
properties of the underlying scaffold, thus heightening
the need to assess the cell-material interactions of each
particular material. This highlights the need for an in
vitro platform which facilitates the analysis of 3D
scaffolds under physiological wall shear stress (WSS)
profiles.
2. Bioreactor Characterisation
The original cone and plate bioreactor design (Fig 1),
with the capacity to deliver a controlled uniform SS
distribution [1], was modified and validated to allow for
the analysis of 3D cell-seeded materials [2]. The effect
which porous materials induce on the WSS distribution
across the complex surface was investigated.
Figure 1. Cone and plate bioreactor for 3D substrate
analysis.
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The fluid flow and resulting WSS distributions were
visualised using computational models of alternating
geometries to correspond with height variations present
within porous topographies. To determine the biological
effect of the varying height parameter; monolayers of
HAECs were subjected to steady shear stress under five
geometric parameters to determine critical tolerance of
the bioreactor design. HAEC monolayers were analysed
using microscopy and RNA was extracted. Through
PCR analysis, RNA expression of the established WSS
biomarker (VCAM) was investigated against a reference
gene and subsequently normalised against static
controls.
3. Study Outcomes
The PCR data suggests a critical height tolerance
exists, outside of which the spatial WSS gradient
significantly influences VCAM expression which is
exacerbated by a corresponding decrease in mean WSS.
Following extensive studies, the maximum potential
WSS gradients induced by the local topography height
variations of the materials studied were within the
critical range deemed suitable from the computational
and PCR data obtained. Thus the cone and plate test
facility was deemed suitable for ECM and other porous
3D substrates. Consequently ECM materials were
seeded with endothelial cells and exposed to
physiological WSS to determine the shear resistance of
the formed endothelial layer. The metabolic activity pre
and post shear was analysed using Alamarblue ® reagent
and visualised using confocal microscopy.
4. Conclusions
The methodology described and the experimental
system employed provides an ideal platform for analysis
of various materials. This test methodology may serve
to enhance the graft material selection process prior to
clinical testing through the facilitation of accurate in
vitro simulation and analysis to predict the in vivo
performance of materials.
8. References
[1] O’Keeffe (et al.), J Biomech Eng 131(8): 081003.2009
[2] Meaney (et al.), 6th World Congress of Biomechanics
Singapore (WCB 2010) Springer Berlin Heidelberg. 31: 143-
146