Biomedical Engineering Reference
In-Depth Information
Fn gradients have also been generated by Smith et al.
14
using an alka-
nethiol SAM diffusion approach as described previously. Fn was then co-
valently bound through EDC-NHS coupling to the carboxy-terminated
regions of the surface. Similarly to other examples described above, bovine
endothelial cells were found to attach or migrate towards the high Fn density
region of the gradient. A gradient of heparin, a glycosaminoglycan known to
interact with a wide variety of proteins crucial for a range of cellular pro-
cesses,
214
was developed by Robinson et al.
215
using a plasma polymer gra-
dient template. Chemical gradients of allylamine to 1,7-octadiene (OD)
functionality were deposited using the gradientiser, with heparin sub-
sequently adsorbed onto the plasma polymer surface. Using the same allyla-
mine-OD plasma polymer gradient described above, Vasilev et al.
187
created
grafted PEG gradients and subsequently fibrinogen and lysosome protein
gradients exploiting the varying allylamine concentration across the surface.
As discussed previously, cell attachment can be mediated by integrin re-
ceptors present on the surface of anchorage dependent cells on the one hand
and ECM proteins, e.g., Fn, or fragments of ECM proteins on the other. The
RGD motif discovered by Erkki Ruoslahti
216
has been a particularly popular
target for immobilisation on biomaterial surfaces
27,176
as it is recognised by
integrins that are expressed by a variety of cell types.
76,173,217,218
In addition
to enhancing cell attachment, the display of ECM-based ligands affects the
migration, apoptosis and differentiation of adherent cells.
75,176,184,219,220
The
ability to optimise the presentation and density of such signals is vital for the
advancement of implantable devices and regenerative medicine. Using short
peptide sequences has advantages over whole ECM proteins because the
former are fully chemically defined, are not of animal origin and do not pose
any risk of disease transmission. In addition, small peptides are less sus-
ceptible to degradation during sterilisation processes.
Many techniques have been developed to prepare gradients in RGD pep-
tide (or similar) concentration including functionalised SAM gradi-
ents,
173,221
hydrogels,
222,223
microfluidic mixing,
94
polymer brushes,
217
and
biotin-streptavidin coupling.
224,225
Burdick et al.
222
developed a gradient in
RGD peptide concentration using a microfluidic device (Figure 10.16A). Two
monomer/initiator solutions (one containing just the PEG monomer, the
other containing a modified monomer containing an RGD peptide) were
injected into the microfluidic device, with the gradient formed via mixing
within the microfluidic device through the channels repeatedly splitting and
mixing the solutions. The density of endothelial
222
and fibroblast
223
cells
was found to increase with increasing RGD concentration. Likewise, Petty
et al.
94
have utilised microfluidics to create gradients of RGD peptides along
the substrate. The gradients varied from the active RGD peptides (G
RGD
SC)
to an inactive peptide (G
GRD
GSC), which meant that the topography and
surface chemistry of the surface was uniform over the whole substrate.
Mouse melanoma cells attached preferentially to the high-density active
RGD sites. Lagunas et al.
225
developed a RGD gradient using biotin strep-
tavidin coupling (Figure 10.16B). A uniform PMMA surface was converted by
d
n
3
r
4
n
g
|
7
.
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