Biomedical Engineering Reference
In-Depth Information
via plasma treatment, leaving gold nanoparticles of 5-8 nm diameter. The gold
particles were organized in hexagonal patterns and are separated by 28-110 nm.
The space between gold nanoparticles was then covered with PEG (molecular
weight 2000), supposedly to prevent cell and protein adhesion. The interaction
between Au and sulfur was used to attach the thiolated cyclic peptide
c-RGDFK to the gold particle. Cellular adhesion experiments were conducted
using integrin-transfected fibroblasts. The results of this work indicated
strongly that, when exposed to the nanoparticle modified surfaces, the cells
were able to 'recognize' spacing effects. Using a phase-contrast microscopy
protocol it was shown that fibroblasts spread well for the case where nanodots
were spaced by 50 nm, in contrast to the situation with 110 nm spacing where
limited spreading was observed. In addition to this observation it was noted
from the microscopy that quiescent cells are rounded whereas migrating cells
exhibit a polarized shape. The role of integrin a
v
b
3
during adhesion to RGD
nanopatterns at different spacing was also examined via a technique which
involved the antibody-based blocking of receptors prior to seeding cells onto
the surfaces. The number of attached cells was evaluated one hour after seeding
using formaldehyde fixation and toluidine blue staining. Interestingly, the
number of cells adhered to both spacings were almost identical after such a
blocking procedure. In addition to these experiments the authors also studied
the recruitment of integrin-associated molecules in terms of their dependence
on the distance between integrin ligands. Example images from this type of
research are shown in Figure 2.13.
It is not only the fundamental spacing between RGD sites that can influence
cellular behavior. Experiments have indicated that the actual ordering of
nanopatterns on surfaces can mitigate cell adhesion. In an analogous approach
to that described immediately above, ordered and disordered nanopatterns
using the gold particles were produced on a supposedly bio-inactive substrate.
45
The various types of ordering were instigated through the use of an 'inter-
ference reagent' (a copolymer) during nanopattern formation and cellular
adhesion studies involving osteoblasts. Ordering was characterized through an
order parameter in addition to particle diameter and spacing on the surface, as
evidenced from atomic force microscopy. An important aspect of this study
was that the nature of the nanoparticles meant that only one integrin complex
could be attached to an RGD-treated particle. After allowing the fluorescent-
labeled cells to interact with the various substrates, the number of attached cells
and area occupied by osteoblasts were determined. It was found that the
number of cells and area occupied on ordered patterns decreased with
increasing distance between nanoparticles. This was not the case for
disordered patterns, the result being ascribed to the greater variety of ligand
(peptide) densities. Moreover, the crucial nature of interaction or clustering of
integrin moieties in the cell membrane is more likely to be exhibited in the case
of disordered surface nanodots. This effect
d
n
4
t
3
n
g
|
0
n
3
.
is
shown schematically in
Figure 2.14.
Although gold-sulfur functional group SAM chemistry outlined in Chapter
1 was proposed some years ago as a useful structure for studying RGD
