Biomedical Engineering Reference
In-Depth Information
The percentage of dead cells after 24 h in the indirect cell test with medium that had been
conditioned with PEG samples gave values between 7 % and 16 % depending on the sample
composition (variations in the amount of PI and CL) and the solvent used for previous
extraction of the samples. No significant difference in cytotoxicity could be observed
comparing soft, intermediate and stiff samples. Looking at the impact of sample extraction
medium, substrates which had been incubated in water for 24 h prior to the test showed the
lowest cytotoxic effect. Compared with results gained by extraction with organic solvents
(acetone) or without any treatment, the cytotoxicity level detected for the water washed
samples was significantly lower (p<0.05 and p< 0.01, respectively). As a consequence of
these test results PEG samples were stored in sterile water for at least 24 h after the
crosslinking process prior to in vitro application.
2.3 Fibroblast culture on smooth PEG hydrogel substrates
In order to assess the cytocompatibility of the PEG-based hydrogels in direct contact
with cells, L929 cells were cultured directly on the surface of bulk free-standing
substrates. Samples of the three introduced elasticities were applied and cell morphology
was documented by live imaging after 24 h and 48 h in culture (
Figure 5
).
Subsequently, fixed and dried cells were further evaluated by means of electron
microscopy (FESEM).
As can be seen in Figure 5, the cell morphology on the three different PEG substrates did
not vary significantly, all cells displayed a round shape and only little or no stable
cell adhesion was evident. This clearly confirms the cell-repulsive properties of the PEG-
based material. Electron microscopy further showed that the rinsing and fixation of
the samples led to the removal of a large number of cells. This observation underlines that
only a negligible amount of cells was able to establish a contact to the surface.
The majority of the cells tended to form aggregates after longer cultivation times. The
number of cells visible on the images of the different samples cannot be compared directly
as a slight shaking of the medium led to an immediate re-destribution of the cells above
the surface.
Based on the observation that the cells were not able to build stable contacts to the surface
the consequences for intracellular processes were studied as well. Many anchorage-
dependent cell types stop to proliferate or can undergo a programmed cell death (apoptosis)
without the presence of integrin-mediated cell-surface contacts (Frisch & Francis, 1994;
Gilmore, 2005). The amount of the apoptotic markers caspase-3 and caspase-7 after 48 h of
cultivation time on the three different PEG substrates and polystyrene (PS) was assessed
with a commercially available assay and compared to a culture where apoptosis was
specifically induced by staurosporin addition (values set to 100 %). The results are depicted
in
Figure 6
.
As seen in Figure 6, cells cultured on the three different sP(EO-stat-PO) hydrogels did not
show an enhanced level of apoptotic activity compared to those seeded on the control
substrate PS. This observation is in accordance with results from other groups showing that
fibroblasts are not very sensitive to lack of adhesions to a solid substrate if serum is present
in the medium (Ishizaki et al., 1995; McGill et al., 1997). There was also no significant
difference between the samples with the three different crosslinking degrees. Cell adhesion
to PS after 48 h was confirmed by light microscopy.
