Biomedical Engineering Reference
In-Depth Information
The cell number was evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium
bromide (MTT) test [13]. The MTT test is an established method to determine viable cell
number by measuring the metabolic activity of cellular enzymes. Figure 8 shows steady
increases in cell number with culture time on both the e-BC gel and tissue culture plate
(TCP). There was a lag time before steady increase in cell number on the e-BC gel. Semler et
al. reported that cells shows high growth rate on matrices with high mechanical compliance
such as TCP, whereas the cells aggregate three-dimensionally on matrices with low
mechanical compliance such as collagen gel [39]. Therefore, the difference in the mechanical
properties of the surface of the culture substrate might affect the initial rate of cell growth.
Fig. 8. Growth curves of HUVECs cultured on e-BC gels and plastic tissue culture plate.
Values are mean ± SD (n=4).
The SEM images show the distribution and spreading morphology of the HUVECs cultured
on the e-BC gel at day 1 and day 5 after cultivation (Fig. 9). Apparently, the cells at 5d
cultivation were confluent and showed cobblestone-like morphologies (Fig 9b). HUVECs
also grow better on the e-SC gel and shows confluent at day 6 after cultivation [13].
Therefore, the e-BC gel could be used as a cell culture scaffold as well as the e-SC gel. EDC
cross-links collagen molecules by the formation of isopeptides without being incorporated
itself, thus precluding depolymerization and the possible release of potentially cytotoxic
reagents. Furthermore, the by-product of the cross-linking reaction and un-reacted EDC in
the e-BC gel should be completely removed by the drastic shrinkage in hot water. It is
expected that the e-BC gel has good biocompatibility and no cytotoxicity.
To assess the degradability of the e-BC gel in collagenase solution (50 U/ml), protein
content measurement was performed using a bicinchoninic acid protein assay kit as
