Scanning electron microscopy observations.
At the end of the various incubation times, the non-attached cells on
the different substrates were removed from the substrates by rinsing
twice with 0.1 M sodium cacodylate buffered solution, dehydrated in a
graded series of ethanol (50%, 70%, and 90%), and then placed in
100% ethanol. The dehydrated samples were then immersed in tert-butyl
alcohol under vacuum (FDU-1200, EYELA, Tokyo, Japan). The freeze-dried
samples were mounted on aluminum stubs and were then sputter-coated with
a gold-palladium alloy. Scanning electron microscope (SEM, S-2150,
Hitachi, Tokyo, Japan) observations of each sample were then performed
at numerous magnifications at 10 kV.