Biomedical Engineering Reference
In-Depth Information
(a)
(b)
(c)
(d)
12.2
Photomicrographs of normal human skin cells in selective culture.
(a) dermal fibroblasts, (b) epidermal keratinocytes, (c) dermal micro-
vascular endothelial cells, and (d) epidermal melanocytes. Scale bar =
500 µm.
density and grown for 5-6 days. Once cells have reached approximately 80%
confluence they are harvested and are then inoculated into the expansion culture in
large-scale static culture vessels such as roller bottles or cell factories, or in
automated bioreactors to reduce labor requirements,
41-43
to generate the cell
populations needed for inoculation onto biopolymer sponges.
12.3.2
Fabrication of biopolymer substrates
Collagen-glycosaminoglycan (GAG) sponges are fabricated from bovine skin
collagen and chondroitin-6-sulfate from shark cartilage to generate biopolymer
substrates with a controlled thickness and pore diameter. Collagen is solubi-
lized in acetic acid (0.60% wt/vol) and coprecipitated by the addition of
chondroitin-6-sulfate at a controlled rate.
44
The mixture is homogenized, cast
into a sheet with a given thickness and area and frozen by submersion in a bath
of 95% ethanol (EtOH). The reticulated network of collagen is formed as the
collagen precipitate is displaced by developing ice crystals.
45,46
Subsequent
sublimation of the ice crystals generates a highly porous sponge. The pore size
and structure depend on the nucleation and growth rate of ice crystals during
the freezing process. Thus the desired pore size and porosity can be controlled
by the freezing rate and the concentration of protein in the homogenized solu-
tion. The freeze drying process used for the cultured skin substitute model
generates collagen sponges that are approximately 200 µm thick with an aver-
age pore diameter of 60 µm (
Fig. 12.3
). The lyophilized sponges are
