Multistep production of bioengineered skin substitutes: Sequential modulation of culture conditions

Many studies are being conducted to define the role of growth factors in cu taneous physiology in order to add cytokines in a timely fashion for optima l tissue engineering of skin. This study is aimed at developing a multistep approach for the production of bioengineered skin substitutes, taking into account the effects of various growth factors according to the culture tim e. The use of a serum-supplemented medium throughout the whole culture peri od of skin substitutes was compared to the sequential use of specific addit ives at defined culture steps. Histological analysis revealed that serum wa s necessary for keratinocyte proliferation and migration on dermal substitu tes during the first 2 d after their seeding, However, the serum-free mediu m presented some advantages when supplemented with different additives at s pecific culture steps. Interestingly, ascorbic acid added to the dermal sub stitutes before and after keratinocyte seeding maintained their cuboidal mo rphology in the basal epidermal layer. In the absence of serum, collagen ma trix degradation slowed down, and a better multilayered epidermal organizat ion was obtained, notably with retinoic acid. Stratum corneum formation was also enhanced by fatty acids. Thus, sequential addition of exogenous facto rs to the medium used to produce skin substitutes can improve their structu ral features and functional properties in vitro.