Keratin expression in cultured skin substitutes suggests that the hyperproliferative phenotype observed in vitro is normalized after grafting

Cultured skin substitutes, consisting of fibroblasts and keratinocytes in a biopolymer matrix, are an adjunctive treatment for full thickness burn wounds. Previous studies revealed that cultured skin substitutes in vitro exhibit a gene expression profile similar to hyperproliferative skin or wounded normal skin. In the present study, we sought to determine whether this hyperproliferative phenotype is maintained after healing of grafted cultured skin in vivo. Immunohistochemistry was used to localize multiple keratin proteins in native human skin, and in cultured skin substitutes in vitro and after grafting to athymic mice. Keratin 6, keratin 16, and keratin 17, which are known to be upregulated during keratinocyte activation and in hyperproliferative epidermis, were highly expressed in cultured skin substitutes in vitro. These proteins were low or undetectable in native human skin, and were reduced in cultured skin after grafting. Conversely, keratin 15, which is downregulated in activated keratinocytes, was not detected in cultured skin substitutes in vitro but was upregulated after grafting to mice. The results confirm previous observations suggesting a hyperproliferative or activated phenotype in cultured skin substitutes in vitro, similar to wounded native skin. After grafting to athymic mice, the expression patterns suggest a normalization of cultured skin substitutes to a phenotype more closely resembling uninjured human skin.