A dermal equivalent developed from fibroblast culture alone: effect of EGF and insulin.

We have recently developed a new dermal equivalent without exogenous materials by culturing dermal fibroblasts alone in serum-containing medium treated with several supplements. In this study, we investigated the effects of epidermal growth factor (EGF) and insulin on the formation of a dermal equivalent. After cultured dermal fibroblasts reached a confluence in serum-containing medium, they were treated with EGF or insulin. The combined effects of EGF and insulin were also studied. Macroscopically, in contrast to the culture without supplement, the addition of EGF or insulin produced a fibrous sheet. The combination of EGF and insulin showed a more marked effect than a single factor. Histologically, EGF or insulin alone induced a three-dimensional tissue containing several layers of fibroblasts. The combination of EGF and insulin produced a thicker tissue. It was composed of abundant extracellular matrix containing fibroblasts, suggesting a dermis-like tissue. It revealed collagen fibers by Masson-trichrome staining. Immunohistochemically, the components of dermal extracellular matrix such as type 1 collagen, elastin, and fibrillin-1 were diffusely expressed. Ultrastructurally, a large number of collagen fibrils with cross-striated patterns were found around the fibroblasts. These results showed that a dermal equivalent could be formed by culturing dermal fibroblasts alone postconfluently in serum-containing medium with EGF and insulin. They suggest that the two factors play an important role in the formation of a dermal equivalent.