Immunochemistry of a keratinocyte-fibroblast co-culture model for reconstruction of human skin

Abstract

Our purpose was to determine differentiation markers of an in vitro co- culture model in which fibroblasts grown in a three-dimensional nylon mesh were recombined with human keratinocytes. The cultures were kept for 5 weeks and then processed for electron microscopy and immunochemistry. The specimens revealed an epidermis, a basal lamina, an anchoring zone, and a dermis. Epidermal differentiation was confirmed by the presence of K10-keratin, trichohyalin, and filaggrin. The basal lamina contained Type IV collagen, laminin, nidogen, and heparan sulfate. Type IV collagen, laminin, and nidogen were also noted in the extracellular matrix. Type VI collagen was present in the anchoring zone and also gave a reticulated pattern in the rest of the dermis. There was a heavy signal for tenascin and fibronectin throughout the dermis. Osteonectin was restricted to the epidermis and dermal fibroblasts. Fibrillin stained at the anchoring zone and dermis but elastin and vitronectin were negative, suggesting early formation of elastic fibrils. Collagen fibrils stained for Types I, III, and V, as well as the amino propeptide of Types I and III procollagen, suggesting newly synthesized collagen. Decorin was present throughout the dermis. The model described appears suitable for in vitro reconstruction of the skin and may be useful to study the development of various supramolecular skin structures.