Biologic attachment, growth, and differentiation of cultured human epidermal keratinocytes on a graftable collagen and chondroitin-6-sulfate substrate

Abstract

Repair of full-thickness burns requires replacement of both the dermal and the epidermal components of the skin. Use of tissue culture methods allows very large expansions of surface area to be covered by cultured normal human epidermal keratinocytes (HK). Porous and resorbable materials, such as collagen and chondroitin-6-sulfate membranes, may be expected to adhere to wounds and promote fibrovascular ingrowth better than grafts of cultured epidermal keratinocytes alone. This article demonstrates the in vitro formation of biologic attachments between HK and a collagen and chondroitin-6-sulfate dermal skin replacement. Dermal membranes are prepared as generic acellular sheets and stored in the dry state for extended periods. Subconfluent HK cultures in logarithmic phase growth can attach quickly to dermal membranes in vitro, form a confluent epithelial sheet on the surface of each membrane, and exhibit mitotic cells for at least 1 week in vitro. Transmission electron microscopy demonstrates the formation of hemidesmosomes, extracellular matrix, and banded collagen at the interface of the epidermal cells and the dermal membrane. By comparison, HK cultures as confluent sheets released enzymatically with Dispase do not attach to the dermal membranes in vitro, under the conditions tested, although complete coverage of the membrane by the cell sheets is obtained. Growth assays show that subconfluent HK cells retain sufficient growth potential to maintain logarithmic phase growth, but that HK cells disaggregated from confluent sheets become growth arrested in comparison. The composite material has discrete dermal and epidermal compartments, has total thickness comparable to split-thickness skin graft, and can be applied to full-thickness skin defects in a single procedure.