Incubation of cultured skin substitutes in reduced humidity promotes cornification in vitro and stable engraftment in athymic mice

Cultured skin substitutes have been used successfully for adjunctive treatm ent of excised burns and chronic skin wounds. However, limitations inherent to all models of cultured skin include deficient barrier function in vitro , and delayed keratinization after grafting in comparison to native skin au tografts. Experimental conditions for incubation of skin substitutes were t ested to stimulate barrier development before grafting, and measure respons es in function and stability after grafting. Cultured skin substitutes cons isted of human keratinocytes and fibroblasts attached to collagen-glycosami noglycan biopolymer substrates. Parallel cultured skin substitutes were inc ubated at the air-liquid interface in ambient (48-61%) or saturated (79-91% ) relative humidity, and grafted to athymic mice on culture day 14. Additio nal cultured skin substitutes were incubated in the experimental conditions for a total of 28 days. Cadaveric human skin and acellular biopolymer subs trates served as controls. Epidermal barrier was evaluated as the change in surface hydration by surface electrical capacitance with the NOVATM Dermal Phase Meter. Cultured skin substitutes and cadaveric skin incubated in amb ient humidity had lower baseline surface electrical capacitance and less ch ange in surface electrical capacitance than parallel samples incubated in s aturated humidity at all ti me points in vitro. Data from healing cultured skin substitutes at 2, 4, 8 and 12 weeks after grafting showed an earlier r eturn to hydration levels comparable to native human skin, and more stable engraftment for skin substitutes from ambient humidity. The data indicate t hat cultured skin substitutes in ambient humidity have lower surface electr ical capacitance and greater stability in vitro, and that they reform epide rmal barrier more rapidly after grafting than cultured skin substitutes in saturated humidity. These results suggest that restoration of functional ep idermis by cultured skin substitutes is stimulated by incubation in reduced humidity in vitro.