Rapid and effective healing of burn wounds with cultured analogs of human skin is the central objective of this proposal. Medical benefits from improved healing may include, but not be limited to: reduced requirements for split-thickness autograft, shorter hospitalization time, and reduced long-term morbidity after recovery. However, anatomic and physiologic deficiencies of all current models of cultured skin have restricted realization of these benefits. Major deficiencies result from culture conditions that do not generate an epidermal analog with functional barrier properties, and from absence of a vascular plexus in the dermal analog. The current model of cultured skin in this laboratory is a collagen-based sponge populated with cultured human keratinocytes and fibroblasts. In vitro studies will regulate physiology by: a) media (e.g., nutrient, mitogen) and biophysical conditions that affect cellular metabolism of keratinocytes, fibroblasts and melanocytes; b) composition (structure and biochemistry) of implantable biopolymer substrates for cell delivery; and assess c) expression of barrier lipids, cytokines and matrix molecules by skin analogues before grafting. Evaluation of these factors will be performed by : a) measurements of cell growth (DNA synthesis) and epidermal differentiation (barrier ultrastructure, lipids, surface hydration); b) molecular and ultrastructural analysis of skin substitutes; and, c) ELISA and northern blot analyses of expressed xenograft, autograft, no graft) for efficacy (wound contraction, epidermal barrier, HLA-ABC), pigmentation (hue & chromaticity by chromameter) and expression of cytokines and adhesion proteins by grafting to full-thickness wounds in athymic mice. Clinical studies will focus on stimulation of angiogenesis, and maximum survival of transplanted cells during engraftment of skin substitutes. Excised burns treated with cultured skin substitutes will be studied in the clinic by paired-site comparison to treatment with meshed, split-thickness skin autograft. Comparative parameters will include: a) quantitative wound closure with skin substitutes (incidence of engraftment, ratio of healed area to biopsy area, time to wound closure, frequency of regrafting); and, b) qualitative outcome (scarring, contraction, function and cosmesis). The investigators claim to possess all of the required expertise in cell biology, skin biochemistry and biophysics, wound physiology, and clinical burn care to perform these studies successfully. Accomplishment of these objectives may contribute to reduced mortality and morbidity from burns, improved materials for plastic and reconstructive surgery, and development of other tissue and organ substitutes.
