Rapid and effective healing of burn wounds with cultured analogues 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 skin autograft, shorter hospitalization time, and reduced long-term debilitation 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 produce an epidermal analogue with true barrier properties, and from absence of a vascular plexus in dermal analogues. The current model of cultured skin in this laboratory is a collagen-based sponge populated with cultured human keratinocytes and fibroblasts. Proposed studies will address and correct some or all of the deficiencies of cultured skin by integrated studies in vitro, in animals, and in burn patients. In vitro studies will address: 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 c) expression of cytokines and adhesion proteins 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, trans epidermal water loss, surface hydrophobicity); b) electron microscopy and biochemical assay of biopolymer substrates; and c) western blot analyses of expressed cytokines and adhesion proteins. Skin analogues with optimal composition will be compared to controls (human xenograft, autograft, no graft) for efficacy (wound contraction, HLA-ABC expression), skin pigmentation (melanin content, tyrosinase) and expression of cytokines and adhesion proteins by grafting to full-thickness skin wounds on athymic mice. Experimental wounds will also be treated to stimulate vascularization during initial engraftment by topical delivery of angiogenic agents (bFGF, TGF-beta, and nutrients to improve cell survival. Angiogenesis will be studied versus time after grafting by: a) acrylic casts of the neovascular plexus; b) transcutaneous oxygen; and c) laser Doppler of capillary blood flow. Clinical studies will focus on stimulation of angiogenesis, and control of microbial contamination of the skin substitute during engraftment. Excised burns treated with composite cultured skin will be studied in the clinic by paired-site comparison to treatment with meshed split-thickness skin autograft. Comparative parameters of outcome will include: a) engraftment of cultured skin (incidence of failure, degree of infection, time to wound closure, need for reconstruction); and b) long- term results (function, contraction and cosmesis). The investigators possess all of 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, validated models as alternatives to animal testing of consumer products, and development of other tissue and organ substitutes.
