Rapid burn wound closure improves patient survival. Multi-center studies have shown that mortality rates decrease with increasing speed of full-thickness burn wound closure. The purpose of this study is to demonstrate that replacement of injured skin with a combination of prosthesis and cultured skin cells will be faster, safer, less expensive and will decrease morbidity and mortality compared to current techniques of dressing, debridement, and autografting. The syngeneic Buffalo rat will be employed as the experimental animal. A living skin equivalent will be made from a prosthetic dermis and Buffalo rat keratinocytes grown in cell culture. Current cell culture techniques require several weeks to produce clinically useful quantities of synthetic skin. Our experimental plan involves manipulation of the culture medium using recognized methods that accelerate the proliferative rate of keratinocytes. These manipulations include the isolated addition of cholera toxin, 8-bromo c-AMP, and insulin to culture medium. The reduction of medium calcium concentration will also be done to accelerate cell growth. The proliferative rate of keratinocytes will be measured by [3H]-thymidine pulse labeling, DNA determination, Lowry protein assay, autoradiography, and scintillation counting. Progression of differentiation will be assessed by Kreyberg staining, [3H]-leucine pulse labeling, extraction of proteins from epidermal keratinocyte cultures, determination of protein concentration (Lowry) assay, slab gel sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. Once the characteristics of keratinocytes grown at an accelerated rate are established, a living skin substitute will be fabricated. This epidermis will consist of rat keratinocytes. Biologic and synthetic dermal substitutes will be tested. This skin equivalent will be grafted onto full-thickness wounds created on the backs of adult Buffalo rats. Adherence of the grafts will be determined, being the most important characteristic of a skin substitute. The wounds will be observed for graft take and healing. Biopsy and histologic examination of the skin substitute will be performed at appropriate intervals. The ultimate goal of our efforts is to develop a living skin substitute useful in treating the severely burned patient. This skin equivalent produced using tissue culture techniques will provide rapid burn wound closure.
| Morykwas, M J; Stevenson, T R; Marcelo, C L et al. (1989) In vitro and in vivo testing of a collagen sheet to support keratinocyte growth for use as a burn wound covering. J Trauma 29:1163-6;discussion 1166-7 |
