Skin Anatomy and Antigen Expression after Burn Wound Closure with Composite Grafts of Cultured Skin Cells and ~ i o ~ b l ~ m e r s Steven T. Boyce, Ph.D., David G. Greenhalgh, M.D., Richard J. Kagan, M.D., Terry Housinger, M.D., J. Michael Sorrell, Ph.D., Charles P. Childress, M.A.T., Mary Rieman, R.N., and Glenn D. Warden, M.D. Ciitriilitnti nil(/ Clnl~lnilrl, Ohio Closure of large skin wounds (i.e., burns, congenital giant nevus, reconstruction of traumatic injury) with split-thickness skin grafts requires extensive harvesting of autologous skin. Composite grafts consisting of col- lagen-glycosaminoglycan (GAG) substrates populated with cultured dermal fibroblasts and epidermal kerati- nocytes were tested in a pilot study on full-thickness burn wounds of three patients as an alternative to split-thick- ness skin. Light microscopy and transmission electron microscopy showed regeneration of epidermal and der- mal tissue by 2 weeks, with degradation of the collagen- GAG implant associated with low numbers of leukocytes, and deposition of new collagen by fibroblasts. Complete basement membrane, including anchoring fibrils and an- choring plaques, is formed by 2 weeks, is mature by 3 months, and accounts for the absence of blistering of healed epidermis. All skin antigens tested (involucrin, filaggrin, laminin, collagens IV and VII, fibronectin, and chondroitin-sulfate) were expressed by 16 days after grafting. This cultured skin analogue provides an exper- imental alternative to split-thickness skin graft that de- velops histiotypic markers of skin anatomy and antigen expression after wound closure. (Plast. Reconstr. Surg. 9 1 : 632, 1993.) Closure of full-thickness skin wounds is accom- plished safely and effectively by grafting of split thickness autologous skin as sheets or However, inadequate availability of skin au- tograft in large total body surface area burns has led to development of alternative materials from ex vivo and in vitro sources4-" for repair of epidermal and/or dermal skin. These materials are applied in either one or two surgical proce- dures. Cultured epidermal auto graft^^.^'^ are ad- ministered over fascia, granulation tissue, or a110 dermis, but are known to blister and ulcerate from slow development of dermal-epidermal junction (DEJ) for several months after graft- ing.7,"-13 By contrast, skin analogues with mes- enchymal and epithelial components that are ap- plied in one procedure are reported not to blister after regeneration of epidermal t i s ~ u e ~ ~ ' ~ in greater similarity to split-thickness skin au- tograft. T o provide maximum availability of skin sub- stitutes for permanent closure of full-thickness burns, composite grafts consisting of cultured autologous keratinocytes and fibroblasts at- tached to collagen-glycosaminoglycan (GAG) substrates can be prepared consistently and stored conveniently in large q~antities.~.'~-" This pilot study describes initial evidence that normal markers of epidermal differentiation (in- vol~crin,~~ filaggrinlg), DEJ (collagens IV and VII,20 laminin21), and dermis (fibrone~tin,~~ chondroitin-sulfate23)are expressed by 16 days after grafting of this cultured skin analogue onto full-thickness excised burns in pediatric patients. Microscopic analyses of patient biopsies begin- ning 2 weeks after grafting confirm develop- ment of skin anatomy and ultrastructure corre- sponding to antigenic markers of normal human skin. Together, these qualitative data demon- strate that a cultured cell-biopolymer implant From the Shriners Burns Institute, the Department of Surgery at the University of Cincinnati, and Case Western Reserve University. Received for publication June 19, 1991; revised March 25, 1992. Presented 04 Apr 91 at the 23rd Annual Meeting of the American Burn Association. Supported by the Shriners of North America, grants #I5893 and #15837. 632