Cultured epidermal allografts promote rapid healing of partial-thickness wounds. We developed procedures for cryopreservation of cultured epidermal allografts which, when retrieved from storage, were successfully grafted onto burn patients. We also used sodium n-butyrate (NaB) (a strong promoter of keratinocyte differentiation) to induce a stratum corneum in vitro prior to grafting. The NaB - induction protocol has evolved into a model system which allowed us to identify potential positive regulators (in particular, plasminogen activator inhibitor type-1 [PAI-1], transforming growth factor beta [TGF-beta] and c-fos) of terminal differentiation in keratinocytes. Cultured epidermal allografts do not persist on the wound bed. Their effectiveness may be due, in part, to production and delivery of growth factors and PAI-1 to the wound bed. We propose to continue analysis of the molecular mechanisms which regulate differentiation of cultured epidermis and to determine how molecular and biochemical perturbation of PAI-1 and c-fos expression affect the subsequent ability of cultured grafts to enhance wound healing in vivo. Our long-term goal is to improve availability and quality of transplant-suitable cultured epidermal allografts.
Our specific aims are: I.To conduct a clinical study on the effectiveness of cryopreserved cultured epidermis. II.To begin clinical assessments on the ability of NaB-differentiated cultured epidermal grafts to function as transplants in vivo. III.To determine the extent to which exogenous PAI-1 can induce: (A) keratinocyte growth and differentiation; (B) reorganization of cytoskeletal actin and stabilization of the extracellular matrix; and (C) transplant- appropriate cultured epidermal grafts. IV.To utilize molecular genetic approaches to specifically alter expression of PAI-1 and fos in cultured keratinocytes and to assess the effects of such perturbations on keratinocyte differentiation in vitro and in vivo.
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