The co-application of sprayed cultured autologous keratinocytes and autologous fibrin sealant in a porcine wound model

Abstract

We have explored the potential for cultured autologous keratinocytes to form an epidermis when delivered as a spray intermixed with autologous fibrin sealant. Twelve full-thickness wounds in Large White pigs (six wounds in each of two pigs) were isolated from the surrounding skin by 4 cm diameter polytetrafluoroethylene chambers, and grafted with Integra artificial skin (Ethicon). Autologous fibrin sealant was produced 10 days later, using an automated processor unit (Vivostat System, ConvaTec, Bristol Myers Squibb), from 120 ml of autologous citrated blood taken 30 min before keratinocyte application. Nine wounds were sprayed, using a Vivostat System automated applicator unit, with a mixture of the sealant preparation and freshly trypsinised cultured autologous keratinocytes in growth medium, at a density of 1-3 x 10(5) cm(-2). Three control wounds were sprayed with the same mixture without cells. The sealant-cell mixture polymerised and adhered to the wound surface immediately. Histological analysis of biopsies taken following sealant-cell application showed that isolated spherical keratinocytes were distributed throughout the sealant at between 3.1 x 10(4) cm(-2) and 7.6 x 10(4) cm(-2). After 4 days discreet colonies of keratinocytes were observed on the wound bed. At 14 days a multi-layered undulating epidermis was formed, punctuated by sporadic epidermal cysts; the mean area of epithelium was 50.1% (s.d. = 19.7%, n = 9). There was no epithelium in the controls (s.d. = 0, n = 3). The difference was statistically significant (P=0.016). This study suggests that co-sprayed cultured keratinocytes and autologous fibrin sealant may be an effective means of delivering epithelial cells to assist wound healing.