Employing human keratinocytes cultured on macroporous gelatin spheres to treat full thickness-wounds: An in vivo study on athymic rats

Abstract

Providing cutaneous wounds with sufficient epidermis to prevent infections and fluid loss is one of the most challenging tasks associated with surgical treatment of burns. Recently, application of cultured keratinocytes in this context has allowed this challenge to be met without several of the limitations connected with the use of split-thickness skin grafts. The continuous development of this novel approach has now revealed that transplantation of cultured autologous keratinocytes as single-cell suspensions exhibits several advantages over the use of cultured epidermal grafts. However, a number of methodological problems remain to be solved, primarily with regards to the complexity of culturing these cells; loss of viability and other negative effects during their preparation and transportation; the relatively long period of time required following transplantation to obtain a sufficiently protective epidermis. In the present investigation we attempted to eliminate these limitations by culturing the keratinocytes on macroporous gelatin spheres. Accordingly, the efficacies of normal human keratinocytes in single-cell suspension or growing on macroporous gelatin spheres, as well as of split-thickness skin grafts in healing wounds on athymic rats were compared. Human keratinocytes were found to adhere and proliferate efficiently both on the surface and within the pores of such spheres. Transplantation of such cells adherent to the spheres resulted in significantly more rapid formation of a stratified epidermis than did transplantation of single-cell suspensions or spheres alone. Twenty-three days after transplantation, the epidermis formed from the cells bound to the spheres was not as thick as the epidermis on wounds covered with split-thickness skin grafts, but significantly thicker than on wounds to which single-cell suspensions, spheres alone or no transplant at all was applied. Furthermore, fluorescence in situ hybridisation revealed that the transplanted keratinocytes, both those adherent to gelatin spheres and those in single-cell suspension, were components of the newly formed epidermis. These findings indicate that application of biodegradable macroporous spheres may prove to be of considerable value in designing cell-based therapies for the treatment of acute and persistent wounds.