Background. Burn excision followed by immediate wound coverage has become the clinical standard for managing extensive burn injuries in much of the world. When sufficient autograft skin to achieve permanent wound closure is unavailable, cell culture technology has made the use of cultured human keratinocyte (HK) sheets clinically feasible. Whereas previous techniques have focused on development of multilayered, differentiated HK sheets, our attention has been drawn to using HK in a highly proliferative, less differentiated state. Time requirements for preparation of multistratified cultured HK are high, and preparatory steps may destroy important integrin adhesion molecules. Methods. We describe the use of HK cultured to single layer confluence on a polyurethane membrane (HD), with serum-free medium. HK-HD grafts were transplated to full-thickness wounds on athymic mice (n=31). A second group of mice (DG-HK-HD, n=28) received a living human dermal replacement containing cultured fibroblasts before placement of HK-HD. Control mice received HD alone (n=4). Basement membrane proteins on healed wounds and surface integrins on cultured HK were identified by means of immunostaining and direct microscopic visualization. Results. HK cultured just to the confluent state on polyurethane membrane were positive for integrins alpha(5) and alpha(6), major integrins on proliferating HK. Histologic analysis showed epithelialized wounds in all groups after 21 days. Using an anti-human involucrin antibody we demonstrated the presence of HK in 64.5% of the HK-HD group, 61% of the DG-HK-HD group, and 0% in the HD group. Mice that received the living human dermal replacement containing cultural fibroblasts in combination with HK-HD grafts developed a thick, well-vascularized neodermis. Strong laminin and collagen IV staining was observed in wound areas covered with HK. Conclusions. These data show that full-thickness wounds can by closed by application of a single layer of proliferating HK cultured on a biocompatible polyurethane membrane. This technique is an alternative to the use of multilayered, differential HK sheets. Preparation times for HK-HD grafts should be significantly shorter than required for multilayered HK sheets, technical efforts should be less, and more extensive wound areas could be covered.
