WOUND CLOSURE WITH HUMAN KERATINOCYTES CULTURED ON A POLYURETHANE DRESSING OVERLAID ON A CULTURED HUMAN DERMAL REPLACEMENT

Background. Burn excision followed by immediate wound coverage has bec ome the clinical standard for managing extensive burn injuries in much of the world. When sufficient autograft skin to achieve permanent wou nd 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, diffe rentiated HK sheets, our attention has been drawn to using HK in a hig hly proliferative, less differentiated state. Time requirements for pr eparation of multistratified cultured HK are high, and preparatory ste ps may destroy important integrin adhesion molecules. Methods. We desc ribe the use of HK cultured to single layer confluence on a polyuretha ne membrane (HD), with serum-free medium. HK-HD grafts were transplate d to full-thickness wounds on athymic mice (n=31). A second group of m ice (DG-HK-HD, n=28) received a living human dermal replacement contai ning cultured fibroblasts before placement of HK-HD. Control mice rece ived HD alone (n=4). Basement membrane proteins on healed wounds and s urface integrins on cultured HK were identified by means of immunostai ning and direct microscopic visualization. Results. HK cultured just t o the confluent state on polyurethane membrane were positive for integ rins alpha(5) and alpha(6), major integrins on proliferating HK. Histo logic analysis showed epithelialized wounds in all groups after 21 day s. Using an anti-human involucrin antibody we demonstrated the presenc e 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 replaceme nt containing cultural fibroblasts in combination with HK-HD grafts de veloped a thick, well-vascularized neodermis. Strong laminin and colla gen IV staining was observed in wound areas covered with HK. Conclusio ns. These data show that full-thickness wounds can by closed by applic ation of a single layer of proliferating HK cultured on a biocompatibl e polyurethane membrane. This technique is an alternative to the use o f multilayered, differential HK sheets. Preparation times for HK-HD gr afts should be significantly shorter than required for multilayered HK sheets, technical efforts should be less, and more extensive wound ar eas could be covered.