Keratinocyte-driven contraction of reconstructed human skin

We have previously reported that reconstructed human skin, using deepidermi zed acellular sterilized dermis and allogeneic keratinocytes and fibroblast s, significantly contracts in vitro. Contracture of split skin grafts in bu rns injuries remains a serious problem and this in vitro model provides an opportunity to study keratinocyte/mesenchymal cell interactions and cell in teractions with extracted normal human dermis. The aim of this study was to investigate the nature of this in vitro contraction and explore several ap proaches to prevent or reduce contraction. Three different methodologies fo r sterilization of the dermal matrix were examined: glycerol, ethylene oxid e and a combination of glycerol and ethylene oxide. While the nature of the sterilization technique influenced the extent of contraction and thinner d ermal matrices contracted proportionately more than thicker matrices, in al l cases contraction was driven by the keratinocytes with relatively little influence from the fibroblasts. The contraction of the underlying dermis di d not represent any change in tissue mass but rather a reorganization of th e dermis which was rapidly reversed (within minutes) when the epidermal lay er was removed. Pharmacological approaches to block contraction showed fors kolin and mannose-6-phosphate to be ineffective and ascorbic acid-2-phospha te to exacerbate contraction. However, Galardin, a matrix metalloproteinase inhibitor and keratinocytte conditioned media, both inhibited contraction.