OBJECTIVETo constitute a composite skin substitute that can proliferate well with epidermal stem cells and fibroblasts on collagen sponge.

METHODSEpidermal stem cells were selected by rapid attachment to collagen IV for 10-15 min and cultured on 3T3 feeder layers. Collagen was extracted from rat tail. The matrix lattice was fabricated by freeze-dryer and cross-linked with glutaraldehyde. Fibroblasts were inoculated on collagen sponge and cultured for 4 days prior to inoculation of epidermal stem cells to construct composite skin substitute. The composite skin substitute were examined by means of histology, immunohistochemistry and electron microcopy, the histologic appearance was similar to that of normal epidermis.

RESULTSThe epidermal stem cells formed large colonies at 7-8 days, expressed K19 antigen. The percentages of cells at G0/G1 phase of cell cycle and the percentage of alpha6 briCD71dim cells in ESC groups were higher than those in the control group. The skin substitute had epidermis and dermis, the histologic appearance was similar to that of normal skin. The artificial skin expressed keratin antigen by immunocytochemical methods.

CONCLUSIONSEpidermal stem cells proliferated well and differentiated properly on this artificial skin dermis which contained fibroblasts. It seemed that the composite skin to be a good equivalent.