| Objective:To constitute a tissue engineering skin substitute that take human epidermal stem cells and human bone marrow stem cells as seed cells and take acellular derma matrix as stent in vitro and then transplant tissue engineering skin on rabbit dorsal full thickness dermal wounds to repair. Investigate the clinical application possibility of repairing full-thickness skin defect with tissue engineering skin by observing wound healing.Methods:The skin samples came from the operation of burn and plastic patients. The epidermal stem cells were separated by trypsin digestion and EDTA united digestion. The typeⅣcollagen is used to isolate and purify the human epidermal stem cells and the epidermal growth factor and the keratinocyte serum free medium are used to culture the cells in vitro. Observe cell morphology and growth. Keratinocytes are used as control.The bone marrow comes from the health people. Separate bone marrow-derived mesenchymal stem cells with density gradient centrifugation separation and mesenchymal stem cells are cultured with DMEM/Low Glucose medium. Observe cell morphology and growth.Constitute tissue engineering skin substitute with epidermal stem cells, human bone marrow stem cells and acellular derma matrix in vitro.Make four full-thickness skin defect models on the back of New Zealand White rabbits. The models then are randomly grouped which are covered respectively with composite skin (Group A)which is builded with mesenchymal stem cells, epidermal stem cells and acellular dermal, composite skin (Group B) which is builded with epidermal stem cells and acellular dermal, and acellular dermal matrix (Group C). The blank is Group D. Observe the full thickness dermal wounds healing. Separately in each group after 15days and 21 days in some subjects Wound Routine HE staining are observed in each group in the process of wound healing into the situation of the organizational structure.Results:1,The epidermal stem cells which are isolated and purified by rapid adhesion to typeⅣcollagen are observed by inverted phase contrast microscope. It revealed colony growth and gradually confluence. When the cells are observed by TEM (transmission electron microscope), we can see that nucleus is big, while cytoplasm and cytoplasmic organelles are less, and proportion of nucleoplasm is large.2,Bone marrow-derived mesenchymal stem cells, which are isolated and purified by the methods of using density gradient centrifugation separation are observed by inverted phase contrast microscope. The cell shape is long spindle and cell is gradual integration connected as radial shape. We will see poorly differentiated naive cells in TEM (transmission electron microscope) containing different cell morphology, nucleoli, highly nucleoplasm ratio, secreted vesicles in cytoplasm and less cell organelles.3,The four full-thickness skin defect models are covered with different skin substitutes. The blank is used as control. Postoperative wound healing time were: 14.56±1.06d,16.21±1.32d,18.25±1.16d,23.0±1.37d, inter-group difference was significant (P <0.05). Conventional HE staining shows the alteration of the wound: (1) group A and B wounds after 15 days had epithelia and cell stratification is obvious. After 21 days collagen become mature with the rules. But group C and D wounds after 15 days wound epithelium is late or incomplete and early wound inflammatory cells can be seen much. After 21days epidermal is thicker than the groups which contain epidermal stem cells. (2) At the same time, there are many capillaries and fibroblasts in granulation tissue in group A, and the number of capillaries in other groups are less than group A.Conclusion:Epidermal stem cells can be isolated and purified by rapid adhesion to typeⅣcollagen and bone marrow-derived mesenchymal stem cells can be separated by density gradient centrifugation separation.Repairing the full skin defect on the rabbit back by the compound skin with human epidermal stem cells, mesenchymal stem cells and acellular derma matrix, two stem cells have function in defect repairing, wound epithelia, wound neovasculariza- tion. The both stem cells play an important function in accelerating the wound healing and reconstruction and improving the the quality of wound healing.
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