| Objectives:The epidermal stem cell, which was isolated and purified through rapid adhesion to typeⅣcollagen, was as the seed cell; the acellular amniotic membrane was as dermal matrix, the compound skin has been constructed In vitro and grafted to repair full skin defect on the rabbit back . The information of wound repair was observed, then, the function of the compounded skin in repairing wound was discussed. We wished to establish the foundation of further clinical practice.Methods:The skin samples came from the operation of burn and plastic patients. The sample was disposed with Trypsin-EDTA and got the epidermis, then digested the epidermis and had the epidermal cells, the typeⅣcollagen was used to isolate and purify the human epidermal stem cells, the epidermal growth factor and the keratinocyte serum free medium were used to culture the cells. Then, the cell appearance and growth information was observed and the colony forming efficiency was calculated. The epidermal cells were used as the control groups. In vitro, the separated, purified human epidermal stem cell was implanted on the acellular amniotic membrane, and the compound skin with human epidermal stem cell and acellular amniotic membrane has been constructed.Three full skin defect models, which had been made on the New Zealand white rabbit back, were divided into three groups randomly. Separately, the compound skin with human epidermal stem cell and acellular amniotic membrane or acellular amniotic membrane was used to cover the corresponding wound, the blank wound were used as the control groups. The healing information of the wound was observed. After operation, the materials from the part wound in every group was drawn at the time of one week, two weeks, three weeks and four weeks and detected by routine hematoxylin and eosin stain and immunohistochemistry stain, in order to observe the rebuild information of wound in every group in the process of the wound healing. Results:The epidermal stem cell, which was isolated and purified by rapid adhesion to typeⅣcollagen, was observed by inverted phase contrast microscope. It revealed colony growth and gradually confluence. The colony forming efficiency of the epidermal stem cell is 11.67% and the colony forming efficiency of the epidermal cell is 4.67%, and there is significantly different between groups, P<0.05.When observed by transmission electron microscope, the nucleus is big, the kytoplasm is few, the proportion of the nucleus and kytoplasm is big , the organelle in the kytoplasm are few.The full skin defect on the rabbit back were covered by the compound skin with human epidermal stem cell and acellular amniotic membrane or acellular amniotic membrane, the blank wound were used as the control groups. In each group, the time of the wound healing after operation is 13.78±1.06d,20.89±1.28d,28.67±1.37d,and there is significantly different in three groups, P<0.05. In each group, when the wound healing, the rate of contraction of wound is 22.6±1.54%,29.3±1.41%,30.4±1.58%, there is significantly different between the compound skin group and acellular amniotic membrane group or the bland wound group P<0.05,but no different in acellular amniotic membrane group and the bland wound group, P﹥0.05. The wound revealed by routine hematoxylin and eosin stain index: when the time was at the seventh day after operation, the wound, which was covered by the compound skin with human epidermal stem cell and acellular amniotic membrane, demonstrated that epidermis was thin, interface between epidermis and dermis was smooth, skin nail was not obvious, the fusiform fibroblast in dermis was confused; at the time of the fourteenth day after operation, epidermis got thick, the cell layer was obvious, had emerged cornified cell layer , granular cell layer, spinous cell layer and basal cell layer, skin nail emerged, the fusiform fibroblast in dermis was parallel with epidermis. The rebuild time of the wound covered by acellular amniotic membrane group or the blank wound group was later, at the same time, the wound tissue rebuild was not consummate as the compounded skin group. The wound revealed by immunohistochemistry stain index: in the epidermis of the wound covered by the compound skin ,the Ck19 and HLA-DR expressed at the seventh day after operation, but not in the epidermis of the wound covered by acellular amniotic membrane and the blank wound. The typeⅣcollagen expression was linear and continuous surrounding the basal cell at the seventh day after operation in the epidermis of the wound covered by the compound skin. but was fraction and discontinuity in the epidermis of wound covered by acellular amniotic membrane and the blank wound. Conclusions:Repairing the full skin defect on the rabbit back by the compound skin with human epidermal stem cell and acellular amniotic membrane, the wound healing was promoted, the time of wound healing was short. At the different time after operation, the wound tissue, which were revealed by the routine hematoxylin and eosin stain or the immunohistochemistry stain, index that the epithelization of full skin defect was quicker, the ordering of the fusiform fibroblast in dermis was quicker and there was the human epidermal stem cell in the epidermis of the wound. Possibly, this hinted that the epidermis stem cell can autonomously participate in the process of the skin wound repair and quick the wound healing and rebuild. |
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