| Auto-skin grafting will be fist used when extensive skin loss occurs, when suffer a deficient of donor sites available for skin graft, other methods always used in clinic such as small stamps of autografting, mesh skin grafting, intermingle transplantation of auto-allografting, micro-skin-grafting and cultured epidermal Auto-grafting, because lack of dermis, can't resolve the problems regarding the cosmesis and function satisfactorily. Tissue-engineering skin can help resolving this problem, but there are some trouble in all kinds of tissue-engineering skin now, some are just dermis scaffold, some may lead to reject reaction because of the variaton of epidermal cells. Therefore, it will have clinical importance to composite a permanent skin substitute with ideal functions and no reject reaction.Objective: Investigate the feasibility constructing living composite skin of tissue-engineering. To construct tissue-engineered skin with cultured epidermal stem cells and acellular dermal scaffold and investigate the feasibility of repairing animal skin defection with the tissue-engineered skin. To research the construction and function of the tissue-engineered skin.Methods: Epidermis was separated from dermis of child's forskin,and dissociated into single cells. epidermal cells were incubated for 10-15min at 370C on dishes coated with collagen typeⅣ,then the adherent and nonadherent cells were cultured respectively in the K-SFM medium, supplemented with 100g/L fetal calf serum (FBS, for stem cell). The cells were examined for phenotypes under microscope, the expressions of Cytokeratine 15 (CK15) , Cytokeratine 19 (CK19) andβ1-integrin were detected with immunohistochemical method, andCD71 were detected with flow cytometry. Acellular dermal scaffolds were prepared by using of trypsin .Well-growing human epidermal stem cells which isolated were seeded onto the surface of acellular dermal scaffold which covered by collagen typeⅣto construct living tissue-engineered skin. This kinds of skin were transplanted to full skin wound in SD rats, compared with transplatation of acellular dermal scaffold only. The wounds were oberserved after operation. Tissue samples were harvested and examined by means of histology and immunohistochemistry method.Results:1,The epiderm stem cells can be adhered rapidly by collagen typeⅣ; 2,the adhered cells formed more and bigger colonies than that of nonadhered cells. The positive expressions by immunohistochemical method of CK15 are50.80%+1.81, CK19 are 51.40%+2.22,β1-integrin are51.40%+1.90, and the positive expressions by flow cytometry method of CD71 is1.91%+0.21. 3. The acellular xeno-dermis prepared firstly has been removed of the epidermis and all cellular components of the dermis. but the basement membrane complex and the three-dimensional structure of the collagen scaffold appeared intact. 4, ESCs can soon adhered to the surface and seed on the acellular dermal scaffold which voverd by collagen typeⅣ. A living tissue-engineered skin include 1-2 layers of epithelium was constructed in vitro after 2 weeks Co-cultivation. 5, After the tissue-engineered skin being transplated onto the wounds, it healed well with no rejiection and only a little contraction. The tissue histology structure also demonstrated that the wounds had healed. The neoepithelium was 1-2 layers thick consisting of epidermal cells, but there is no auxiliary structure of skin can be seen . The positive expression of broad-spectrum cytokeratine demonstrated that the neoskin was regenerated from grafted human cells. But in control group, the acellular dermal scaffolds transplated become dry because of no epithelium. In the end, the acellular dermal scaffolds seperated from the wounds and the wound contraction , 3 weeks later, the wounds heal with scar, tissue histology show that the expression of broad-spectrum cytokeratine is negative.Conclusion:1,The character of epiderm stem cell can be adherent by collagen typeⅣcan be used to separate ESCs, ESCs can grow rapidly in K-SFM medium, and produce massive daughter cells, so can be used as the seeds of tissue-engineered skin.2,The acellular dermal scaffolds prepared by method of Trypsin had been demonstrated that it had been removed of the epidermis and all cellular components of the dermis. but its basement membrane complex and the three-dimensional structure of the collagen scaffold appeared intact. Can be used as scaffold of TE.3,The ESCs can be seeded onto the surface of scaffold after amplification and passage, after 2 weeks'growth and Co-cultivation in K-SFM medium, can construct a living tissue-engineered skin with human epidermal stem cells and acellular dermal scaffold in vitro .4,After transplantation onto animal wounds,the composite skin was demonstrated to be a good skin substitut to repair skin defection, tissue history show that the ESCs can differentiate to epithelium, duration the observation there is no auxiliary structure of skin can be seen. |
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