Preparation And Application Of Tissue Engineering Of Skin Substitute

Objective: The purpose of this study was to prepare PADM without cytotoxicity. Experiment on use of composite skin graft consisting of PADM combined with aotologous micro-skin and allogenic skin graft to cover the deep burn wound were studied, and its clinical effect was observed. Experimental study on tissue engineering of skin containing epidermal cell,and living dermal matrix prepared by Fibroblasts-Collagen glue-PADM to cover the deep burn wound were carried out.Methods: 1. The split-thickness skin graft of 0.3~0.4mm were first obtained. The PADM without cytotoxicity was prepared by removing cells from the skin with Hyperosmotic Saline and Sodium Hydroxide. They were cross-linked and sterilized. Bacteria and porcine virus were examined. The PADM were embedded beneath the Sprague-Dawley (SD) rat's skin, change in general and histological were observed post transplantation. SD rats with full-thickness skin defect on the back were covered with PADM prepared by two different methods, together with auto-microskin and allo-skin, we got test group I and II respectively. The conditions of the grafts were observed in 2, 4, 6 weeks post transplantation. Ethical permission was obtained, PADM with auto-microskin and allo- skin was used to cover the deep burn wound, the transplatation results were observed periodically. 2. The Epidermal cell and fibroblasts culture in vitro were conducted by enzyme digestion. Epidermal cell primary culture was described by growth graph and MTT method was used in subculture cell growth. The fibroblasts were directly or mixed with collagen inoculated on the surface of PADM to get living dermal matrix. The TGF- β 1 and bFGF contents in the supernatant fluid of the culture medium were measured with ELISA method. The epidermal cells were inoculated on the collagen surface of the living dermal matrix. Tissue engineeringof skin substitute was observed. Using RT-PCR method, the influence of differentiation and gene expression of epidermal cells were observed by air-liquid culture. SD rats with full-thickness skin defect on the back were randomly divided into two groups, then tissue engineering of skin and epidermal cell sheet were adopted to cover the skin defect respectively.Results: None cellular components were observed in PADM. The bacteriology and the virus examination results of all specimen were negative. PADM prepared by Hyperosmotic Saline and Sodium Hydroxide method beneath rats skin could maintain their general structure over 24 weeks. There was no statistical difference between test group I and II in wound healing quality. Histological examination revealed that there was full differentiation of epithelium, orderly collagen arrange and intact basal membrane. Clinical observation revealed that PADM with auto-microskin and allo-skin could be used to cover deep burn wound, and the quality of composite transplation was better than that of auto-microskin with only allo-skin graft. Fibroblasts and epidermal cells in vitro culture take on the form of attachment growth. At the same seeding concentration, during primary culture, foreskin epidermal cell growth ability was the best, then that of the scalp, while the slowest growth ability in armpit area was observed. However, there were no statistical significant difference in the proliferation speeds of subculture (P>0.05). The TGF- ft 1 and bFGF contents of test group I reached a stable level at the eighth day after inoculation, while the contents of test group II reached a stable level at the fourth day after inoculation, and the TGF- ft 1 and bFGF contents of test group II were higher than that of test group I (P< 0.05). Epithelium and dermis were observed in the equivalent skin, and desmosome existed in cells of epithelium. Epithelial cell differentiation was better using air-liquid interface culture. The bFGF, TGF- ft 1 and KGF expressions were higher in air-liquid interface culture. Compared with epidermal cell sheet transplation, the graft survival rate was no significant difference between composite skin grafting group and epidermal cell sheet transplation(P >...