Construction, Application And Evaluation Of Tissue-Engineered Skin Composite Utilizing Autogeneic Plasma Fibrin As A Scaffold

Skin defects,especially full-thickness skin defect and chronic ulcer are majoy problems in plastic and burns surgery all along.Usually,autograft,aUograft or skin flap transplantation is used to repair skin defects.However,this therapy has many disadvantages,such as insufficient autografts,a severe hurt of supply section,immune elimination towards allografts.Along with thorough tissue-engineered technology research,the application of tissue-engineered skin to repair skin defects is one of hot spots.Researchists have invented many methods for constructing tissue-engineered skin which give us new prospects for wound healing.Present existing tissue-engineered skin are similar with the structure and the barrier function of normal skin,which has obtained a certain effect,but the tissue-engineered skin has not to be mature still.Most of these tissue-engineered skin products have their disadvantages, such as scarce of autologous seed cell,immune exclusive reaction towards xenogenous seed cells,long cultruring time in vitro,scarce of blood vessal and expensive cost.So it is necessary to invent a new method for constructing tissue-engineered skin which will be usefull in clinical practice.Objective:To prepare a kind of new tissue-engineered skin composite utilizing autogeneic plasma fibrin as a scaffold,and to seed fibroblasts and keratinocytes from autologous skin in the scaffold to construct one kind of tissue-engineered skin composite.To transplant this tissue-engineered scaffold in the animal fascia level,and to research the tissue compatibility and blood capillary growth situation of scsffold. To discussing the feasibility of this tissue-engineered skin composite to repair the full-thickness skin defect.Methods:1.Collecting the rat plasma and fibrin to prepare a scaffold used for morphology observation and the evaluation of physics and biology.2.Using rat autologous skin to primary culture fibroblasts and keratinocytes by isolation of epidemal and dermal tissue from thickness skin and enzyme digestion.Using immunostaining of Monoclonal mouse anti-rat cytokeratin and Monoclonal mouse anti-rat vimentin to identify fibroblasts and keratinocytes.3.Seeding the 3-5^th generation fibroblasts and keratinocytes in the scaffold to construction of tissue-engineered skin composite.The tissue-engineered skin composite was evaluated by HE staining and SEM.The TGF-β1 and bFGF contents in the supernatant fluid of the fibroblasts culture medium were measured with ELISA method.4.transplanting the tissue-engineered scaffold in the animal fascia level to research the blood capillary growth situation of scsffold.The full-thickness skin obtained from 3,5,7,14,21,28th day were measured with histology and immunostaining.Results:1.The prepared plasma fibrin scaffold was clear,salmon pink and glossy.The scaffold could be got down from the round slide easily.The shape of scaffold was round,with the diameter approximately 1.3cm.The HE staining of the scaffold was eosinophilic,which was even.SEM could observe the remarkable netted vesicular structure.The bacteriology and the virus examination results of all specimen were negative.2.fibroblasts and keratinocytes in vitro culture take on the form of attachment growth.The immunostaining of Monoclonal mouse anti-rat cytokeratin and Monoclonal mouse anti-rat vimentin were positive.3.fibroblasts and keratinocytes cultrued in the scaffold grow well.Under the light microscope,the cell junction was close.HE staining showed cell overspreads the scaffold.The SEM showed the cell entered in the scaffold.4.After transplanting the tissue-engineered scaffold in the animal fascia level,the injured area repaired well.It was not seen the reject reaction.HE staining showed the little inflammatory cell infiltration.Five days later,the transplants appeared newborn endothelial cell.Seven days later,the transplants appeared the tube structure of blood capillary.The growth of endothelial cell in the scaffold with VEGF is better than in the scaffold without VEGF.Conclusions:The prepared autogeneic plasma fibrin is one kind of new tissue-engineered scaffold,because of nontoxicity,convenient source,simple preparation and inexpensive cost.fibroblasts and keratinocytes in vitro culture take on the form of attachment growth,which explained that scaffold has provided the advantageous matrix for the seed cell's growth.After the tissue-engineered scaffold imbeded in the animal fascia level,the transplants appeared the tube structure of blood capillary,which confirmed the feasibility of repairing full-thickness skin defect.