Preliminary Studies On Heterogenous Bioderived Material (Sheep Acellular Dermal Matrix)

Objective To widen source of heterogeneous bioderived materials and try to develop a new xeno-dermis scaffold material and a new biomedical dressing -sheep acellular dermal matrix (SADM) by tissue engineering technology. The construction of skin tissue engineering following with the development of tissue engineering provides a new approach to treat full thickness skin defects by the burns and other injures and also provides new type materials for covering of wounds. In the study of skin tissue engineering,the emphasis is the extracellular matrix scaffold material which can provide nutrition for the seeding cell adhesion,proliferation and differentiation. Scaffold material may act as the place of cellular metabolism,but that synthetic polymers scaffold material may cause the immune exclusion effect and infection because the synthetic polymers are foreign body, and application of human allo-scaffold materials were limited for many reasons . So that,it is attached importance to the finding of natural heterogeneous scaffold materials in the field. For efficiently protecting wounds, development of new type biomedical dressing also be paid high attention. The purpose of this study was to get a new natural heterogeneous bioderived dermal scaffold material and a new type biomedical dressing by xenogeneic sheepskin after acellular process. The histological ingredients after acellular process were analyzed by histological and ultrastructural study. The experiment of sheep xeno-dermis as biomedical dressing for covering of wounds were carried out,and the histocompatibility of xeno-scaffold material were analyzed of scaffold material by in vivo and in vitro experimental studies. The comparation of the content of Type I and Type III collagen of sheepskin after and before sheepskin splited was made by SDS-polyacrylamide gel electrophoresis. Methods (1)One white sheep was anesthetized and disinfected, the hair of trunk were taken off by 8% barium sulfide.Split-thickness skin, 0.4mm thick, was obtained from sheep'sfull-thickness skin with a dermatome, and sheepskin were cell-extracted to obtain natural biomedical scaffold material by the high osmotic salt and chemical detergent and enzyme. (2)The effects of different concentrations of TritonX-100 on cell-extracted degree of sheepskin were compared. (3)The histological ingredients of the bioderived scaffold material were analyzed by histological study. The ultrastructural structures were also observed by transmisson electron microscope. (4) The histocompatibility test of sheep acellular dermal matrix to the body was analyzed by implanting subcutaneouslly .(5)Determination of the content of Type I and Type III collagen of sheepskin after and before splited was made by SDS-polyacrylamide gel electrophoresis. (6) The experiment of SADM as biomedical dressing : the full-thickness skin defect on the back of Sprague-Dawley rats was covered with auto-microskin, then covered with SADM, allo-skin and Paraffn gauze dressing, respectively. The histological changes and also covering results were observed in 1,2, 4 weeks after experiment. Results (1)Analysis of the histological ingredients of sheep acellular dermal matrix(SADM):①H istomorphological observation observed by microscope: deepidermis of sheepskin was very hard, the sheepskin cellular ingredients were not easily deleted. For completely cell-extracted, action time of chemical detergent with sheepskin was prolonged and concentration of chemical detergent are increased.SADM after cell-extracted by 1%TritonX-100,the figures existed,but cells arranged out of order,crowded,and the nucleus were irregular that some concentrated,some broke,the shapes of cells hadn't obvious variation. However,SADM after cell-extracted by 3%TritonX-100,the sheepskin cellular ingredients disappeared,the shapes couldn't be seen,the pink collagen fibers arranged loosely,the splits were big and appeared network structure.②Ultrastructural observation observed by transmission electronmicroscope:SADM after cell-extracted by 1%TritonX-100,the cell membranes broke,the cell organs destroyed and the chromatin concentrated,that became some scattered fragments,some nucleus concentrated,the semilunar apoptosis bodies. However,SADM after cell-extracted by 4 % TritonX-100,cell organs and nucleus disappear, only the figures of cells existed,and sections of collagen fibers distributed in different directions. Basement membrane complex was normal.③Analysis of collagen in sheepskin before and after sheepskin splited with SDS-polyacrylamide gel electrophoresis: Type I and Type III collagen was determined by SDS-polyacrylamide eletrophoresis in sheepskin before and after sheepskin-splited, and results showed reduction of Type I and Type III collagen in the spliting process. In SADM,the remained network frame lose some type I and III type collagen proteins during skin-splited course. (2)Implanting subcutaneously tests of SADM :the expremental effects were normal,and there were no death and poisonous effect. non-crosslinked SADM group: one week after implanting,the cuts healed well, there are some cells (granulovytes,macrophages) infiltrating in the (SADM) sheepskin acellular scaffold material implanted with slight imflammatory, but SADM hadn't been wrapped and could be extracted easily; After two weeks,a thin layer of transparent fibrous membrane bound up the implanted material; macrophages were beginning to increase , more neutrophilic granulocytes and some lymphocytes could be seen also, SADM had been wrapped by the fibrous membrane and connected tightly to wound base with the proceeding ingrowth of host cells and tissue from the wound margins;three weeks after implanting,observed by microscope,some fibroblusts and microvessel and no inflammatory cells develop evidently between collagen ,neutrophilic granulocytes and lymphocytes increased a little,the boundary between the fibrous membrane and its peripheral tissue was still clear,the structure of collagen changed little. After four weeks , the fibrous membrane get thicker,and continunous ingrowth of fibroblusts and microvessel can be seen. From 8 weeks,the SADM implanted was gradually replaced by rat's connective tissue,the fibrous membrane didn't get thicker any more and begin to be thinner,the fibrous membrane density increased,the neutrophilic granulocytes and lymphocytes decreased,polynucleate macrophagocyte could be seen occasionally. 12 weeks after implanting, structure of non-crosslinked SADM became vague, during 16～32 weeks after implanting, non-crosslinked SADM were gradually degraded and trace of SADM cannot be found; Crosslinked SADM group: the boundary between the crosslinked SADM and its peripheral tissue was always clear, there were slight imflammatory reaction. Until 36 weeks, the crosslinked SADM can be subcutaneouslly palpated. (3)The observation and test of the SADM as biomedical dressing: wound grafted with SADM contracture rate was more lower than that of wound without SADM. Conclusion (l)The SADM which was obtained from the high osmotic salt﹣chemical detergent and enzyme method has normal Basement membrane complex and collagen fibers. It's soft , ductility, extensibility , spring and. undisrupted ,but sheepskin structural characteristic has obvious variation to porcine skin and human skin, deepidermis of sheepskin was very hard ,the sheepskin cellular ingredients were not easily deleted. For completely cell-extracted, mechanically scraping method was used for deepidermis of sheepskin prolongation of action time of chemical detergent with sheepskin and increase of chemical detergent concentration are necessary . (2) Main ingredients of the SADM were type I and type III collagen proteins, sheepskin shouldn't be splited too thin because most of sheepskin collagen located in deep layer of sheepskin. (3)The bioderived scaffold material made from SADM had good histocompatibility. (4)SADM has slow degradation rate and can evidently delay wound contraction. (5)SADM can served as a scaffold for ingrowth of host cellular elements such as fibroblast and capillary vessel . (6)SADM has good moisture-maintaining capability and can served as good biomedical dressing for wound, but SADM want to be a new substitute of dermis for tissue engineering of skin, there are still lot of technology problems to be resolved before SADM can be used for medical application. .