Research Of Biocompatibility In Decellularized Tracheal Matrix

Research Objective:Long tracheal lesions as a common disease in clinical can be traumatic, congenital disease or from tumors involving the trachea. The kind of lesion can be repaired and reconstructed by Tracheal surrenal.Tissue Engineering is a new way for it. Decellularized Tracheal Matrix material provide well Scaffold for Tissue Engineering because its composion and structure similar with normal tissue. The objectives of this study were to evaluate the biocompatibility of decellularized tracheal scaffold getting by NaC104 and compare biocompatibility of decellularized tracheal scaffold, genipin cross-linking agent and glutaraldehyde the cross-linking agent.Reserch method:1.Taking the young newzealand rabbit (NZR) as donor of mesenchymal stem cells(MSCs): extracting marrow from the rabbit tibial plateau after anestheting it.using Whole blood cell culture method to identify its ability of proliferation and adherent.2. Taking the young NZR as donor of MSCs. MSCs were decellularized using 5% NaC104 and stained by HE,DAPI.Observing the results using scanning electron microscopy and electron microscopy.3. Dividing decellularized tracheal matrix Scaffold into small squares 0.5 * 0.5cm,and randomly dividing into three groups:decellularized tracheal matrix Scaffold groups (group A), 1% genipin cross-linking agent group (group B),0.625% glutaraldehyde the cross-linking agent group (group C).Seeding three generation MSCs into outside surface of three decellularized tracheal matrix and placing in 24-well plates to culture in vitro.Finally,assessing the biocompatibility by observing activity, extracts cytotoxicity test,and scanning electron microscopy.Reserch Results:1.Culturing and identification of MSCs:MSCs has a characteristic of adherent, grow in clusters, different forms, fusiform appearance, polygonal. MSCs present bright red stained by oil red O after 10 days and dyed orange stained by Alizarin Red S suggesting MSCs has multiple potential of differentiation.2. Identification of acellular matrix trachea:Tracheal epithelial cells have been removed by HE staining, the microstructure basic of mucosa basal layer, submucosa and tracheal cartilage keep keep integrity, the outer wall of structure keep loose. The nucleus of epithelial cells and submucosal cell has been significantly reduced through DAPI staining. But the nucleus of cartilage cells retain active presenting blue fluorescence. Under the electron microscope, the structure of tracheal epithelium and basement membrane retain intact.3. Inoculability and cell activity identification of stem cells:The material of group A and B has a better security.Adherent cells around it can be observed and no suspension cells. Group C Material has poor security, death cell around it can be observed.4. Cytotoxicity test of leach liquor cell:the leach liquor of groupA and B can promote cell proliferation, group B has a better effective than group A. Group C has toxic making cell death.5. Observation of Scanning electron microscopy:the tissue material of group A has well biocompatibility. the cells attached to the outer wall of the trachea matrix material is good, form a monolayer sheet, covering tracheal wall gap matrix material.Exposed areas can be observed in loacation. Tissue material of group B has a best biocompatibility of all groups.The outside wall and gap of trachea matrix material is filled with cell; Group C has little cell.Reserch conclusion:1. The mesenchymal stem cells provide source of seeds for the construction of tissue engineering trachea because of multi-directional differentiation potential.2. Decellularized tracheal matrix treated with 5% NaC104 could support the seed cells adhesion and growth for its well biocompatibility.3. Genipin as a kind of natural protein cross-linking agent, can effectively improve the biocompatibility of decellularized trachea matrix materials.