Study Of Genipin Cross-linked Acellular Tracheal Scaffolds For Allogenic Transplantation In Situ

Congenital malformation, trauma, tumor and infection. Injury after prolonged endotracheal intubation and some autoimmune diseases, can cause tracheal stenosis or necrosis need tracheal reconstruction or transplantation. The main treatment method for benign or malignant tracheal lesions in patients with various tracheal resection. However, when the gas pipe length of lesions in children with 30% or 50% in adults, direct end-to-end tracheal resection of the lesion agreement will bring a huge risk. Tissue engineering is an emerging research combined with engineering technology and biological principle applied in functional tissue replacement, in order to promote the regeneration of damaged tissue, is expected to become the long segment trachea Reconstruction is an effective method to treat.In this study, New Zealand rabbits as the object, through the acellular and genipin crosslinked method of tracheal scaffolds, based on the cultivation of autologous bone marrow mesenchymal stem cells, with growth factor, orthotopic transplantation of trachea, comprehensive evaluation of in situ transplantation, monitoring its immunogenicity and rejection. On this basis, to obtain autologous bone marrow mononuclear cells by gradient centrifugation, avoid cultured stem cells in vitro, and the use of green fluorescent protein labeled autologous bone marrow mononuclear cells. Containing green fluorescent protein labeled autologous bone marrow mononuclear cells in cell removal of tracheal scaffolds differentiated into cartilage cells, this process avoids the stem cells in vitro, greatly shorten the organization the tracheal preparation cycle, save time and economic costs, for more treatment time for tissue engineering tracheal transplantation patients.Part 1. Genipin cross-linked acellular tracheal scaffolds with autologous bone mesenchymal stemcells allotransplantation in situObjectiveThe orthotopic transplantation of genipin crosslinked off cells of the trachea scaffolds and on the surface of the bracket out of the planting of autologous bone marrow mesenchymal stem cell research, the study of bone marrow mesenchymal stem cells in vivo differentiation of chondrocytes and regeneration of epithelial cells and transplantation after inflammation rejection was evaluated.Methods1.The preparation of acellular scaffold.2. The test of acellular scaffold in vitro.3. Culture and identification of autologous bone marrow mesenchymal stem cells.4. Orthotopic tracheal transplantation;5. The detection of the trachea after orthotopic transplantation.Results1.After the cell was removed, the epithelial cells were removed completely, and most of the chondrocytes were cleared.2. Bone marrow mesenchymal stem cells were obtained by the whole bone marrow culture method and adherence screening method, and the morphological rules of bone marrow mesenchymal stem cells were cultured to the third generation.3. After acellular genipin crosslinked tracheal scaffolds combined with bone marrow mesenchymal stem cells transplantation was similar in degree of inflammation and in situ autograft transplantation and implantation of bone marrow mesenchymal stem cells in vivo can differentiate into cartilage cells.Conclusion1. The decontamination agent combined with enzymatic get off cells of tracheal scaffolds, using natural crosslinking agent genipin crosslinked by genipin crosslinked cells of tracheal scaffolds removal;2.Autologous bone marrow mesenchymal stem cells were isolated and purified by the whole bone marrow adherent culture method, and the autologous bone marrow mesenchymal stem cells were identified by the morphological characteristics and multi differentiation potential of bone marrow mesenchymal stem cells;3.Autologous bone marrow mesenchymal stem cells were successfully implanted in the orthotopic transplantation experiment, and autologous bone marrow mesenchymal stem cells differentiated into chondrocytes in vivo, and no obvious rejection occurred.Part 2. Genipin cross-linked acellular trachealscaffolds planting autologous bone marrow mononuclear transplantation in situ.ObjectiveTo investigate the green fluorescent protein of bone marrow mononuclear cells containing the genipin crosslinked tracheal cells in situ acellular tracheal scaffolds, green fluorescent protein content of bone marrow mononuclear cells were observed to assess planting of autologous bone marrow mononuclear cells differentiation of chondrocytes, and on the use of growth factor in peripheral blood mononuclear cells were evaluated, to investigate the effect of autologous bone marrow mononuclear cells in the tissue engineering tracheal transplantation.Methods1.To obtain the genipin cross-linked acellular tracheal scaffolds.2.Resuscitation and culture of 293T cells.3. To obtain the plasmid of transfection.4.Transfection of 293T cells.5. To obtain the autologous mononuclear bone marrow.6. Slow virus and cell infection.7. Orthotopic tracheal transplantation.8.The content of mononuclear cells and the differentiation of mononuclear cells into chondrocytes were detected after tracheal transplantation.Results1.293T transfected cells expressing green fluorescent protein containing green fluorescent protein, and can synthesize the slow virus containing green fluorescent protein which can infect other cells.2. The slow virus containing green fluorescent protein can infect bone marrow mononuclear can be seen in the cells with green fluorescence under the fluorescence microscope.3. Planting containing green fluorescent protein of autologous bone marrow mononuclear cells of the genipin crosslinked after removing cells of tracheal scaffolds orthotopic transplantation and implantation of bone marrow mononuclear in vivo can differentiation into chondrocytes and in differentiated into cartilage cells containing green fluorescent protein.Conclusion1. Green fluorescent protein can be packaged in the 293T cells within the virus, in the case of other cell activity does not affect the green fluorescent protein into other cells;2. Autologous bone marrow mononuclear cells obtained by gradient centrifugation can be infected with the green fluorescent protein, and the green fluorescent protein can be labeled without affecting the bone marrow mononuclear cells;3.The autologous bone marrow mononuclear cells were traced by the slow virus infection, and the regeneration of cartilage cells was identified by the transplantation of autologous bone marrow mononuclear cells;4. Autologous bone marrow mononuclear cells in orthotopic transplantation of the genipin crosslinked removal of tracheal and cell differentiation into chondrocytes, which avoid stem cells cultured in vitro, saving time and economic costs. And bone marrow mesenchymal stem cells compared. Bone marrow mononuclear cells is more ideal, a kind of stem cells.