Biological Properties Of 3D Printing Biomimetic Material And Decellularized Tracheal Graft

Congenital tracheomalacia collapse stenosis or atresia,acquired tracheal injury caused by trauma,tumors,large area burns and other reasons can cause severe dyspnea or even asphyxia.End-to-end anastomosis after resection of the diseased trachea is generally considered to be the most effective treatment.However,with the deepening of tracheal reconstruction research,the emergence of tissue engineered trachea brings dawn for tracheal reconstruction.Tissue engineering effectively combines seed cells,scaffolds and cytokines to repair tissue defects and reconstruct organs.As the main framework structure of tissue engineered trachea and the soil for seed cell growth,scaffold material is the basis for successful construction of tissue engineered trachea.At present,primary allogeneic tracheal matrix materials and synthetic materials are widely used in tissue engineering tracheal scaffolds.Our laboratory mainly studies decellularized tracheal matrix materials and 3D printing materials.The purpose of this study is to compare the biomechanical properties and biocompatibility of three-dimensional printed tracheal material and acellular tracheal matrix material in vitro;to evaluate the biocompatibility,immune rejection and inflammation of the two materials through in vivo implantation experiments;and the last,orthotopic transplantation of two kinds of patches was carried out to evaluate the effect and shortcomings of the transplantation.Finally,the advantages and disadvantages of the two materials are obtained through the above comparative study,which provides a basis for the construction of more reasonable and ideal tissue engineering trachea.Part 1.Biomechanical properties and biocompatibility of two matrix materials in vitroObjectiveTo evaluate the biomechanical properties and biocompatibility of three-dimensional printing tracheal material and acellular tracheal matrix material.Methods1.Using 3D printing technology and sol gel method to prepare PCL porous tracheal scaffolds modified by nano silica.2.Decellularized rabbit tracheal matrix material was prepared by detrgent-enzymatic method.3.The biomechanical properties of the scaffolds were evaluated by longitudinal tension,radial compression and three-point bending tests.4.CCK-8,scanning electron microscopy,Giemsa test and contact test were used to detect the proliferation,adhesion and cytotoxicity of co-cultured scaffolds and cells.Results1.The biomechanical properties of 3D printed PCL tracheal stent are better than that of decellularized tracheal stent.2.Scanning electron microscopy showed that the cells adhered well on both groups of scaffolds.Giemsa test showed that decellularized tracheal scaffolds had less cytotoxicity.CCK-8 test showed that acellular tracheal scaffolds were more conducive to cell proliferation.Conclusion1.3D printed PCL tracheal stent has good biomechanical properties.2.Both groups of scaffolds were conducive to cell adhesion.Decellularized tracheal scaffolds had better cell proliferation environment and lower cytotoxicity.Part 2.In vivo experiment:subcutaneous implantation ObjectiveThe biocompatibility,immune rejection and inflammation of the two materials were evaluated.Methods1.Two groups of tracheal stents were implanted subcutaneously.2.Dynamic monitoring of blood routine and serum immunoglobulin changes in recipients;3.Histological analysis of buried plants;4.Immunohistochemical staining was used to detect the expression of CD68,MHC-I and II antigens in buried plants.Results1.The white blood cells of experimental animals reached the peak about one week after operation and then decreased.The peak of IgM appeared earlier than that of IgG,which accorded with the rule of humoral immune response antibody production.In the acute stage of inflammation,the monitoring indexes of the three-dimensional printed PCL tracheal stent group were higher than those of the decellularized tracheal stent group,and the levels of the two groups were similar after the acute stage.2.HE staining showed that eosinophil infiltration was the main inflammatory response in PCL tracheal group,while inflammatory cell infiltration was less in decellularized tracheal stent group.3.Immunohistochemical examination showed that there was no significant infiltration of macrophages in the scaffolds in both groups..Conclusion1.Acellular tracheal stent has little stimulation to receptor inflammation.2.The immune rejection of recipients in PCL tracheal stent group and decellularized tracheal stent group was less than that in PCL tracheal stent group.Part 3.Experiments on tracheal defect repairObjectiveTo evaluate the effect and shortcomings of two groups of tracheal stent patches in repairing tracheal defects.Methods1.The recipient tracheal defect was repaired and transplanted with two groups of tracheal patches respectively.2.Regular tracheoscopy and sputum aspiration were performed after repair.3.Histological analysis of the repaired tissue was performed after harvesting.4.Immunohistochemical staining was used to detect CK-18 antibody in tissues.Results1.The fresh tracheal patch group died of asphyxia due to acute inflammatory rejection and tracheal collapse in the acellular tracheal patch group.The recipients in the 3-D printed PCL tracheal stent patch group survived until the grafts were harvested after regular sputum aspiration and anti-infection treatment.2.After 30 days of transplantation,the anastomotic healing and patency were observed by fiberoptic bronchoscopy.No obvious granulation tissue was found,and epithelialization was observed.No obvious narrowing or secretion was found in the distal lumen.3.HE staining showed neocilia on the surface of the scaffold,complete PCL material structure,disordered tissue structure below cilia and infiltration of inflammatory cells 30 days after operation.4.Immunohistochemical detection of CK-18 antibody showed that the epithelium regenerated on the graft surface,the cilia arranged neatly,and a small amount of gland regeneration could be seen.Conclusion1.The effect of acellular tracheal patch for tracheal defect repair is not satisfactory.2.After 30 days of orthotopic transplantation of PCL tracheal stent patches,the anastomotic stoma healed completely,the lumen was completely unobstructed,and the patch surface was epithelialized,which had a certain function of primary gas tube.