| Tracheal resection and end-to-end anastomosis has been the standard clinical approach for the treatment of most airway diseases which may result from stenosis,tracheomalacia,and tumors.However,when the length of the diseased trachea exceeds more than one-half of the total length in adults or more than one-third of the total length in children,tracheal replacement or reconstruction seems to be the only possible solution.Tissue engineering,introducing seeding cells,scaffold materials and cytokines for the regeneration of the desirable native tissue,has distinct virtues.Organs and tissues printed by 3D printing technology have better biomechanical properties and cellular biocompatibility.They make up shortcomings and deficiencies of traditional tissue engineering,which can be applied to clinical therapy in the future.With the development of tissue engineering,the research and applications of absorbable biomaterials become particularly important.In recent years,polycaprolactone(PCL)has attracted more and more attention in the area of 3D printing technology.However,the hydrophobicity of PCL greatly influences the interaction between cells and materials,and then affects the proliferation behavior of the cells on the surface of the material.Surface modifications can improve the cytotropism without affecting the original properties of the material.The porous structure of the material can help deliver the appropriate signal and necessary nutrition for seeding cells and strengthen intercellular communication,so that it can increase the cell viability.The purpose of this study was to prepare 3D printed porous tracheal graft fabricated by PCL and to select the appropriate pore size and surface modification techniques,in order to explore its effect on cell behavior.What's more,the cellular biocompatibility and immune rejection of 3D printed porous tracheal graft were evaluated via implantation into rabbits.This study provides the theoretic and experimental basis for further research and practice in tracheal reconstruction.Part 1.Preparation and biomechanical test of 3DP tracheal graftObjectiveTo prepare 3DP tracheal graft fabricated by PCL and study its biomechanical properties.Methods1.The PCL porous tracheal graft was prepared by 3D printing technology;2.Biomechanical properties of 3DP tracheal graft were measured by means of longitudinal tension,radial compression and three-point bending test.Results1.The 3DP porous tracheal graft has similar morphology with the native tracheas;2.Compared to native tracheas,the 3DP porous tracheal graft has better biomechanical properties,which is inversely proportional to the pore size.Conclusion1.The PCL porous tracheal graft was successfully prepared by 3D printing technology;2.3DP porous tracheal graft shows favorable biomechanical properties.Part 2.Selection of the appropriate pore size and surface modification techniques for 3DP tracheal graftObjectiveTo select the appropriate pore size and surface modification techniques.Methods1.The porous grafts were surface-modified through hydrolysis,amination and nanocrystallization treatment;2.The surface properties of modified grafts were characterized by energy dispersive spectroscopy(EDS)and scanning electron microscopy(SEM);3.The effect of different pore sizes and surface modifications on the cell proliferation behavior was evaluated by CCK-8;4.Micrographs of the 3DP tracheal graft and BMSCs were observed by SEM.Results1.It is more suitable for cell adhesion and proliferation when the pore size is 200 ?m;2.Compared to hydrolysis and amination,the surface of the nano-modified scaffold is more glossy,and the effect is better;3.Nanocrystallization treatment successfully improved the cytotropism of the 3DP tracheal graft.Conclusion1.The appropriate pore size of the 3DP porous tracheal graft is 200 ?m;2.The appropriate surface modification techniques is nanocrystallization.Part 3.Evaluation of cellular biocompatibility and immune rejection for 3DP tracheal graftObjectiveTo evaluate the cellular biocompatibility and immune rejection of surface-modified 3D printed porous tracheal graft.Methods1.The cellular biocompatibility of surface-modified 3DP tracheal graft was measured by in vitro cellular toxicity test;2.Dynamic monitoring of blood routine and serum immunoglobulin in the receptor;3.Histological analysis of harvesting samples was analyzed to identify the immune rejection;4.Immunohistochemistry detection of harvesting samples was done to show the presence of CD68,MHC-? and MHC-? antigens.Results1.Cellular contact toxicity test exhibited that BMSCs grew well around the surface-modified graft,which showed good cellular biocompatibility.2.The leukocyte of the experimental animals reached the peak about one week after the operation and then subsided;the peak of IgM appeared earlier than IgG,it's conformed to the general law of humoral immunoresponse.3.Histological evaluation of native trachea by HE staining at 30 days after implantation showed clear signs of rejection.The cartilage matrix structure distorted and disordered,and there was macrophagocyte and lymphocyte infiltration in the outer surface,furthermore the inner surface was covered with hemorrhagic necrosis and inflammatory cells.On the contrary,inflammatory reaction obviously faded in 3DP porous tracheal graft.The arrangement of the fiber in granulation tissue was looser,and the number of inflammatory cells significantly decreased.4.Immunohistochemistry detection of native trachea at 30 days after implantation showed that tracheal matrix folded and fractured,and the nucleus in cartilage excavatio disappeared.The number of the cells with positive expression of CD68,MHC-? and MHC-?antigen increased.The detection of 3DP porous tracheal graft indicated that the number of the cells with positive expression of antigen decreased.There was neovascularization in the granulation tissue,the nucleus of vascular endothelial cells were round-like.ConclusionThe 3D printed tracheal graft with a diameter of 200 ?m has good cellular biocompatibility and low immunogenicity after nano-silica modification. |
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