In Vivo Immuno-reactivity Analysis Of Three-dimensional Chitosan/SIO₂/hydroxyapatite Hybrids

Part one:Preparation of chitosan/SIO2/hydroxyapatite 3D hybrids and its local immuno-inducing effect.Objective:Preparation of CS-SIO2 and CS-SIO2-HA 3D hybrids;The immune reactivity of CS-SIO2 and CS-SIO2-HA hybrids was compared and analyzed.Methods:Reparation of CS-SIO2 or CS-SIO2-HA precursor mixture,3D-Bioprinter tm system printing,respectively for and CS-SIO2 and CS-SIO2-HA hybrids,natural drying,and freeze drying.Materials were cut into pieces of 0.1x0.1x0.2 cm3 into and then inserted the C57BL/6 mouse gastritis under aseptic conditions.The rats were sutured directly after the blunt separation of the gastritis.The muscle tissue containing the implant was extracted from 14d,28d,42d and 56d after the implantation,and the frozen section was made,and the local inflammatory exudation induced by the implant was analyzed by HE or immunofluorescence.Results:CS-SIO2 and CS-SIO2-HA hybrids were successfully constructed using sol-gel process and 3D printing technology.The geometry of the two materials was uniform and the nanoparticles and hydroxyapatite particles were scattered on the surface of chitosan.Histological examination and immunofluorescence observation confirmed that CS-SIO2 and CS-SIO2-HA hybridization materials implantation in the muscle can rapidly trigger local mononuclear(CD1lb+)/macrophage(F4/80+)infiltration of the implant.The inflammatory exudate was basically subtracted within 2 months after implantation.Compared with freeze-dried materials,the inflammatory response induced by natural drying is more significant.Dendritic cells(CD11c+)and CD3+/CD4+ T cells were clustered around the material.Conclusions:After CS-SIO2 and CS-SIO2-HA 3D hybrids were implanted into the body,it could induce local inflammatory seepage,but the inflammatory response rapidly subsided in the short term.Compared with natural drying,freeze-dried mixed compounds have better internal compatibility,and local inflammatory responses are more transient.Part two:Chitosan/SIO2/hydroxyapatite 3D hybridized material triggers the damage and regeneration of muscle.Objective:The damage and regeneration of the adjacent muscle tissue that were triggered by the analysis of the hybrids of chitosan-SIO2(CS-SIO2)and chitosan-silica-hydroxyapatite(CS-SIO2-HA).Methods:C57BL/6 mice were implanted with CS-SIO2 and CS-SIO2-HA hybrids(0.1x0.1x0.2cm3).In implantation after 14d and 28d,42d,56d harvesting contains muscular tissue implant,frozen section,HE and Masson staining and Dystrophin immunofluorescence staining analyzing the collapse of implant adjacent muscle tissue necrosis,degree of stromal fibrosis,and muscle fibers regeneration process.Results:HE and Masson staining results showed that both CS-SIO2 and CS-SIO2-HA hybrids had certain muscular toxicity,leading to the necrosis of the implant adjacent to myocyte.With the extension of implantation time,the fiber content in the surrounding matrix increased significantly.Compared with CS-SIO2,CS-SIO2-HA hybrids induces more severe collagen accumulation in the inflammatory region.In addition,the muscle necrosis and fibrosis induced by freeze-drying are more obvious than that of natural drying.Dystropin staining observed further evidence that the degeneration of the muscle fibers began to regenerate.As the inflammation in the muscle subsides,the regenerative repair of the damaged muscle tissue is highly effective,and the muscle repair is completed within 2 months after implantation.Conclusions:CS-SIO2and CS-SIO2-HA hybrids has certain toxic effects in the body,may induce the implant adjacent tissue necrosis and fibrosis,with the degradation of materials,intramuscular inflammation subsidise,muscle regeneration,effectively repair muscle fibers.The addition of HA into CS-SIO2 hybrids or freeze-drying technology will affect the compatibility of the hybridized materials.Part three:The activation of chitosan/SIO2/hydroxyapatite 3D hybrids on complement C3.Objective:Systemic immune response was induced by the analysis of the embedded CS-SIO2and CS-SIO2-HA.Methods:C57BL/6 mice were implanted with CS-SIO2 and CS-SIO2-HA hybrids(0.1x0.1x0.2cm3).The spleen cells were extracted from 14d,28d,42d and 56d after the implantation,and the spleen cells were isolated from the spleen cells,or the blood was extracted from the eyes in aseptic conditions.Flow cytometry analyzed the difference of T cells,B cells,monocytes/macrophages and dendritic cells in spleen cells.The activation of complement C3 in peripheral blood was detected by enzyme-linked-immuno-sorbent-assay.Results:Hybrids composition only induce degradation of the spleen in monocyte/macrophage increased,did not see T cells,B cells and dendritic cells number changed.ELISA analysis confirmed that the plasma complement C3 level of the receptor mice increased rapidly after implantation and subsequently decreased.The increase and duration of C3 induced by freeze-drying hybrids were more significant than that of natural drying materials.Conclusions:In mice,degradation of CS-SIO2 and CS-SIO2-HA 3D hybrids was not susceptible and it was difficult to trigger a sustained systemic immune response.