| Objective:Implant denture is the preferred method to repair missing teeth.The success of implant implantation requires sufficient height and width of alveolar ridge in the missing area.Patients with insufficient alveolar ridge need to receive different degrees of alveolar ridge augmentation surgery.Onlay bone grafting uses massive bone implanting on the surface of alveolar ridge in the recipient area to increase the height or width of severely absorbed alveolar ridge,so that the amount of alveolar ridge can meet the basic requirements of implantation.Studies on the use of autogenous tooth roots as bone augmentation material for Onlay bone grafting have confirmed that autogenous tooth roots have similar histological properties and osteogenic effects as bone,and can participate in the process of bone remodeling.Compared with the limited autogenous tooth source,the allogeneic tooth root source is extensive,but there are immunogenicity problems.The extracellular matrix(ECM)is an ideal biomaterial to remove the cell components in tissues and organs,and retain the complex three-dimensional structure and physicochemical properties of tissues and organs while removing the immunogenicity.Therefore,this study explored the use of acellular matrix material of tooth root as bone augmentation material for Onlay bone grafting to solve the problem of insufficient alveolar ridge bone mass.The purpose of this experiment is as follows:1.Explore the decellularization method suitable for tooth root and obtain the bone augmentation material from decellularized tooth root;2 、 Evaluate the cytocompatibility,blood compatibility and systemic compatibility of acellular root derived bone augmentation materials in vitro and investigate the effect of acellular root derived bone augmentation material on phenotypic polarization of macrophages;3、Evaluate the osteogenic effect of acellular root derived bone augmentation materials for Onlay bone grafting in vivo.Methods:1、To explore the decellularization method for tooth root and optimize the process of tooth root decellularization to obtain decellularized rat tooth root and decellularized human tooth root.HE staining and Masson staining were performed on the histological sections of fresh and acellular tooth roots to evaluate the effect of decellularization of rat and human teeth.The surface morphology of rat root and human root before and after decellularization was observed by scanning electron microscopy.SDS residue kit is used to detect the residual status of acellular reagent and evaluate the damage degree of acellular reagent to tissue cells.2、The cytotoxicity test,hemolysis test,intradermal reaction test and acute toxicity test were used to detect the cytocompatibility,blood compatibility and systemic compatibility of the acellular rat tooth root materials.The effect of acellular human tooth root material on the phenotypic polarization of mouse peritoneal macrophages was detected by immunofluorescence to evaluate the effect of inflammatory regulation of acellular human tooth root material on bone repair.3 、 The acellular rat tooth roots were implanted into the allogeneic rat tibia,and the binding effect between the material and the recipient bone was observed and evaluated by Micro-CT scanning and HE staining of hard tissue sections 8 weeks after surgery.Acellular human tooth roots were implanted into the mandible of New Zealand white rabbits.24 weeks after surgery,micro-CT scanning and hard tissue grinding HE staining were performed to observe and evaluate the binding effect between the material and the recipient bone.Through the analysis of rabbit peripheral blood cells,the toxic reaction of the material to the blood was detected.The systemic toxicity of the material after long-term implantation was evaluated by observing the visceral toxicity.Results:1、(1)Acellular rat roots: The surface of fresh rat roots(rat-FTR)was rough and covered by periodontal ligaments,while the surface of acellular rat roots(rat-ATR)was smooth and showed little change in size and hardness compared with that of rat-FTR.The surface morphology of rat-FTR and rat-ATR was observed by SEM.A large number of fibrous structures and cementum were found on the surface of rat-FTR,while the surface of rat-ATR is exposed to the cementum,showing a natural porous structure.HE staining of cross sections of rat root before and after decellularization showed that rat-ATR removed the periodontal membrane,pulp and cementum cells of rat-FTR,while the structure of hard root tissue was relatively intact.Masson staining of cross sections of rat root before and after decellularization confirmed further that there was no significant difference in the hard tissue structure of tooth root before and after decellularization.(2)Acellular human root: Fresh human root(h-FTR)was attached with a large number of periodontal membrane fibers.SEM showed that the cable fibers covered the surface of the root and penetrated into the cementum.There was no soft tissue on the surface of the decellularized human tooth root(h-ATR),and cementum was exposed.SEM showed that the fibers on the surface of the tooth root disappeared,and the exposed cementum became porous structure.HE staining on the longitudinal section of human tooth root before and after decellularization showed that cementum cells were contained in the cementum of h-FTR,and periodontal membrane fibers were attached to the outer surface of cementum.The periodontal membrane on the outer surface of cementum of h-ATR completely disappeared,the cellular components in cementum were removed,and the structure of hard tissue of tooth root was relatively intact.(3)After thorough rinsing,SDS residues in rat-ATR and h-ATR were 0.0012% and 0.0014%,respectively.2、(1)Cytotoxicity test: CCK-8 assay showed that rat-ATR extract had no toxicity to MC3T3-E1 cells,suggesting that rat-ATR can support cell growth and proliferation.(2)Hemolysis test: There was cell precipitation at the bottom of the tube of rat-ATR extract,and the upper fluid was similar to that of the negative saline control group.The rat-ATR extract has a HR of 1.25% and can be considered as a non-hemolytic material.(3)Intradermal reaction test: The clinical manifestations of all animals were normal.No erythema or edema was observed at all injection sites 72 hours after injection.The results showed that the rat-ATR extract had no obvious irritation to the skin after intradermal injection,and was a safe and compatible biomaterial.(4)Acute toxicity test: The clinical reaction of all animals was normal during the experiment,and no abnormal toxicity symptoms were found.No weight loss was observed within 3 days after rats were injected with rat-ATR extract,and no death was observed within 7 days after rats were injected.There was no significant difference in body weight between the experimental group and the normal saline control group(P < 0.05).No ascites,organ edema,adhesion and necrosis were found in the abdominal cavity after all animals were killed 7 days after injection.HE staining of liver,spleen and kidney showed no inflammatory reaction in the experimental group.These results suggest that rat-ATR has no systemic toxicity.(5)Macrophage polarization test : Immunofluorescence staining showed that CD86 and CD206 were almost not expressed in M0 group,which could be considered as non-polarized cells.In the M1 group,a large number of cells expressed CD86 and a small number of cells expressed CD206.The positive ratio of CD86 and CD206 was about 0.79 and 0.1 respectively,indicating that macrophages differentiated into M1 phenotype under the action of LPS and IFN-γ.The expression level of CD206 in M2 group was higher than that in M1 group,and the positive ratio of CD86 and CD206 in M2 group was about 0.14 and 0.83 respectively,indicating that macrophages differentiated into M2 phenotype under the action of IL-4.The h-ATR group and the Bio-oss group showed higher expression of CD206.The positive ratio of CD86 and CD206 in the h-ATR group was about 0.18 and 0.7 respectively,and the positive ratio of CD86 and CD206 in the Bio-oss group was about 0.32 and 0.6 respectively.These results indicate that both h-ATR and Bio-oss can stimulate the differentiation of macrophages into M2 phenotype.The positive ratio of CD206 expression in h-ATR group was higher than that in Bio-oss group,but there was no statistical significance(P <0.05).Changes in macrophage phenotype have potential influence on bone repair.3、(1)Acellular rat root implantation experiment:Rat-ATR material was implanted into the tibial defect of the allogeneic rat.8 weeks after implantation,high-density images between rat-ATR and tibia could be seen at most levels of micro-CT.HE staining of hard tissue sections showed no obvious boundary between rat-ATR and tibia,and normal bone trabeculae were connected.The results showed that rat-ATR had good osseointegration with tibial bed.(2)Acellular human root implantation experiment:Blood cell test results of New Zealand white rabbits at 12 and 24 weeks after h-ATR implantation showed that the number of white blood cells and the percentage of neutrophils in the experimental group and the control group were within the normal reference range,indicating no bacterial inflammatory infection after surgery.The animals were sacrificed 24 weeks after implantation.No ascites,organ edema,adhesion and necrosis were observed.HE staining of liver,spleen and kidney showed that there were no abnormal cells in the organs of the experimental group,and the structure was similar to that of the control group.These results indicate that long-term implantation of h-ATR in vivo has no systemic toxicity and has good biocompatibility.At 24 weeks after surgery,Micro-CT showed high density images between the material and the mandible.Hard tissue grinding HE staining showed that there was a boundary between the material near the bone marrow cavity and the bone tissue,and the material at cancellous bone was connected with the bone trabecula,without rejection reaction.Conclusion:The combined decellularization protocol established in this study can effectively remove the cellular components in rat root and human root tissues,and successfully obtain decellularized root derived bone augmentation materials.The material has good biocompatibility,can promote the polarization of macrophages to M2 phenotype,and can form good bone binding with bone.Long-term implantation of the material has no toxic effect on the body.Therefore,we believe that acellular root material can be used as a potential bone augmentation material for clinical application. |
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