| Part Ⅰ: To establish the BRONJ model on osteoporosis SD ratsObjective: To establish a BRONJ animal model of osteoporosis SD rats after tooth extraction,which lays a foundation for further exploration of preventive measures.Method: Twelve Sprague-Dawley(SD)rats underwent bilateral ovariectomy 3 months after subcutaneous injection of alendronate 1.0 mg/kg/d.After 60 days of continuous administration,the left lower jaw of the rat was removed.Second molar.The incidence of BRONJ in rats was detected by gross observation,Micro CT examination and histopathology.Results: After 8 weeks of tooth extraction,4 rats in the drug-administered group showed unhealed extraction,gingival redness and bone surface exposure.Imaging and HE staining showed osteonecrosis,with an incidence rate of 66.7%.In the control group,the extraction sockets of all the rats were completely healed,no obvious redness of the gums and bone surface exposure.Imaging and HE staining showed that the extraction sockets healed well.Conclusion: Osteoporosis SD rats can establish BRONJ animal model by subcutaneous injection of alendronate after tooth extraction.The method is stable,reliable and can be used to explore the mechanism and the preventive measures of BRONJ.Part II: PDGF-BB and BMP-2 prevent the occurrence of BRONJ in osteoporosis SD ratsObjective: To explore the preventive effect of PDGF-BB/BMP-2 gelatin sponge on BRONJ in osteoporosis SD rats and to explore the prevention method of BRONJ.Method: Twenty-four SD rats were divided into the first part to establish an osteoporosis animal model and subcutaneously injected with ALN.After the end of the administration,the rats were randomly divided into 4 groups,and the first and second molars of the left mandible were removed.Group 1: The tooth is filled with a gelatin sponge and the suture is reduced.Group 2: The extraction of the gelatin sponge loaded with PDGF-BB was performed after the extraction was performed.Group 3: The teeth were filled with a gelatin sponge loaded with BMP-2 and then sutured.Group 4: The teeth were filled with a gelatin sponge loaded with PDGF-BB+BMP-2 and then sutured.The incidence of BRONJ in rats was detected by gross observation,Micro CT examination and histopathology.The mandibular vascular density was measured by immunohistochemistry and immunofluorescence in rat mandibular mucosa.The levels of VEGF and CTX Ⅰ in serum of rats were detected by ELISA.Results: After 8 weeks of tooth extraction,the incidence of BRONJ in the prevention group was significantly lower than that in the control group.Micro CT scan showed that the extraction group was well healed compared with the control group,and the bone volume of the extraction was significant.Increase.Immunohistochemistry and fluorescence detection showed that the periosteal neovascularization in the PDGF-BB prevention group was significantly higher than that in the control group.There were no significant difference in CTX I and VEGF between the treatment groups and the control group.Conclusion: The subcutaneous injection of ALN osteoporosis SD rats with the extraction of PDGF-BB and BMP-2 absorbable gelatin sponge can significantly reduce the incidence of BRONJ,and can increase the new bone synthesis and increase bone mass.PDGF-BB is best for preventing BRONJ prevention after tooth extraction,which may be related to PDGF-BB promoting local neovascularization.Part Ⅲ: Experiments to explore the mechanism of prevention BRONJ by PDGF-BB in vitroObjective: To study the effects of PDGF-BB on the function of osteoblasts and vascular endothelial cells after ALN pretreatment in vitro.In order to explore the mechanism of PDGF-BB prevention of BRONJ.Method: Osteoblasts and vascular endothelial cells were cultured and pretreated for 48 h in medium containing different concentrations of ALN.The culture medium was changed and incubated for 48 h in PDGF-BB containing medium.The osteogenesis was detected by CCK-8 method.Cell and vascular endothelial cell proliferation activity,vascular endothelial cell migration ability was detected by scratch test and Transwell chamber test.In vitro angiogenesis assay of matrigel was used to detect the angiogenic ability of vascular endothelial cells and co-cultured cells(treated osteoblasts and normal vascular endothelial cells)in vitro,and analyzed by Image J software.The concentrations of VEGF in the final culture medium of osteoblasts were detected by ELISA.The levels of FGF-2 and VEGFA protein expression in vascular endothelial cells were detected by WB assay.Results: PDGF-BB could promote the proliferation,migration,in vitro angiogenesis,VEGFA and FGF-2 protein expression of ALN pretreated vascular endothelial cells,and also increase osteoblast proliferation,angiogenic ability and exocrine VEGF protein levels.Conclusion: PDGF-BB can reverse the inhibitory effect of ALN on proliferation,migration,angiogenesis,osteoblast proliferation and angiogenesis of vascular endothelial cells.This process may be related to the expression of VEGFA,FGF-2 protein and extracellular VEGF protein by osteoblasts in PDNF-BB-promoted ALN-pretreated vascular endothelial cells. |
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