| Objective: The study used lentivirus-mediated silencing of P75 nerve growth factor receptor(P75NTR)combined with nerve growth factor(NGF)overexpression to transfect rat bone mesenchymal stem cells(BMSCs)composite demineralized bone matrix(DBM)co-culture to construct tissue engineered bone and explore new ways to treat bone defects.Methods : BMSCs from 4-week-old SD rats were isolated and cultured by whole bone marrow adherence method,and BMSCs-specific surface markers were detected by flow cytometry.The rat BMSCs were divided into 4 groups,group A was the untransfected group,group B was the transfection group with silenced P75 NTR,group C was the transfection group with overexpression of NGF,and group D was the co-transfection group of silenced P75 NTR combined with overexpression of NGF.The fluorescent protein expression of the target gene in the double-gene transfection group was observed by fluorescence microscope on the 6th day after transfection.The relative protein expression of P75 NTR and NGF in each group of cells after transfection was examined by Western blot experiment.CCK-8 method was used to detect the changes of proliferation activity of cells in each group from 1 to 6 days after transfection.Scanning electron microscopy was used to observe the adhesion of cells in the silenced P75 NTR combined with NGF overexpression transfection group after co-culture with DBM scaffold material.The above-mentioned cells of each group were co-cultured with DBM scaffold material to make tissue engineered bone.8-week-old SD rats were taken to prepare a rat femoral bone defect model,and the rats were randomly divided into 4 groups.Group A was implanted with untransfected BMSCs-DBM,group B was implanted with silent P75NTRBMSCs-DBM,and group C was implanted with Overexpression of NGFBMSCs-DBM,group D was implanted with silencing of P75 NTR combined with overexpression of NGF-BMSCs-DBM.Femoral defect healing in each group 4 weeks after Micro CT scanning.Three rats were selected from each group at 4 weeks,8 weeks and 12 weeks after the operation,respectively,and the defects were observed by HE staining.Results: The third-generation BMSCs were observed by inverted microscope,which were swirling-like adherent,long-fusiform,triangular,and spindle-shaped growth,closely arranged,and partially protrudes pseudopodia.The cultured cells were extracted as BMSCs.On the 6th day after transfection,the expression of red and green fluorescence was observed by fluorescence microscope,and the transfection efficiency could reach about 75%.Western blot results showed that the relative expression of NGF protein in group C and group D was significantly higher than that in group A and group B,and the relative expression of P75 NTR protein in group B and group D was significantly lower than that in group A and group C,and the difference was statistically significant.Significance(P<0.05).The results of CCK-8 detection showed that the cells in each group grew slowly on the 1st to 2nd day after transfection.From the 3rd day,the growth rate and activity of groups B,C,and D increased,which were higher than those of group A.There was statistical significance(P<0.05),and the increase in group D was the most obvious,which was higher than that in groups B and C(P<0.05).Scanning electron microscope observation showed that the cells adhered well to DBM.Micro CT test results showed that the bone volume/total volume(BV/TV)and trabecular number(Tb.N)in groups B,C,and D were higher than those in group A,and the average trabecular distance(Tb Sp)was lower than that of group A,and the difference was statistically significant(P<0.05).At the same time,it was found that BV/TV and Tb.N in group D were higher than those in groups B and C,while Tb Sp was lower than that in groups B and C,the difference was significant.There was statistical significance(P<0.05).The results of HE examination showed that at 4,8,and12 weeks after operation,the new bone tissue in group D was higher than that in the other three groups,and that in groups B and C was higher than that in group A,and there was no significant difference between the two groups.Conclusions: Silencing P75 NTR combined with NGF overexpression transfection BMSCs composite DBM scaffold material to construct tissue engineered bone has good osteogenic properties,promotes bone regeneration,and has a good repair effect on bone defects. |
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