Experimental Study On The Repair Of Femoral Defect In Rats By Over-expression Of MiR-378a Modified RBMMSCs Combined With Collagen Sponge Scaffold

Objective:The purpose of this study is to observe the growth,adhesion and proliferation of rat bone marrow mesenchymal stem cells（r BMMSCs）modified by over-expression of mi R-378a on collagen sponge（CS）scaffold,and the effect of over-expression of mi R-378a cell scaffold complex in repairing femoral defects in SD rats.To explore the feasibility of over-expression of mi R-378a as a gene therapy for bone tissue regeneration.Methods:r BMMSCs were isolated and cultured,and the cell phenotype was identified by flow cytometry,and the osteogenic and adipogenic differentiation ability of r BMMSCs was identified.r BMMSCs were transfected with lentivirus over-expressing mi R-378a and negative lentivirus,and the transfected cells were divided into three groups:over-expressing mi R-378a transfected r BMMSCs group（LV-mi R-378a group）,negative control lentivirus transfected r BMMSCs group（LV-mi R-NC group）and non-transfected r BMMSCs group（Blank control group）.The transfection efficiency of lentivirus was detected by flow cytometry.The effect of over-expression of mi R-378a on the migration ability of r BMMSCs was observed by scratch test.The complex of transfected cells and CS scaffold was prepared,and the adhesion and proliferation of cells in each group on CS scaffold were detectedby cell adhesion rate detection and CCK-8 method.Scanning electron microscope and fluorescence microscope were used to observe the compatibility of cells in each group with CS scaffold.The model of femoral defect replantation in SD rats was established.Forty-Five male SD rats were randomly divided into three groups（n=15）:LV-mi R-378a/CS group（r BMMSCs composite CS scaffold transfected with lentivirus over-expressing mi R-378a）,LV-mi R-NC/CS group（r BMMSCs composite CS scaffold transfected with negative no-load lentivirus）and CS group（CS only）.At the 4th week,6th week and 8th week after operation,SD rats were killed by CO₂inhalation.Femoral samples from the operation side were taken for gross observation,X-ray observation and analysis,Micro-CT,quantitative analysis of bone mineral density（BMD）and bone volume/total volume（BV/TV）,and HE,Masson staining and OPN were used.Results:（1）The phenotype of r BMMSCs was detected by flow cytometry:the positive expression of cell surface antigen CD44 was 95.5%,the positive expression of CD29 was 94.7%,the negative expression of CD45 was 0.8%,and the negative expression of CD34 was 0.7%.（2）Identification results of cell differentiation ability:After 14th days of osteogenesis induction,several red-stained calcium salt nodules were observed;After the 10th day of adipogenesis induction,many fat drops with different sizes appeared in the form of grape beads.（3）Flow detection of lentivirus transfection efficiency Results:Compared with LV-mi R-NC group,LV-mi R-378a group had the same transfection efficiency,and the transfection efficiency reached more than 85%,with no significant difference between the two groups（P>0.05）.（4）Observation results of scratch experiment:The scratch repair rate of LV-mi R-378a group was obviously better than that of the other two groups at 24 hours and 48 hours（P<0.0001）,but there was no difference between LV-mi R-NC group and blank control group at 24 hours（P>0.05）,but the scratch repair rate of LV-mi R-NC group was slightly higher than that of blank control group after 48 hours of scratch（P<0.05）.（5）Test results of cell adhesion rate:The cells of each group were co-cultured with CS scaffold for 24 hours,and the average cell adhesion rate of the three groups was all over85%,with no statistical difference among the three groups（P>0.05）.（6）CCK-8 test results:On the 3rd day of co-culture with CS scaffold,the cell proliferative activities of LV-mi R-378a group were significantly higher than those of LV-mi R-NC group and blank control group（P<0.05）,while there was no significant difference between LV-mi R-NC group and blank control group（P>0.05）.（7）SEM observation results:The CS scaffold material has a good three-dimensional cavity structure.After the three groups of cells were co-cultured with CS scaffold for 7 days,the spreading area of cells on the surface of the scaffold material in LV-mi R-378a group was larger than that in the other two groups,and the cells grew in clusters,with more pseudopods protruding,while the extension an adhesion of cells on the CS scaffold material in LV-mi R-NC group were less different from those in the blank control group.Results of fluorescence microscope observation:LV-mi R-378a group expressed more fluorescence on CS scaffold and covered more cells than LV-mi R-NC group.（8）Gross observation results:At the 4th week,6th week and 8th week after operation,the amount of new bone deposits in the bone defect area of each group increased gradually and the scaffold gradually degraded.However,in the LV-mi R-378a/CS group,the bone defect area was repaired quickly and to a good extent,while the incomplete mineralized new bone was still observed in the bone defect center of the LV-mi R-NC/CS group and CS group until the 8th week.（9）X-ray observation and analysis,Micro-CT and quantitative analysis all showed that the effect of repairing bone defect area in LV-mi R-378a/CS group was better than that in the other two groups,and the X-ray score,BMD and BV/TV values were significantly higher than those in the other two groups（P<0.05）.（10）HE and Masson staining results:At the 4th and 6th week after operation,the remodeling speed of bone tissue in LV-mi R-378a/CS group was faster than that in the other two groups.By the 8th week,compared with LV-mi R-NC/CS group,the scaffold material in LV-mi R-378a/CS group was completely degraded,and more mature trabeculae were produced in the bone defect area,and new bone and host bone were formed.（11）Immunohistochemical staining results:At the 4th and 6th week after operation,the positive expressions of OPN and Osx in LV-mi R-378a/CS group were higher than those in the other two groups,but there was little difference between LV-mi R-NC/CS group and CS group.Eight weeks after operation,the positive expression of OPN and Osx in LV-mi R-378a/CS group was significantly lower than that in the other two groups,while the positive expression of OPN and Osx in CS group was the strongest among the three groups.Conclusion:Over-expression of mi R-378a can enhance the growth,adhesion and proliferation of r BMMSCs on CS scaffolds,and over-expression of mi R-378a cell scaffold complex can accelerate the formation of new bones in vivo.