Objective To explore the effect of miR-378a overexpression in modifying bone marrow mesenchymal stem cells (BMMSCs) combined with collagen sponge (CS) scaffold in repairing femoral defects of rats. Methods The BMMSCs were isolated and cultured, and the cell phenotype was identified by flow cytometry. The BMMSCs were transfected with an overexpressed miR-378a lentivirus and a negative unloaded lentivirus, and the transfected cells were divided into three groups: a BMMSCs transfected with overexpressed miR-378a group (LV-miR-378a group), BMMSCs transfected with negative control lentivirus group (LV-miR-NC group) and an untransfected BMMSCs group (control group). The transfection efficiency was detected by flow cytometry. The composite of transfected cells with CS scaffold was prepared, and the compatibility of the cells with the scaffold was observed by scanning electron microscopy. A femoral defect replantation model was established in SD rats, and 15 SD rats were randomly divided into three groups (n=5): LV-miR-378a+CS group (with the lentivirus complex CS scaffold overexpressing miR-378a implanted), LV-miR-NC+CS group (with the BMMSCs complex CS scaffold transfected with negative non-load lentivirus),and CS group (with the CS scaffold implanted only). At the 8th week after operation, the SD rats were sacrificed by CO₂ inhalation.The femur on the operation side was taken for gross observation and micro-CT scanning reconstruction. Bone mineral density(BMD) and bone volume/total volume (BV/TV) were quantified, and HE, Masson and osteopontin (OPN) immunohistochemical staining were used to observe bone repair. Results The phenotyping results of BMMSCs by flow cytometry showed that the positive expression rate of cell surface antigen CD44 and CD29 were 95.5% and 94.7%, respectively, while the positive expression rate of CD45 and CD34 were 0.8% and 0.7%, respectively. Transfection efficiency detected by flow cytometry: compared with the control group, the transfection efficiency of LV-miR-378a group and LV-miR-NC group increased obviously (P<0.05). The lentivirus transfection efficiency of LV-miR-378a group was consistent with that of LV-miR-NC group with no statistically significant difference (P>0.05). The results of scanning electron microscopy showed that the CS scaffold material had a good three-dimensional cavity structure. After the two groups of cells were co-cultured with the CS scaffold for 7 days, the cells in LV-miR-378a group had a larger spreading area on the surface of scaffold material than those in LV-miR-NC group, and the cells grow in clusters with more pseudopod. The gross observation results showed that the bone defect area in LV-miR-378a+CS group was completely repaired 8 weeks after surgery. The incompletely mineralized new bone could be observed in the bone defect centers of both LV-miR-NC+CS group and CS group, and the rough outline of the defect area could still be seen in CS group. The results of micro-CT three-dimensional reconstruction and quantitative analysis showed that 8 weeks after surgery, the repair effect of the bone defect area in LV-miR-378a+CS group was better than in other two groups, and there was more new bone deposition. The BMD and BV/TV values were significantly higher than those in other two groups (P<0.05). The defective areas in both LV-miR-NC+CS group and CS group were not completely repaired, and less new bone deposition and uneven density in CS group. HE and Masson staining results showed that the stent material in LV-miR-378a+CS group degraded completely 8 weeks after surgery, and more mature trabeculae could be seen, the junction area of new bone and host bone showed good continuity, and the trabeculae were connected with each other and had regular morphology. In LV-miR-NC+CS group, mature trabeculae were relatively few, and the junction area of new bone and host bone was not completely connected. In CS group, the stent materials were not completely degraded, and there were still more trabeculae in remodeling and irregular shape. Immunohistochemical staining showed that the expression rate of OPN in LV-miR-378a+CS group was significantly lower than that in other two groups 8 weeks after surgery, (P<0.05), and the expression rate of OPN in LV-miR-NC+CS group was lower than that in CS group (P<0.05). Conclusion Over-expression of miR-378a modified BMMSCs combined with CS scaffold can promote new bone formation.