Abstract
OBJECTIVE: To investigate the role of microRNA miR-488-5p, which showed increased expression after the disconnection of the inferior alveolar nerve, in promoting the osteogenic and neurogenic differentiation of rat bone marrow mesenchymal stem cells (rBMSCs), as well as its effect on promoting the neuralized tissue engineered bone regeneration. METHODS: rBMSCs were subjected to in vitro neural or osteogenic differentiation induction cultures. The expression levels of miR-488-5p at different time points (days 0, 2, 4, and 7) were detected by quantitative real-time polymerase chain reaction (qRT-PCR). miR-488-5p overexpression or low expression in rBMSCs was achieved by transfection with miR-488-5p mimics or inhibitors. Four groups, the miR-488-5p mimics, the miR-488-5p inhibitor, and their respective negative controls (NC), were established to investigate the effects of miR-488-5p on the neural differentiation and osteogenic differentiation of rBMSCs.A 5 mm diameter, full-thickness circular critical bone defect was created in the rat calvaria. The rats were treated with light-cured gelatin methacryloyl (GelMA) seeded with rBMSCs. The rats were divided into four groups: ①BLANK group: GelMA; ②BMSCs group: GelMA + rBMSCs; ③NC-BMSCs group: GelMA + rBMSCs transfected with miR-488-5p mimics NC; and ④miR-488-5p-BMSCs group: GelMA + rBMSCs transfected with miR-488-5p mimics. Specimens were obtained 4 and 8 weeks after surgery, and micro-CT was performed to measure and analyze bone mineral density (BMD), bone volume/total volume (BV/TV), bone surface area/total volume (BS/TV) and trabecular number (Tb.N). The effects of neuralized tissue engineering bone formation in the defect area were assessed using Hematoxylin-Eosin (HE) staining, Masson staining, and tissue immunofluorescence staining of the nerve-specific protein soluble protein-100 (S100). RESULTS: As rBMSCs progressed toward neural or osteogenic differentiation, miR-488-5p expression increased significantly from day 0 to day 7. Regarding neural differentiation, the mimics group showed increased expression of neural-related genes and proteins compared with the mimics NC group, while the opposite result was observed in the inhibitor group. As for osteogenic differentiation, the mimics group showed increased expression of osteogenic genes and proteins, more intense alkaline phosphatase (ALP) and alizarin red staining (ARS) staining, and enhanced ALP activity compared with the mimics NC group, while the opposite result was observed in the inhibitor group. 4 and 8 weeks after critical calvarial defect construction in rats, the BLANK group had the least amount of new bone formation, while the BMSCs group and the NC-BMSCs group had similar and intermediate amounts of new bone formation. The miR-488-5p-BMSCs group had the most new bone formation. At 4 weeks, the BMD [(0.63±0.05) g/cm(3) vs. (0.51±0.03) g/cm(3)], BV/TV (33.17%±6.43% vs. 18.11%±1.52%), BS/TV [(3.43±0.69) /mm vs. (2.46±0.20) /mm], and Tb.N [(0.92±0.21) /mm vs.(0.59±0.07) /mm] in the miR-488-5p-BMSCs group were significantly higher than those in the NC-BMSCs group. At 8 weeks, the BMD [(0.80±0.04) g/cm(3) vs. (0.68±0.04) g/cm(3)], BV/TV (56.69%±6.22% vs. 42.36%±3.86%), and the number of S100-labeled nerve cells around the new bone (46.33±4.04 vs. 26.00±3.61) in the miR-488-5p-BMSCs group was also significantly higher than that in the NC-BMSCs group. CONCLUSION: miR-488-5p promoted the osteogenic and neurogenic differentiation of rBMSCs and promoted the formation of neuralized tissue-engineered bone in rat calvarial defects.