Promoting effect of osteopractic total flavone combined with nano-bone materials on proliferationand differentiation of MC3T3-E1 cells / 中国组织工程研究

ABSTRACT

BACKGROUND: Preliminary study has found that osteopractic total flavone can promote osteogenic differentiation of MC3T3-E1 cells on the surface of nano-bone material, but the underlying mechanism needs to be studied in depth. OBJECTIVE: To investigate the actin mechanism of osteopractic total flavone combined with nano-bone material on MC3T3-E1 cells. METHODS: MC3T3-E1 cells were co-cultured with nano-bone material, and 100 mg/L and 250 mg/L osteopractic total flavone were treated as drug intervention, including 10 μg/L transforming growth factor-β as positive control. The samples were divided into eight groups: (1) Normal group; (2) DKK1 group: Wnt pathway inhibitor DKK1 (0.1 mg/L) blocks Wnt/β-catenin signaling pathway; (3) DKK1+transforming growth factor-β group; (4) DKK1+100 mg/L osteopractic total flavone group; (5) DKK1+250 mg/L osteopractic total flavone group; (6) DKK1+ nano-hydroxyapatite/collagen+transforming growth factor-β group; (7) DKK1+nano-hydroxyapatite/collagen+100 mg/L osteopractic total flavone group; (8) DKK1+nano-hydroxyapatite/collagen+250 mg/L osteopractic total flavone group. Cells in each group were harvested after 24 and 48 hours of intervention. Immunofluorescence labeling was used to observe the binding of Wnt and LRP in osteoblasts in the Wnt/β-catenin pathway. The expression of β-catenin, LRP 5, GSK-3β, Cyclin D1, and RUNX2 was detected by real-time polymerase chain reaction and western blot assay. RESULTS AND CONCLUSION: (1) Confocal laser scanning microscope showed that obvious brown and yellow staining was shown in the DKK1+transforming growth factor-β group, DKK1+250 mg/L osteopractic total flavone group, DKK1+nano-hydroxyapatite/ collagen+transforming growth factor-β group, and DKK1+nano-hydroxyapatite/collagen+250 mg/L osteopractic total flavone group, indicating that Wnt and LRP combined better than other groups. (2) Real-time polymerase chain reaction and western blot assay results showed that osteopractic total flavone could promote the expression of β-catenin, LRP5 and RUNX2, and downregulated GSK3β expression. These findings confirm that osteopractic total flavone can promote the differentiation and proliferation of osteoblasts by activating the Wnt/β-catenin signaling pathway. Gene activation induced by osteopractic total flavone was dose-dependent.