Effects Of Total Flavonoids Of Drynaria On Proliferation And Osteogenic Differentiation Of Rat Dental Pulp Stem Cells

Objectives:To explore the effects of total flavonoids of drynaria on proliferation and osteogenic differentiation of rat dental pulp stem cells (DPSCs), and to evaluate the potential role of PI3K/Akt pathway in the aforementioned effects.Methods:1. Dental pulp cells were cultured by explants combined with enzyme digestion technique. Single cell line of DPSC was isolated by limited dilution of cultured cells and identified by immunohistochemistry.2. DPSCs were cultured in osteoconductive cultured media with different concentrations of total flavonoids of drynaria (0.01 g/L, 0.05 g/L and 0.1 g/L). Cells in control group were cultured without total flavonoids of drynaria. Cell proliferation of each group was assayed at 3th, 5th, 7th and 9th day by MTT. The ALP activities were tested after 9 days, and forming of calcified nodules of each group was tested after 3 weeks.3. The expression of pAkt in each group was analyzed by Western blotting after 9 days.Results:1. Most of the cultured cells were characterized by a typical fibroblast-like morphology, but a bit of them were irregular. DPSCs by colony formation showed a high proliferation rate and arranged closely. As the extension of cultivating time, the cells expanded as spindle-shape gradually. Immunohistochemical analysis showed that DPSCs expressed CD44 and CD29 while CD34 was absent.2. Compared with the control group, the results of MTT assay showed that the OD490 of DPSCs with the total flavonoids of drynaria with the concentration of 0.01 g/L, 0.05 g/L and 0.1 g/L increased as a function of time (P<0.05). The OD490 of 0.01 g/L group was significantly increased when compared with the 0.1 g/L group (P<0.05), while the OD490 of 0.1 g/L and 0.05 g/L groups were similar (P﹥0.05). The OD490 of all groups at 5th, 7th and 9th day was higher than that of 3th day (P<0.05). The ALP activities of treated groups obviously increased with the concentrations of total flavonoids of drynaria (P<0.05). The volume and number of calcified nodules in treated groups was significantly increased than control group (P<0.05).3. The expression level of pAkt increased with the concentrations of Drynaria total flavonoid (P<0.05).Conclusions: The total flavonoids of drynaria could promote the proliferation and osteogenic differentiation of rat DPSCs and this effect might rely on the PI3K/Akt pathway.