Fig. 6

NBP treatment promoted hBMSCs osteogenic differentiation through the activation of PI3K/AKT pathways. A, B The relative mRNA expression of hmTOR, PIK3CA, PIK3R1 and PIK3R2 is upregulated in hBMSCs quantified via qRT-PCR. Total RNA was extracted from hBMSCs at two-different points: combination treatment of NBP at 4 and 7min treatment time and mineral supplement (0.08 g/ml of ascorbic acid, 0.30611 g/ml of β-glycerol phosphate, 0.051 M dexamethasone) and without mineral supplements following NBP at 4 and 7min treatment time. GAPDH was used as the internal control. C, D Western blot analysis was performed to detect PI3K, p-AKT, and PTEN protein expression with the administration of NBP treatment at 4 and 7min on day 2. D The quantification of band intensities is represented in the graphs. GAPDH was used for the normalization. E, F Representative images of Ca deposition in hBMSCs via Alizarin Red Staining at day 2 under the light microscope. Scale bars, 60 µm. F Quantified areas of ARS staining measured by ImageJ software. The mean and standard deviation values are used to represent the experimental results. The statistical significance of the data points was assessed using the student’s t-test, and significant differences were indicated by *p < 0.05; **p < 0.01, and ***p < 0.001 vs the untreated control