Fig. 4

Anti-ROS effects and inhibition of abnormal osteogenic differentiation of hMSCs in vitro. (A) Cellular flow cytometry analysis of total ROS after treatment with PBS, TNF-α (10 ng/mL), CH6-MF NPs (10 µg/mL) + TNF-α and CH6-MF NPs/BMP2 siRNA nanocomplexes (CH6-MF-Si NPs, 10 µg/mL) + TNF-α for 48 h. (B) Quantitative analysis of ROS via detection of the mean fluorescence intensity (MFI) of DCF. (C) Western blot analysis of components of the nuclear factor kappa-B pathway on day 2 of PBS, TNF-α, TNF-α + CH6-MF NPs or TNF-α + CH6-MF-Si NPs stimulation. (D) Relative mRNA expression of OPN, RUNX2 and BMP2 was distinguished by qRT-PCR on day 12 of hMSC osteogenic differentiation after being treated with PBS, TNF-α, TNF-α + CH6-MF NPs or TNF-α + CH6-MF-Si NPs. (E) Western blot for OPN, RUNX2 and BMP2. The right panel shows the data quantification. (F) ARS staining of hMSCs treated with PBS, TNF-α, TNF-α + CH6-MF NPs or TNF-α + CH6-MF-Si NPs on day 12. (G) ALP staining of hMSCs treated with PBS, TNF-α, TNF-α + CH6-MF NPs or TNF-α + CH6-MF-Si NPs on day 12. (H) Quantitative analysis of ARS staining on day 12. (I) Quantitative analysis of ALP activity on day 12. Data were analysed using one-way ANOVA. The outcomes are presented as the mean ± SD. ns = statistically nonsignificant, *P < 0.05, **P < 0.01, and ***P < 0.001; n = 3. Scale bar = 100 μm