Fig. 3

Osteogenic effect of C₂S NP (0, 10, 50, 100 μg/mL) nanoparticles in vitro. a The cell cycle was detected by flow cytometry. BMSCs were incubated with 50 and 100 μg/mL C₂S NPs for 24 h. b Cell proliferation was evaluated using CCK-8 assays at different times points (2, 3, and 7 days). ALP activity (c) and the formation of mineralized nodules (d) were detected by BCIP/NBT and Alizarin red S staining accompanied by increasing concentrations of C₂S NPs at different time points (7, 14, 21 days). e The total mRNA levels of COLI, OSX (Osterix), RUNX2, and BMP2 in BMSCs treated with C₂S NPs (0, 10, 50, 100 μg/mL) at different time points (3, 7, 14, 21 days) was detected by RT-PCR. f The total protein levels of β-catenin, RUNX2, BMP2, OPN and AXIN1 were detected by western blotting, and the cells were treated as in (e). Densitometry data were normalized to GAPDH. The relative optical density was analyzed using ImageJ software (below). Values are expressed as the mean ± SEM. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001. Compared with the control group