Rvl 100 m

The procedures of cell seeding, medium replacement, and incubation followed the same steps as the ALP staining. After 14 days of osteogenic induction, the samples were fixed using 4% paraformaldehyde, washed with deionized water, and then stained for 10 min with 2% alizarin red S solution at pH 4.2. The samples were then repeatedly washed with deionized water to remove any residual alizarin red S. Finally, we observed the calcium deposits under a microscope (RVL-100-M, ECHO). Calcium phosphate in the I-EMC samples can interfere with ARS staining. Therefore, we cultured BMSCs in a blank plate and co-cultured them with the different samples. After 14 days of osteogenic induction, the cells at the bottom of the plate were stained with alizarin red. After thoroughly removing unbound dyes with deionized water, we captured images using a microscope (RVL-100-M, ECHO). The Alizarin Red-stained calcium nodules were then eluted from the cultures using the cetylpyridinium chloride method (Xiao et al., 2020 (link))), and their absorbance at 540 nm was assessed for quantification.

After 14 days of osteogenic induction, immunocytochemical staining was performed to detect the expression of osteocalcin (OCN) and runt-related transcription factor 2 (RUNX-2). The samples were fixed with 10% paraformaldehyde at room temperature for 10 min, followed by cell permeabilization with 0.1% Triton X-100 at room temperature for 20 min. To block nonspecific binding, cells were incubated with 10% goat serum at room temperature for 30 min. Subsequently, primary antibodies against OCN (A14636, AB clonal) and RUNX2 (A2851, AB clonal) diluted at 1:180 were added and incubated overnight at 4°C. Secondary antibodies (AS053, AB clonal) diluted at 1:400 were then added and incubated for 1 h at 37°C. Subsequently, phalloidin and DAPI staining were performed as described. Throughout the entire experiment, the samples were rinsed with PBS-Tween between every step. Finally, the images were observed and captured using a fluorescence microscope (RVL-100-M, ECHO), and the fluorescence intensity of each bone-related protein was semi-quantitatively analyzed using ImageJ software.