Mineral deposition was monitored using alizarin red as a vital stain for calcium.

Mineralized matrix formation was visualized using alizarin red S staining.

Approach and results: Arterial calcium accumulation was evaluated using alizarin red stain and atomic absorption spectrometry.

The results of alizarin red staining and ALP detection showed that SpA treatment suppressed the osteogenic differentiation of hBMSCs.

Cell counting kit-8 activity was examined to test cell proliferation, and alkaline phosphatase assays and alizarin red S staining were conducted to examine differentiation.