Axio 40

DPCs were seeded into 12-well plates and cultured in OS medium for 7 days. After fixation in 4% paraformaldehyde for 30 min, DPCs were stained with 1% alizarin red staining solution (ARS; Cyagen, Suzhou, China) for 5 min at room temperature and were then rinsed with distilled water. The matrix calcium deposition was scanned using an inverted phase-contrast microscope (Axio 40; Zeiss, Jena, Germany). To quantify the mineralized nodules, the stained cells were incubated in 0.1M hexadecylpyridinium chloride monohydrate (Sigma-Aldrich, St. Louis, MO, USA) for 30 min. The absorbance of the supernatant was then measured at a wavelength of 562 nm.

To evaluate the mineralization level, cells cultured in osteogenic medium were induced for 7 days. After washing with PBS, the cells were fixed in 4% paraformaldehyde for 20 min and then stained with alkaline phosphatase (ALP) (Yeasen, Shanghai, China) or 1% alizarin red S (GL Biochem, Shanghai, China) solution for 10 min. The ALP activity and matrix calcium deposition were photographed and analyzed under an inverted phase contrast microscope (Axio 40; Zeiss, Jena, Germany). The cells stained by alizarin red were incubated with 10% cetylpyridinium chloride (Sigma, St. Louis, MO, USA) for an hour. The absorbance was then measured at 562 nm.