Quantitative Analysis of Early and Late Mineralization

The early mineralization was determined based on the instructions of BCIP/NBT alkaline phosphatase chromogenic kit (Beyotime, Shanghai, China). Cells were fixed in 4% paraformaldehyde (Beyotime) for 10 min, washed three times with PBS, and then incubated with BCIP/NBT staining solution for 30 min at 37 °C in the dark. After that, the treated cells were washed twice with distilled water and photographed. Then, ALP concentration was quantitatively analyzed. Late mineralization was measured according to the instructions of the Osteoblast Mineralized Nodule Staining Kit (Beyotime). The cells were fixed with 4% paraformaldehyde for 10 min, washed with PBS for another three times, and stained with alizarin red for 10 min. Subsequently, the treated cells were washed with distilled water to be observed and photographed microscopically, and the calcium nodules level was then quantitatively analyzed.