Measurement of alkaline phosphatase activity

The quantitative alkaline phosphatase activity assay was performed using the SensoLyte pNPP Alkaline Phosphatase Assay Kit (AnaSpec, USA). Briefly, the treated MSCs of each condition on day 3, 7, 14, 21, and 28 were washed with 1 × assay buffer. Subsequently, 200 µl of 0.02% Triton X–100 in 1 × assay buffer was added and the adherent cells were scraped off. The cell lysate was incubated at 4 °C for 10 min with shaking. Then, the cell lysate was centrifuged at 2500×g for 10 min at 4 °C to collect the supernatant for alkaline phosphatase activity assay. After centrifugation, 50 µl of supernatant was added into each well of 96-well plates (Costar, Corning, USA). A serially diluted alkaline phosphatase solution, 0–10 ng/ml in 1 × assay buffer, was used as a standard. Then, 50 µl of p-nitrophenyl phosphate (pNPP) substrate solution was added into each well, mixed by gently shaking the plate for 30 s and incubated at room temperature for 60 min. Subsequently, ALP activity was measured on a microplate reader (BioTex, USA) using absorbance at 405 nm. The ALP activity in each sample was calculated by comparing the measured OD values against the standard curve. Total protein levels were determined by the Bradford assay (Bio-Rad, USA). ALP activity was calculated as the amount of phosphorylated nitrophenol release in ng/μmol/min and was further normalized to the total cellular protein. Each assay condition was assessed in triplicate. MSCs cultured in complete DMEM medium were used as a control.