Osteogenic Differentiation Characterization

Cells osteogenically induced for 14 days were washed with precooled PBS for three times before being stained with ARS to examine the matrix concentrations of calcium according to a previously described procedure [3 (link)]. The ALP Quantitative Analysis Kit (Beyotime, Shanghai, China) was used to analyze the ALP activity in cells according to the manufacturer's instructions. Definition of the ALP activity unit is as follows: hydrolysis of para-nitrophenyl (unit: μmol) per minute at 37°C in a diacetamine (DEA) buffer pH 9.8. The absorbance was measured at 405 nm. ALP staining was performed using the Gomori Staining Kit (Nanjing Jiancheng Institute, China). Finally, the samples were washed with deionized water and visualized by phase microscopy using an inverted microscope. ARS reverse quantification was by stain extraction, and the absorbance was measured at 405 nm.