Alp kit

Osteoblastogenesis of COBs and BMSCs were initiated when cells reached 75–85% confluency using osteogenic induction media [(consisted of complete αMEM, 50μg/mL ascorbic acid, and 8mM beta-glycerophosphate (Sigma, St. Louis, MO)]. Medium was changed every two days. To assess osteoblasts and their mineralization, alkaline phosphatase (ALP) and Alizarin Red staining (ARS) were performed on d7 and d14 after differentiation, respectively. ALP kit was obtained from Sigma (St. Louis, MO) and staining was performed according to the manufacturer’s instructions after cell fixation with 10% neutral buffered formalin. For ARS, one percent Alizarin Red solution diluted in dH2O (Sigma; pH 4.2) was used to stain the cells for 30 minutes at room temperature. Cells were then washed a couple of times with water before being visualized under the microscope (Leica DM IRB, TV camera) and taking pictures (ZEISS Efficient Navigation, blue edition).

For alkaline phosphatase (ALP) staining, after induction, cells were fixed with 4% paraformaldehyde and incubated with a solution of 0.25% naphthol AS-BI phosphate and 0.75% Fast Blue BB (Sigma-Aldrich, St Louis, MO, USA) dissolved in 0.1 M Tris buffer (pH 9.3). ALP activity assay was performed using an ALP kit according to the manufacturer's protocol (Sigma-Aldrich, St Louis, MO, USA) and normalized based on protein concentrations. To detect mineralization potential, cells were induced for 2–3 weeks, fixed with 4% paraformaldehyde and stained with 2% Alizarin Red (Sigma-Aldrich, St Louis, MO, USA). To quantify the calcium mineral deposition, Alizarin Red was destained with 10% cetylpyridinium chloride in 10 mM sodium phosphate for 30 min at room temperature.