A2356

Osteoblast precursor cells were isolated and then inoculated into a cell culture dish at a density of 3 × 10³ cells/cm². Upon reaching confluence, the cells were added with osteoblast induction medium: BGJB medium (Thermo Fisher Scientific Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS), 50 μg/mL l-ascorbic acid (A4544, Sigma-Aldrich Chemical Company, St Louis, MO, USA) and 5 μg β-glycerolphosphate (G9891, Sigma-Aldrich Chemical Company, St Louis, MO, USA). Then, the 5-bromo-2′-deoxyuridine (BrdU) labeling method was used to measure osteoblast proliferation. Next, the cells were colored using 3,3′-diaminobenzidine (DAB) and observed under a microscope, with positive cells presenting with brownish yellow color. At the 14th day, alkaline phosphatase (ALP) staining (A2356, Sigma-Aldrich Chemical Company, St Louis, MO, USA) was adopted to detect cell differentiation. At the 21st day, von Kossa staining (Thermo Fisher Scientific Inc., Waltham, MA, USA) was applied to test the mineralization of osteoblasts.