Osteogenic Differentiation Assays

Cells were seeded into 24-well plates at a concentration of 5 × 10⁴ cells/well. After 24 h, the media were replaced with 25% of the different extracted osteogenic mediums and cultured for 14 days. Alkaline phosphatase staining (ALP) was performed at day 10, and Von Kossa and alizarin red s staining were performed at day 14 [11 (link), 14 (link)]. For ALP staining, the cells were fixed and stained with TRACP & ALP double-stain kit (Takara Bio USA Inc., CA, USA). For alizarin red s staining, the cells were fixed with cold methanol and washed with deionized water. Alizarin red s solution (1% w/v) was incubated with the samples for 3 min, removed, and washed with DI water 3 times. The staining was solubilized with 10% cetylpyridinium chloride monohydrate (Sigma-Aldrich, MO, USA) solution and the absorbance was measured at 570 nm using a spectrophotometer. For Von Kossa staining, the cells were fixed with 4% paraformaldehyde (Sigma-Aldrich, MO, USA) in PBS for 10 min. After rinsing with deionized water, 5% silver nitrate (Sigma-Aldrich, MO, USA) was added and the samples were exposed to UV light for 60 min. The cells were washed and rinsed with 5% sodium thiosulfate (JT Baker) 3 times before counterstaining with methyl-green (Takara Bio USA Inc., CA, USA).