Quantification of Osteogenic Mineralization

Following the 15-day culture period, the cells underwent PBS (phosphate-buffered saline) washing and fixation with 10% formaldehyde at room temperature for 15 min. Subsequently, the cells were rinsed thrice with ddH₂O and stained with a 40 mM alizarin red solution (Sigma-Aldrich, Merck Group) at room temperature for 30 min. After staining, the cells were washed four times with ddH₂O. For quantification, the stained mineralized nodules were subjected to a 30 min incubation with 10% cetylpyridinium chloride (Sigma-Aldrich). The resulting solutions were collected, and absorbance at 562 nm was measured using a Tecan Infinite M200 microplate reader. Sample quantification was performed based on the alizarin red standard curve. The inclusion of alizarin red staining, a widely accepted method for assessing calcium deposition, complemented the quantitative measures by providing a qualitative and visual evaluation of mineralization [68 (link)].