0.5 m hcl solution

The mineralization capabilities of cultures were evaluated on day 14 by colorimetry-based assays. The cultures were washed with ddH₂O and incubated overnight in 1 mL of 0.5 M HCl solution (Sigma-Aldrich) with gentle shaking. The solution was mixed with o-cresolphthalein complexone in an alkaline medium (Stanbio LiquidColor, Stanbio, Boerne, TX, USA) to produce a purple calcium–cresolphthalein complexone complex. Color intensity was measured by a multi-detection microplate reader (Synergy^TM HT, BioTek Instruments, Inc.) at 550 nm wavelength. The mineralization capability of each titanium disk was also confirmed by visualizing mineralized nodule areas via Alizarin red staining. On day 14 of culture, the specimens were washed twice with 1× PBS at 37 °C and stained for 5 min using 1% Alizarin red (pH 6.3–6.4). The titanium disks were then rinsed twice with ddH₂O and air-dried.

After 21 days of osteogenic differentiation, samples were incubated with a 0.5 M HCl solution (Sigma-Aldrich) by shaking overnight at 4 °C. Total calcium content was determined using a calcium colorimetric assay kit (Sigma-Aldrich), according to manufacturer’s instructions. Calcium standard solutions were prepared in parallel. Absorbance at 575 nm was measured in triplicate for each condition and normalized to the metabolic activity. Three samples were used for each condition.