Cpc solution

To evaluate mineralization qualitatively, Alizarin red staining was performed. 5 × 10⁴/well HOBs were seeded and treated with 1d and 1h. After 14 days of culture, HOBs were rinsed three times with PBS and fixed with a glutaraldehyde solution (2.5%) for 2 h. An Alizarin staining solution (Sigma-Aldrich) was then added for 1h at room temperature and the excess dye was removed using deionized water. The presence of calcium deposition was qualitatively evaluated by observing the intensity of the red color. Images were taken by a stereomicroscope connected with a camera at a magnification of 12× (Leica, Wetzlar, Germany). Furthermore, to quantify calcium deposition, Cetylpyridinium Chloride (CPC) was used. Upon a washing with deionized water, cells were treated with 1 mL of 10% CPC solution (Sigma-Aldrich) for 1 h to chelate calcium ions. After incubation, the absorbance was read at 540 nm by a microplate reader (Synergy H1 Hybrid BioTek Instruments) and normalized with the cell number.

To quantify calcium deposition, cetylpyridinium chloride (CPC) was added to the bottom of the plate after the qualitative observation upon ARS, as reported in the previous paragraph. Well plates were washed with deionized water and were treated with 1 mL of 10% CPC solution (Sigma-Aldrich) for 1 h to chelate calcium ions. After incubation, the absorbance was read at 540 nm in a microplate reader (Synergy H1 Hybrid BioTek Instruments) and normalized with cell number.

The quantification of calcium deposition was measured by the addition of the cetylpyridinium chloride (CPC) to the cover slips after the qualitative measurement by ARS, as described in the previous paragraph. A total of 1 mL of 10% CPC solution (Sigma-Aldrich) was added to chelate calcium ions. After 1 h, the absorbance was taken over at 540 nm in a microplate reader (OD540) (Synergy H1 Hybrid BioTek Instruments) and normalized with cell number.