Alizarin red s solution

Three days after treatment, cells were washed twice with phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde for 5 min at room temperature, and then washed three more times with PBS. For staining, an ALP substrate solution (Roche Diagnostics, Basel, Switzerland) was added to fixed cells for 30 min at room temperature. Cells were then washed three times with distilled water, and images were scored. ALP activity was quantitatively measured as follows. The cells were washed twice with PBS and lysed with lysis buffer (10 mM Tris-HCl (pH 7.5), 150 mM NaCl, complete protease inhibitor mixture, and 1% Nonidet P-40). The protein concentration was measured with a DC protein assay kit (Bio-Rad, Marnes-la-Coquette, France) according to the manufacturer's instructions. ALP activity was assayed using p-nitrophenyl phosphate as a substrate, and calculated as micromoles of p-nitrophenol/min/mg of protein.
To detect calcium deposits in mineralized tissue, cells were fixed by the same method described above and then stained with Alizarin Red S solution (pH 6.38; Wako Pure Chemical Industries Ltd., Osaka, Japan) for 5 min at room temperature.

Alkaline phosphatase (ALP) activity was measured using a commercially available kit (LabAssay^™ ALP kit, Wako Pure Chemicals, Tokyo, Japan) according to standard procedures. After 3, 7, and 14 days of fluvastatin treatment, the samples were subsequently detached using a cell scraper and sonicated on ice (Branson, MO, USA) (n = 5). Cell debris was removed by centrifugation at 15,000 rpm. ALP activity levels were normalized against total protein using a protein assay reagent (BCA, Pierce Chemical, Rockford, IL, USA). After 14 days of fluvastatin treatment, cells were fixed in 10% neutral buffered formalin. ALP substrate solution (Roche Diagnostics, Basel, Switzerland) was added to fixed cells for staining. Cells were subsequently washed with PBS and images were recorded.
After 21 days of fluvastatin treatment, cells were fixed in 10% neutral buffered formalin and then stained with Alizarin Red S solution (Wako Pure Chemical Industries Ltd., Osaka, Japan) for 5 min at room temperature. Cells were washed with PBS and images were recorded.

For measurement of the osteogenic activity of cells on the scaffolds, alkaline phosphatase (ALP) activity and mineralization assays were performed. Cells were seeded at a density of 1 x 10⁴ and cultured in osteogenic media (α‐MEM with 15% FBS supplemented with 0.2 mM ascorbic acid [Gibco] and 10 mM β‐glycerol phosphate [Gibco]). On days 7 and 14 of culture, ALP activity was measured using an ALP Activity Assay Kit (AnaSpec Inc.,Fremont., CA) and a Protein Assay Kit (iNtRON Biotechnology, Seoul, Korea). ALP activity was normalized as the concentration of p‐nitrophenol (pNP) to the protein amounts. On the day 21, mineralization analysis was performed. Fixation with 95% cold ethanol was done. Cells were stained with a 1% alizarin red S solution (Wako Chemicals, Osaka, Japan) for 5 min.
Cells were fixed in 95% cold ethanol and stained with the images of stained plates were observed. For quantitative measurement, an elution procedure with cetylpyridinium chloride in 10 mM sodium phosphate was performed and measured using an automatic microplate reader at 540 nm wavelength.

DPSCs were subjected to a 14-day culture in an osteogenic differentiation medium. Following this, they were washed with Ca²⁺-free PBS, then fixed in 10% formaldehyde solution in PBS for 10 min at temperature of 4 °C. After rinsing with distilled water, the cells were incubated in Alizarin Red S solution (Wako Pure Chemical Industries, Osaka, Japan) for 15 min at room temperature to detect calcification. The ImageJ software program was used for the quantification of relative staining intensity, with calculations performed using the Prism 9 software program.