*2.6. Alizarin Red S (ARS) Staining Assay*

To evaluate the formation of calcified nodules in GDSCs, we used an ARS assay [25,26]. The powder of each experimental calcium silicate-based cement was mixed with an osteogenic medium at a concentration of 5 mg/mL, and the mixture was placed in a 100% humidity incubator at 37 ◦C for 7 days. The osteogenic medium consisted of complete α-MEM, 50 μg/mL ascorbic acid (Sigma-Aldrich), 0.1 μM dexamethasone (Sigma-Aldrich), and 10 mM beta-glycerophosphate (Sigma-Aldrich). The supernatant fluid was refined through 0.20 μm filters (Minisart; Sartorius Stedim Biotech, Goetingen, Germany). GDSCs were seeded at a density of 2.0 <sup>×</sup> 104 cells/well on 24-well plates (SPL Life Sciences) and cultured for 21 days in calcium silicate-based cement eluate, with changing the eluate every 3 days. Cells were fixed in 4% paraformaldehyde solution and stained with 2% ARS solution (ScienCell, Carlsbad, CA, USA) for 20 min. The stain was treated with 10% cetylpyridinium chloride (Sigma-Aldrich) for 15 min, and the optical density at 560 nm was evaluated using an absorbance microplate reader (Power Wave XS). Each group was evaluated in quadruplicate.
