2.8. Alizarin Red Staining (ARS)

Alizarin red S colored calcium deposits selectively and it has been used for decades to evaluate calcium-rich deposits in cell culture [32]. Well plates and 3D PCL scaffold specimens were seeded with swine DPSCs, fixed with 4% neutral formalin (Sigma-Aldrich, USA) for 5 min and then washed three times with PBS. The fixed cells over scaffolds were further washed with distillated water in order to remove any salt residues. A solution of 2% wt/v Alizarin Red S (Sigma Aldrich, USA), with adjusted a pH 4.2 was added, so that it covered the entire surface of the scaffolds. After 10 min of incubation at room temperature, the ARS excess was washed with distillated water. The ARS staining was imaged using a Leica DM IL LED inverted light-field phase contrast optical microscope.