Mineralization Assay (Alizarin Red Stain):

Calcium depositions in the extracellular matrix were observed using Alizarin Red staining after DPSCs were cultured for 14 days in odontogenic media, and for MDPCs after 1 week. The cells were then washed with PBS and fixed in 70% ethanol for 10 minutes at 4°C. DPSCs were stained with a solution of 1% alizarin red S (Sigma-Aldrich, St. Louis, MO, USA) for 5 mins at room temperature and washed with distilled water. The calcium content was also determined at day 14 using a colorimetric calcium assay kit (Calcium Reagent Set, Pointe Scientific, MI, USA), after disruption and incubation of samples overnight at 4°C with 1 N HCl.