DPSC Culture on Alg/HAp Scaffolds

Alg/HAp scaffolds underwent two cycles of sterilization under an ultraviolet (UV) light (15 W) for 1 h each, and they were rehydrated and conditioned in complete MEM-α overnight as previously reported [13 (link)]. After being expanded up to the sixth passage, DPSCs were trypsinized (trypsin/EDTA 1×, EuroClone, Milan, Italy) and collected by centrifugation (1200 rpm for 10 min at room temperature). DPSCs were then counted, and 5 × 10⁴ cells were resuspended in 130 μL of complete medium and afterward used for seeding drop by drop on each scaffold. Samples were immediately placed at 37 °C and 5% CO₂ for 3 h to allow cell adhesion and interpolation onto/into scaffolds. Next, complete medium or differentiation medium (DM) was added to each sample (named in figures as Alg/HAp and Alg/HAp DM, respectively). Complete DM was supplemented as reported previously [13 (link)], with 100 μM ascorbic acid, 10 nM dexamethasone, 5 mM β-glycerol phosphate disodium salt pentahydrate (all purchased from Sigma Aldrich, MI, USA), and 1.8 mM potassium phosphate (Alfa Aesar Chemicals, Haverhill, MA, USA). DPSCs onto Alg/HAp scaffolds with or without DM were incubated for 1, 3, 7, 14, 21, and 28 days, and medium was refreshed every three days.