Effects of EDTA and saline as the final irrigation in regenerative endodontic procedures on the migration, proliferation, and differentiation of human stem cells from the apical papilla.

Abstract

To evaluate the effect of EDTA and saline as the final irrigation in regenerative endodontic procedures (REPS) on the attachment, proliferation, migration, and differentiation of stem cells from the apical papilla (SCAPs). Dentin specimens from 140 human third molars were irrigated with various protocols-group 1: normal sterile saline (NSS), group 2: EDTA, group 3: EDTA then 5mL NSS, or group 4: EDTA then 20mL NSS. The specimens were used in cell assays. For cell proliferation, SCAPs were seeded on dentin, and the cell viability on days 1, 3, and 7 was determined using an MTT assay. At day 3, the attached cells' morphology was observed using SEM, and cell migration was investigated using a transwell migration assay. The ALP activity and odonto/osteogenic differentiation gene expression were evaluated at days 7, 14, and 21 using an ALP activity assay and RT-qPCR. On days 3 and 7, group 4 demonstrated more viable cells than group 1 (p < 0.01). The amount of migrated cells in groups 2, 3, and 4 was greater compared with group 1 (p < 0.05). Moreover, SCAP differentiation was similar between groups. Irrigating dentin with EDTA alone or with EDTA then NSS promoted SCAP migration. However, a final irrigation with 20mL NSS after EDTA promoted SCAP proliferation without affecting their differentiation. When using a blood clot as a scaffold, a final flushing with 20mL NSS after EDTA could be beneficial for clinical REP protocols.