This study was approved by the Institution Ethics Committee. Fifty human premolars were extracted in accordance with the orthodontic treatment plan and stored in a physiologic saline solution until further use. The teeth were clinically and radiographically examined before and after the extraction procedures. The inclusion criteria for the samples were as follows: free of caries, restoration, cracks, and calcification. Tooth samples that met any of the following criteria were excluded: teeth extracted due to periodontal diseases; teeth with open apices; teeth with root canal fillings, posts, crown restorations, or any metallic restorations; cracked teeth; teeth with any signs of internal or external resorption; and failure to provide informed consent.
Using a high-speed diamond bur with water coolant, roots were resected 3 mm below the cemento-enamel junction. Following this, an access cavity was prepared from the resected root surface in the coronal direction using an Endo Z bur (Dentsply Maillefer, Ballaigues, Switzerland). After preparation of an access cavity, the canals were irrigated by using a 2.5% sodium hypochlorite solution for cavity cleaning.
Teeth were randomly assigned to the following groups:
- ProRoot MTA (MTA group)
- RetroMTA (RMTA group)
- ProRoot MTA with prior application of DBA (MTA-B group)
- RetroMTA with prior application of DBA (RMTA-B group)
- Control group
For the ProRoot MTA (MTA) and RetroMTA (RMTA) groups, MTA plugs were inserted into the cavities, up to the cemento-enamel junction. For the ProRoot MTA with prior application of DBA (MTA-B) and RetroMTA with prior application of DBA (RMTA-B) groups, two layers of dentin adhesive (AdheSE; Ivoclar Vivadent, Schaan, Liechtenstein) were applied to the cavity according to the manufacturer’s instructions; AdheSE primer was applied for 30 seconds, and the excess primer was dispersed with a strong stream of air. Following primer, the AdheSE bonding agent was applied, and the excess was scattered with a soft flow of air. Light cured was done for 40 seconds. Following curing, MTA plugs were inserted into the cavities up to the cemento-enamel junction.
Following cleaning of the cavities, wet cotton pellets were placed over the MTA plugs and cavities were sealed with temporary filling materials (Caviton; GC Corp, Tokyo, Japan). Teeth were then stored in physiological saline for 1 day. The temporary filling materials were then removed, and MTA plugs were inspected to assess curing. Teeth were then restored with composite resin (Tetric ceram A3; Ivoclar Vivadent, Schaan, Liechtenstein). Teeth without MTA were restored with only composite resin (control teeth). All specimens were immersed in saline at room temperature and replenished every week.
Measurement of tooth discoloration
Tooth color changes were recorded using a spectrophotometer (SpectroShade™ Micro; MHT Medical High Technologies, Verona, Italy) at the outset of experimentation, and after 1, 4, and 12 weeks. Before spectrophotometry, an observer calibrated the spectrophotometer according to the manufacturer’s recommendations. The same operator performed measurements under continuous laboratory illumination by positioning the spectrophotometer optimally at the green line and obtaining three measurements. The average of the three measurements was used for subsequent analyses. The data were expressed using the CIE L*a*b* system, where L* represents the shade ranging from black (0) to white (100), and a* and b* correspond to chromaticity from red (+80a*) to green (−80a*) and yellow (+80b*) to blue (−80b*), respectively. Color differences between the baseline and each measurement were expressed as ΔE, based on the following:
Delta values ≥ 3.3 were considered clinically unacceptable .
Internal bleaching of discolored teeth
The MTA and RMTA groups were used to test the effects of bleaching on MTA discoloration. Following 12 weeks of treatment, the composite resin restorations and MTA were removed using a low-speed #1 carbide bur and a microscope (Zeiss OPMI pico; Carl Zeiss, Göttingen, Germany). A sodium perborate and distilled water solution (2:1 ratio) was used as the bleaching agent , which was condensed in the access cavities and sealed with temporary filling materials. Bleach was applied for 3 weeks with replacement each week. Chromatic changes were measured following the removal of MTA and bleaching for 1, 2, and 3 weeks.
Statistical analyses were performed using SPSS software (SPSS statistics 21.0; SPSS Inc., Chicago, IL). A one-way ANOVA was used to assess the differences between the MTAs at each time point and color changes following DBA pre-application in each MTA group. Post hoc Tukey’s tests were used for pairwise comparisons. A p-value of <0.05 was considered statistically significant.