Sample size
The sample size was calculated using the G*Power v3.1 software for Mac (Heinrich Heine, Universität Düsseldorf, Germany), using the ANOVA test to determine the sample size
To determine the effect expected for this study, canal volume and dentin removal data from the studies by De-Deus et al. (2015)[10] and Gagliardi et al. (2015)[11], and volume of remaining filling material with bioceramic sealer data from the study by Romeiro et al. (2020) [6] were considered. The parameters set were an alpha error of 0.05, a beta power of 0.80, and an N2/N1 ratio of 1. A sample of 15 canals per group was stipulated (n = 15), but 18 canals were used considering the possibility of losing samples.
Sample selection
After approval by the local Research Ethics Committee (42870621.2.0000.0102), eighteen extracted mandibular first and second molars with a mesial root with a Vertucci type IV canal configuration and a curvature between 20° and 40° were selected. Micro-computed tomography scanner (SkyScan 1172, Bruker-microCT, Kontich, Belgium) was used to confirm the inclusion criteria with the following parameters: exposure time of 1750 ms, voltage of 80kV, the voxel size of10.89 μm, and filter of 0.5 mm aluminum.
The access cavity was performed using 1012, 1015, and 3205 diamond tips (KG Sorensen, São Paulo, Brazil). The teeth were then sectioned by separating the mesial and distal roots. Only the mesial roots were used.
Root canal preparation
The canals were scouted with manual #08 and #10 C-pilot files (VDW, Munich, Germany) up to the working length (WL), which was defined considering 1 mm from the apical foramen. The canals were prepared using the 15/.03, 25/.01, 25/.04, and 35/.05 instruments (Prodesign Logic 2 System, Easy Equipamentos Odontológicos, Belo Horizonte, Brazil), and the canals were recapitulated with a manual #10 C-pilot file (VDW, Munich, Germany) after the use of each instrument. The root canals were irrigated with 2.5% sodium hypochlorite (NaOCl) (Asfer, São Caetano do Sul, Brazil) using a 5 ml disposable syringe (BD, Curitiba, Brazil) and a 30-gauge NaviTip needle (Ultradent Inc, South Jordan, USA). The root canals were then irrigated with 17% EDTA (Fórmula & Ação, São Paulo, Brazil). The irrigating substances were agitated with Easyclean instruments (Easy Equipamentos Odontológicos, Belo Horizonte, Brazil), and the canals were dried with intracanal suction tips (Ultradent, Indaiatuba, Brazil) and 35/.04 absorbent paper tips (VDW, Munich, Germany).
Root canal filling
The samples were filled using a 35/.05 gutta-percha cone (Easy Equipamentos Odontológicos, Belo Horizonte, Brazil) and the Bio-C Sealer (Angelus, Londrina, Brazil). A plastic needle was inserted 4 mm from the WL, and the sealer was inserted into the root canals with the syringe plunger gently pressed until it flowed back into the pulp chamber.
The buccal and lingual canals were randomly assigned (http://www.openepi.com/Menu/OE_Menu.htm) into 2 groups according to the filling technique:
- Single-cone (n=18): the gutta-percha cone coated with Bio-C Sealer was inserted into the canal up to the WL with brushing movements, spreading the sealer over the canal walls. Then, the gutta-percha cone was cut at the entrance of the canals using a heated hand plugger (Odous de Deus, Belo Horizonte, Brazil) and followed by cold vertical compaction.
- Continuous Wave of Condensation (n=18): the canals were filled using the BeeFill 2in1 system (VDW, Munich, Germany). The gutta-percha cone coated with the sealer was inserted into the canal, then the BeeFill 2in1 thermocompactor was introduced and activated into the root canal up to the root curvature, deactivated, maintained for 10 seconds, reactivated for 1 second, and then removed from the canal. Vertical compaction of the filling was performed with Buchanan hand pluggers (SybronEndo, Orange, USA) in the apical third. Next, using the BeeFill 2in1 thermoinjector, the back-fill was carried out with the gutta-percha heated to 160°C and inserted in the middle third followed by cold vertical compaction with hand pluggers, and then in the apical third following this same protocol.
Periapical radiographs were taken (Phosphor plate system, Durr, Porto Alegre, Brazil) to check the quality of the filling. The canals were then sealed with Coltosol (Coltene, Rio de Janeiro, Brazil), and the samples were kept in an oven at 37ºC and 100% relative humidity for 7 days for the complete setting of the sealer.
The samples were scanned again by micro-CT using the same parameters described before and the images were reconstructed using the NReconv1.6.4.8 software (Bruker, Kontich, Belgium). In the CTan software, the volume (mm3) of filling material was obtained at the 1-3 mm, 3-5 mm, and 5-7 mm segments from the apical foramen, and the dentin thickness (mm3) of the mesial, and distal walls at the 1 mm, 3 mm, 5 mm, and 7 mm segments.
Endodontic Retreatment
The filling material was removed using the Reciproc Blue R25 instrument (VDW, Munich, Germany) and the VDW Silver motor (VDW, Munich, Germany). The instrument was inserted into the mass of the filling material with pecking movements, advancing up to the WL. Every three movements, irrigation was carried out as in the preparation stage. When the WL was reached, movements were made against the canal walls until the filling material was detectably removed. Total removal was determined when no material remained on the instrument and the walls were clean when viewed under an operating microscope at 8x magnification (DFVasconcellos, Rio de Janeiro, Brazil).
The teeth were reprepared using a Reciproc Blue R40 instrument (VDW, Munich, Germany) with small in-and-out movements and gentle apical pressure until the WL was reached. Irrigation was carried out as in the canal preparation stage.
The roots were scanned by micro-CT using the same parameters as the previous scans and reconstructions. With the CT Analyzer software, the volume (mm³) of filling material was measured at the 1-3 mm, 3-5 mm, and 5-7 mm segments from the apical foramen, and the values were converted into the percentage of filling material.
The dentin thickness (mm) of the mesial, and distal walls were measured at the 1 mm, 3 mm, 5 mm, and 7 mm segments to assess the transportation and centering ability of the endodontic repreparation.
The canal transportation (mm) was calculated in each third of the root canal and considered the extent and direction of transportation by measuring the distances between the canal and the root edges in the mesiodistal direction, following the equation (X1-X2) - (Y1- Y2), where X1 is the shortest distance between the mesial edge of the root and the canal before instrumentation, and X2 is the shortest distance between the mesial edge of the root and the instrumented canal. Y1 is the shortest distance between the distal root edge and the canal before instrumentation, while Y2 is the shortest distance between the distal root edge and the instrumented canal (Figure 1). A zero result indicates that there is no root canal transportation. A positive result indicates transportation in the mesial direction, while a negative result indicates transportation in the distal direction.
The centering ability was calculated in each third of the root canal using the equation (X1-X2)/(Y1-Y2). A score of "1" indicates perfect centering, while a score of "0" indicates no centering.
Statistical Analysis
After checking the data normality, the t-test (parametric) or Mann-Whitney test (non-parametric) was applied to the intergroup data, and the ANOVA test (parametric) or Kruskal-Wallis test (non-parametric) was applied to the intragroup data. A significance level of 5% (p < 0.05) was considered for all tests. Statistical analysis was carried out using the GraphPad Prism 9 software (La Jolla, CA, United States).