## 2 Materials and Methods
### Specimen Preparation  
The local research and ethics committee's ethical approval was obtained before the study was carried out (DF-RD-1912/0032-P). Forty-eight single-rooted mandibular pre-molars extracted for orthodontic reasons were collected from patients between 18 and 30 years old. Using a digital caliper (Measuring Tool Enterprise, Shanghai, China), teeth with similar buccolingual (BL), mesiodistal (MD), and root lengths of 22.0 mm (1.0 mm) from the tip of the buccal cusp were compared to control for biological variances. Tooth with caries, open apex or previous root canal treatment were excluded. BL and MD periapical radiographs were made to confirm the presence of a straight single root canal without calcifications or resorption. The chosen teeth were kept in a 0.12% thymol solution and used within three months of storage.  
With a round bur ([PERSON], Ballaigues, Switzerland) the pulp chamber was accessed. The working length was determined to be 1 mm short of the apex by using K-file size 10 (MANI Inc., Utsunomiya, Tochigi, Japan). All teeth were instrumented using REVO-S rotary files (MICRO-MEGA, Besancon Cedex, France). The Revo-S three-step crown-down sequence was used according to the manufacturer's instructions; SC1 (25, 0.06) tapered instrument was used with slow downward movement in a free progression and without pressure to enlarge the coronal two-thirds of the canal, SC2 (25, 0.04) tapered instrument was used with a progressive three-wave movement (up and down movement) to the working length, and SU (25, 0.06) tapered instrument was used to the working length with a slow downward movement in a free progression and without pressure. The root canals were irrigated with 3 mL of 2.5% NaOCl after each file. After completion of instrumentation, the root canals were rinsed with 2 mL of 5.25% NaOCl for 60 s, with 1 mL of 17% EDTA (Pulpdet Corp., Watertown, MA, USA) for 60 s, and with a final rinse of 5 mL normal saline. Just before obturation, the canals were dried with paper points (Brasseler USA, Savannah, GA, USA) and randomly assigned to 2 groups (\(n=24\)): Group 1, AH Plus + GP (Dentsply Maillefer NA, Tulsa, OK, USA) and Group 2, BioRoot RCS + GP.  
The root canals were obturated with matched-taper single-cone (SC) gutta percha coated with the tested sealers, which were handled according to the manufacturers' instructions (Table 1). BL and MD radiographs were made to confirm the adequacy of the root filling in terms of appropriate length, density, and taper (Figure 1). The access opening was sealed with a composite resin (MultCore Flow; Ivoclar AG, Schaan, Liechtenstein). The teeth were stored at 37 \({}^{\circ}\)C with 100% humidity for 7 days to allow the sealers to set.  
\begin{table}
\begin{tabular}{c c c} \hline
**Group** & **Manufacturers** & **Composition** \\ \hline AH Plus & Dentsply-De Trey, Constance, Germany & Paste A: diepoxide, calcium tungstate, zirconium oxide, aerosil, pigment (Fe oxide) \\  & & Paste B: 1-adamantane amine \\ BioRoot RCS & Septodont, St. Maur-des-Fossés, France & Powder: tricalcium silicate, zirconium oxide and povidone \\  & & Liquid: aqueous solution of calcium chloride and polycarboxylate \\ \hline \end{tabular}
\end{table}
Table 1: Manufacturers and composition of the tested sealers.