Sample selection, root canal preparation, and obturation
Thirty bovine mandibular incisors were used. The inclusion criteria were teeth with one canal, straight and with complete root formation, without calcifications, and anatomical diameter equivalent to a #45 K file (Dentsply Sirona, Ballaigues, Switzerland). A double-sided diamond disc (KG Sorensen, Cotia, São Paulo, Brazil) transversally sectioned the roots and standardized their length at 15mm. The working length was determined to be 1mm shorter than the roots. The chemo-mechanical preparation was performed sequentially up to a #80 K File. During all preparation, the canals were irrigated with 10 ml of 2.5% sodium hypochlorite (NaOCl) (Farmácia Marcela, Porto Alegre, RS, Brazil). Next, canals were irrigated with 1 mL of 17% EDTA (Farmácia Marcela) for 3 minutes, followed by 5 mL of 2.5% NaOCl for 60 seconds and 2 mL of saline solution (Farmácia Marcela) and dried with absorbent paper cones #80 (Endopoints Ltda., Paraíba do Sul, RJ, Brazil). With the cold lateral compaction technique, the canals were filled with a #80 main and accessory gutta-percha cones and zinc oxide-eugenol-based sealer (Endofill, Dentsply). Excess filling material was removed with a heated plugger, and vertical compaction was performed. The cervical portion of the roots was sealed with temporary restorative material (Cavit; 3M, Sumaré, São Paulo, Brazil) and kept in 100% humidity at 37oC.
After seven days, apicoectomy of the apical 3 mm roots was performed at an angle of 90o with the long axis using an ultrasonic tip (Bladesonic; Helse Ultrasonics, Santa Rosa de Viterbo, SP, Brazil). Root-end cavities were prepared with 3mm depth using an ultrasonic tip P1 (Helse Ultrasonic). After preparation, the root-end cavities were irrigated with 2 mL of saline solution and dried with paper points.

Computerized microtomography for allocation in the experimental groups

Scanning was performed with a microtomograph (Shimadzu inspexio SMX-090CT Plus, Kiyamachi-Nijo, Kyoto, Japan). The settings were 70 kV X-ray tube voltage, 800 mA anode current, and 0.019mm/pix voxel size. The scan with 1024x1024 pixels originated in 4800 slices, obtained with acquisition intervals of 1° over 360° of rotation, and the average scan time ranged from 45 to 60 minutes. All samples were digitized after preparation of the root-end cavities for volume measurement and allocation into experimental groups by stratified randomization.
Figure 1 shows a sequence of micro-CT images using the CTAn software. The set of images was exported in. DICOM file format for analysis in the CTAn software (CT-Analyser Version 1.13 Bruker micro-CT Konitch Belgium). The volume of the root-end cavity was measured from the most apical layer until the filling material (i.g. gutta percha endodontic sealer) became visible in the axial sections. Then, the regions of interest (ROI) corresponding to the areas to be considered in the volume calculation were determined.
NeoMTA Plus and MTA were handled according to the manufacturers’ instructions. BioRoot RCS was prepared with a powder/liquid ratio of 1.5 spoons for five liquid drops. The root-end cavities filling was performed with a micro applicator. Samples were stored in a humid environment at 37°C for seven days to ensure the complete materials’ setting.