## 2 Materials and Methods  
Mandibular first permanent molars with a fully formed apex and extracted for periodontal disease were considered. The patients signed an informed consent for the treatment and for the use of the extracted tooth for research purposes, and the local scientific committee authorized the in-vitro study. Only teeth free of caries, cracks, and artificial alterations were included in the study. After root surface debridement to remove periodontal debris, plaque, and calculus, specimens were dipped in a 0.01% NaOCl solution at 4 \({}^{\circ}\)C and then, after 24 h, were stored in saline solution at the same temperature. A sample size of 30 molars per group was calculated with G*Power 3.1.4 (Kiel University, Kiel, Germany) to set the study power at 80%. A custom-made support was created in order to scan the specimens. Low-resolution preliminary micro-CT scans were performed to ensure that inclusion criteria were met (SkyScan 1172, Bruker micro-CT, Kontich, Belgium). Preliminary scans were conducted with the following parameters: a total of 450 projections throughout a 225\({}^{\circ}\) rotation (180\({}^{\circ}\) plus cone angle of the X-ray source) using a 1.0 mm-thick aluminum filter, voltage = 100 kV, current = 80 \(\mu\)A, source-to-object distance = 80 mm, source-to-detector distance = 220 mm, pixel binning = 8 \(\times\) 8, and exposure time/projection = 0.2s. Morphological parameters of the mesial canals were obtained. Mesio-lingual separated canals, 12 \(\pm\) 2 mm in length from canal orifice to apical foramen, with moderate curvature (10\({}^{\circ}\)-30\({}^{\circ}\)), and 2 \(<\) r \(\leq\) 6 curvature radius were selected to ensure sample homogeneity. Out of 72 teeth assessed for inclusion, 12 teeth not meeting the inclusion criteria or with prominent intracanal isthmus or significant calcifications were excluded. Sixty samples were randomly assigned to two groups by a blinded operator through random number tables: WaveOne Gold Primary with no brushing technique (NB group) (\(n=30\)) and WaveOne Gold Primary with brushing technique (B group) (\(n=30\)). A blinded operator performed randomization, allocation, and statistical analysis, and a single expert operator with over ten years of experience in endodontics and blinded to the objectives of the study prepared the samples. After access cavity preparation, canal scouting was performed with #10 K-file at working length (WL) using Glyde (Dentsply Sirona) as a lubricating agent (0.80 mg). WL was determined under 10X magnification (OPMI Pro Ergo, Carl Zeiss) by subtracting 0.5 mm from the length at which the tip was visible at the apical foramen. In both groups, glide path was performed with a Proglider single file (size 0.16, taper 0.02 to 0.085) (Dentsply Sirona) using an X-Smart Plus motor (Dentsply Sirona) (300 rpm, 4 Ncm) at WL. In the NB group, root canal shaping was performed with a WaveOne Gold Primary single file (size 0.25, taper 0.07) (Dentsply Sirona) using X-Smart Plus (reciprocating movement settings as suggested by the manufacturer) at WL with an in-and-out motion and no intentional brushing effect. In the B group, root canal shaping was performed with a WaveOne Gold Primary single file (size 0.25, 0.07) using X-Smart Plus (reciprocating movement settings as suggested by the manufacturer) at WL with a brushing motion consisting of four strokes towards the root canal wall opposite to the furcation. Instruments were removed from the canal and cleaned after three pecking motions until WL was reached. Slow in-and-out pecking motions (amplitude less than 3 mm) were used in both groups, and new instruments were employed for each specimen. Irrigation was performed with a 30-gauge needle syringe alternating 5% NaOCl with 10% EDTA during instrumentation for a total of 6 mL of NaOCl and 4 mL of EDTA for each specimen. The selected samples were scanned at a higher spatial resolution before and after root canal preparation (SkyScan, Bruker-microCT).