Maxillary first permanent molars extracted for periodontal disease were selected in accordance with the local ethics committee (Protocol number CS2/1053). A sample size of 15 per group was calculated with G*Power 3.1.4 (Kiel University, Kiel, Germany) considering alpha-error = 0.05 and
ß = 0.95. After the root debridement performed with Gracey curette 7/8 (Hu-Friedy, Chicago, IL), the specimens were dipped into a 0.01% NaOCl solution at 4°C for 24 hours and then stored in saline solution. A total of 41 teeth were selected. The specimens were mounted in the scanner with the occlusal surface against a 2 mm resin customized support fixed on a SEM stub (SkyScan 1172, Bruker micro-CT, Kontich, Belgium) to allow reproducible orientation during pre- and post-instrumentation scans [
23]. Preliminary micro-CT scans were accomplished to attain a root canal anatomy outline and to ensure the respect of the inclusion criteria (SkyScan 1172, Bruker micro-CT, Kontich, Belgium). Preliminary scans were conducted with a total of 450 projections throughout a 180° rotation using a 1.0-mm-thick aluminum filter (voltage = 100 kV, current = 80 μA, source-to-object distance = 80 mm, source-to-detector distance = 220 mm, pixel binning = 8 X 8, exposure time/ projection = 0.2 s). The mesio-buccal (MB1) canals were considered only and their morphological parameters were obtained. Inclusion criteria were the following: root canal length from canal orifice to apical foramen of 12 ± 2 mm, primary canal curvature between 25°- 40° according to Schneider method on the mesio-distal plane [
24], radius of curvature of 4 < r ≤ 8 mm and a point of maximum curvature located within the middle third of the root canal. Teeth with a distinct fourth canal orifice were selected to exclude samples presenting mesial roots with a single flat MB canal. Teeth with significant calcifications or not according to the inclusion criteria were excluded. The teeth were without caries, cracks and extended restorations. Of 41 teeth selected, eleven were excluded due to anatomical features. Thirty samples were randomly assigned to the two groups using a computer-generated randomization system: ProGlider and ProTaper Next rotary shaping system (group PG-PTN) (
n=15) (Dentsply Sirona, Ballaigues, Switzerland) and WaveOne Gold Glider and WaveOne Gold reciprocating shaping system (group WOGG-WOG) (
n=15) (Dentsply Sirona, Ballaigues, Switzerland). A single blind operator checked randomization and allocation and performed statistical analysis. One single expert operator was up-skilled on both instrumentation techniques and previously calibrated for pecking speed and pressure on the handpiece using an endodontic engine with torque measurement. A traditional access cavity preparation was designed following conventional guidelines: outline and cervical dentin were modified as needed until all orifices could be visualized in the same field of view and straight access to canal orifices could be achieved without coronal interferences [
25]. Then, canal scouting was accomplished in all specimens with #10 K-file at working length (WL) using Glyde (Dentsply Sirona, Ballaigues, Switzerland) as lubricating gel (0.80 mg). WL was established with 10X magnification (OPMI Pro Ergo, Carl Zeiss, Oberkochen, Germany) when the tip was visible at the apical foramen and then subtracting 0.5 mm. In Group PG-PTN, glide path was performed with Proglider (PG) rotary single file (size 0.16, taper .02 to .082 at D16) (Dentsply Sirona, Ballaigues, Switzerland). Then, shaping was concluded with ProTaper Next (PTN) X1 (tip size 0.17 mm, taper .04) and X2 (tip size 0.25 mm, taper .06) (Dentsply Sirona, Ballaigues, Switzerland). Both PG and PTN were used with an endodontic engine X-Smart Plus (Dentsply Sirona, Ballaigues, Switzerland) with 16:1 contra angle (300 rpm, 4 Ncm) in continuous rotation up to WL. In Group WOGG-WOG, glide path was performed with WaveOne Gold Glider (WOGG) reciprocating single file (tip size 0.15, taper .017 to .085 at D16) (Dentsply Sirona, Ballaigues, Switzerland). Then, shaping was concluded with WaveOne Gold (WOG) Primary (size 0.25, taper .07) (Dentsply Sirona, Ballaigues, Switzerland). Both WOGG and WOG were used with an endodontic engine X-Smart Plus (Dentsply Sirona, Ballaigues, Switzerland) set in the “WAVEONE ALL” mode until reaching the WL. Rotary and reciprocating instruments were used with in and out motion, with no intentional brushing against canal walls. Instruments were removed from the canal and cleaned each time after three pecking motions until WL was reached. In both groups, the apical gaging was performed with K-Files to confirm the apical preparation diameter. New instruments were used for each specimen. Irrigation was completed with 5% NaOCl (Niclor 5, OGNA, Muggiò, Italy) and with 10% EDTA alternated for a total of 10 mL for each per specimen delivered with a 30-gage needle up to 4 mm from the WL. Recapitulation with a size 10 K-File was conducted between each instrument. The selected samples were scanned at high-resolution before preparation, after glide path and after shaping (100 kV, 100 μA, 16 μm resolution, Al+Cu filter and 360° rotation for a total of 2400 projections). Afterwards, the images were reconstructed with NRecon software (SkyScan 1172, Bruker micro-CT, Kontich, Belgium) using standard parameters for beam hardening and ring artifact correction and the binarized objects were analyzed with CTAn software (SkyScan 1172, Bruker micro-CT, Kontich, Belgium). Two expert operators carried out scans analysis and inter examiner agreement was calculated using weighted kappa statistics (K > 0.90). The increase in canal volume and surface area was calculated for each sample through 3D renderings. The following 2D parameters were measured starting from orthogonal cross sections: the canal centroid shift, the reduction of dentin thickness from the furcation side expressed as a percentage of the difference between pre- and post-instrumentation values, the ratio of diameter ratios (RDR) and the ratio of cross-sectional areas (RA) using ImageJ 1.43u 64-bit software (National Institute of Health, Bethesda) [
13,
16]. RDR represents the instrument tendency to asymmetrically enlarge the root canal in one direction: RDR = (D/d)post/(D/d)pre, where (D/d)post is the post-preparation ratio of the major diameter (D) to the minor diameter (d) and (D/d)pre is the pre-preparation ratio of D to d. Values closer to 1 correspond to a better maintenance of the original canal geometry. RA quantifies the ability of the instrument to enlarge the root canal space: RA = Apost/Apre, where A
post and A
pre are the post-preparation and the pre-preparation cross-sectional areas, respectively. Values closer to 1 correspond to a reduced difference between post- and pre-instrumentation measurements [
26]. Root sections orthogonal to the canal axis were set at 3 different levels: apical (A), 1 mm from the apical foramen; middle (M), set at the point of maximum curvature and coronal (C), set in correspondence to the middle portion of the root canal coronal third defined by 3D calculation of the root canal length from apex to orifice. These levels were selected as most representative of the critical shaping portions [
27]. The bidimensional parameters were analyzed at each level except for the reduction of dentin thickness, which was evaluated only for the M level. An automated minimum threshold was set to avoid manual errors [
28]. The distribution of the data was analyzed with a Shapiro–Wilk normality test. The differences of the root canal curvature at baseline were analyzed with a Kruskal–Wallis and post hoc Dunn’s tests (
P < 0.05). One-way ANOVA and post hoc Tukey–Kramer tests were used to analyze the increase of canal surface area and volume, the centroid shift, the impact of the instrumentation on RDR and RA parameters at each level of analysis and the number of pecking motions (
P < 0.05). All of the statistical analyses were conducted with the Minitab 15 software package (Minitab Inc., State College).