In summary, after approval by the Ethical Committee Department of Dentistry Hospital, Nanjing Medical University (Institutional Review Board) (IRB; approval number PJ2015–001-08), human mandibular first molars freshly extracted for reasons unrelated to this research were collected and stored in distilled water at 4 °C before selection. Only fully developed teeth with two separate mesial roots ending in two fully formed apices were selected. Meanwhile, the teeth also had to have curvatures of 25°-40° according to Schneider’s  method and a maximum curvature located within the middle third of the root canal. A standard endodontic access cavity was prepared, and a number #08 stainless steel manual K-file (Dentsply Sirona) was pre-curved and inserted through the mesial canal to ensure apical patency. To increase standardization, the root canals that could be negotiated with a #10 K-file (or larger) up to the apex without any resistance were excluded, and the crown and distal root of each tooth were flattened. The working length (WL) was measured as 0.5 mm short of the length when the tip of the instrument was just observed at the apical foramen. After the WL measurement, canals with WL shorter than 11 mm were excluded, and canals with WL longer than 11 mm were standardized to 11 mm using a high-speed bur. All of the root selection procedures were performed by one endodontist. Accordingly, 60 mesiobuccal and mesiolingual canals of 39 teeth were selected and randomly assigned into 3 groups for 20 canals each. Then, the balance of canal curvatures among the three groups was analyzed and confirmed by one-way analysis of variance (p > 0.05).
The K-files(KF), PathFiles(PF), ProGlider file(PG), and WaveOne file(WO) were used for the canal preparation.
Group KF + WO: the glide path was prepared using pre-curved stainless steel K-files (15#, 0.02 and 20#, 0.02), and the root canal was subsequently prepared by WaveOne Primary (25#, 0.08) file.
Group PF + WO: the glide path was prepared using PathFiles (13#, 0.02, 16#, 0.02, 19#, 0.02), and the root canal was subsequently prepared by WaveOne Primary (25#, 0.08) file.
Group PG + WO: the glide path was prepared using the ProGlider single file (16#, 0.02 to 0.085), and the root canal was subsequently prepared by WaveOne Primary (25#, 0.08) file.
Both the PF and PG NiTi glide path files were applied with an endodontic motor (X-Smart plus, Dentsply Sirona) operated with a 16:1 contra angle, at 300 rpm and with 5 Ncm torque. The WaveOne file was performed to the WL with the WaveOne program using the same endodontic motor with a slow, in-and-out pecking motion according to the manufacturer’s instructions. After every three pecking motions, the WaveOne file was taken out of the canal and cleaned with gauze. The WL was checked using a #10 K-file, and the canal was irrigated with 10 mL distilled water using a syringe with a 30-gauge side-vented irrigation needle (Wode, Zhenjiang, China) as the file was taken out of the canal each time. Meanwhile, the instrument failure was recorded. To avoid inter-operator variability, the canal preparation was performed by a single experienced endodontist.
Canal transportation and canal volume analysis
The high-resolution micro-computed tomography (micro-CT) scanner SkyScan 1176 (Bruker microCT, Kontich, Belgium) was used to record the canal transportation and canal volume increase. Each of the studied teeth was scanned for three times: before glide path preparation, after glide path preparation and after root canal preparation. The parameters were kept constant: 70 kV, 353 μA, a 0.5-mm-thick aluminum filter, 360° rotations and a 0.5° rotation step, displaying an object with an 18 μm voxel size. After the scanning, the images were reconstructed and the measurements were acquired using CTAn v126.96.36.199 software (Bruker microCT). Root canal transportations were analyzed at three levels: 1 mm, 3 mm and 5 mm from the apical foramen. The following formula developed by Gambill et al.  was used to measure canal transportation: │(m1-m2)-(d1-d2)│, where m1 indicates the thinnest mesial canal wall pre-instrumentation, m2 indicates the thinnest mesial canal wall post-instrumentation, d1 indicates the thinnest distal canal wall pre-instrumentation, and d2 indicates the thinnest distal canal wall post-instrumentation. The root canal volume was measured, then the volume increase was determined by subtracting the volume of the untreated canal from the volume of the treated canal. All of the data were measured and analyzed by an investigator who was blinded to the specimen assignment.
Debris collection and evaluation
The experimental equipment used to collect the apical extruded debris was similar to that described by Myers and Montgomery . Each tooth was fixed on a stopper and then attached to a pre-weighted Eppendorf tube. A 25-gauge needle was used alongside the stopper to balance the air pressure inside and outside of the tube. Then, the tube was fitted into a vial. The apical extruded debris during the glide path preparation and the WaveOne preparation was collected into the tube, and the debris visually adherent to the external surface of the apex was collected into the tube by flushing the apex with 0.5 mL of distilled water. The tube was then stored in an incubator at 70 °C for 5 days to evaporate moisture before finally weighing the tube on a microbalance to 10− 5 g precision (AY 120 Analytic Balance, Shimadzu Corporation, Tokyo, Japan). Each tube was measured three times and the mean value was recorded. The net weight of the dry debris was determined by subtracting the original weight of the empty Eppendorf tube from the gross weight. The evaluation was performed by an experimenter who was blinded to group assignment.
Working time was recorded with an electronic stopwatch, including total active instrumentation phase, cleaning of the flutes of the instruments, checking of the WL and irrigation, while the time required to adjust the rubber stops to the WL was not included.
Normality of variable distribution was evaluated with the Kolmogorov-Smirnov test (SPSS 17.0 software; SPSS Inc., Chicago, IL, USA). Then, the data were statistically analyzed using a one-way analysis of variance. Multiple comparisons were made by using Tukey’s test. The level of significance was set at P < 0.05.