All biological samples were included after obtaining the informed consent from all subjects. This study was carried out in accordance with relevant guidelines and regulations, followed after ethics committee approval (Comité de Ética de Investigación de Galicia).
Sample selection and preparation
After the approval of local ethics committee, a total of 100 single-canal maxillary human teeth, extracted for periodontal reasons, were stored in 2% thymol solution until its use. The specimens were decoronated to obtain a standardized root length of 15-mm using a diamond disc (Komet Dental, Lembo, Germany) and a surgical handpiece (Kavo Dental, Biberach an der Riss, Germany). The external surface of each root, including the apex, was sealed with nail polish to prevent the extrusion of irrigants through the apical foramen, after the placement of a size 10 K-file (Dentsply Sirona Endodontics, Ballaigues, Switerzland) at the working length (WL) to prevent the nail polish from entering the canal .
Root canal instrumentation
The specimens were fixed in the Pro-Train device (Simit Dental, Mantova, Italy) to allow the operator to perform the root canal instrumentation procedures. The WL was determined electronically using a size 10 K-file connected to the Root ZX apex locator (Morita, Osaka, Japan), which was confirmed with radiographs (Ultra-speed E; Kodak, Rochester, NY, USA). A glide path was performed with the PathFile system (Dentsply Sirona Endodontics) using the 0.13, 0.16 and 0.19 instruments. Then, the sample was prepared using the ProTaper Gold system (Dentsply Sirona Endodontics) following the sequence recommended by the manufacturer: S1, S2, F1, F2, F3 and F4 instruments at a speed of 250 rpm and 5.2 Ncm maximum torque. During instrumentation, each root canal was irrigated with 5.25% NaOCl (Parcan; Septodont, Saint-Maur-des-Fosses, France) using a side-vented needle (Max-I-Probe; Hawe Neos Dental SA, Bioggio, Switzerland) by inserting it into the canal as far as possible. Final irrigation was performed with 3 mL of 5.25% NaOCl, 1 mL of 17% EDTA (Coltene Whaledent, Langenau, Germany) for 1 min  followed by 3 mL of 5.25% NaOCl [12, 13].
Final irrigant protocols
After root canal preparation, the sample was randomly allocated into five groups (n = 20), according to the final irrigant protocol used:
PUI group. A 200-µm ultrasonic file (Irrisafe; Satelec, Bordeaux, France) driven by the P5 Newtron ultrasonic system (Acteon; Mount Laurel, NJ, USA), at a power setting of 5, was placed at 1 mm short of the WL and activated in 1-2 mm up-and-down movements with 5.25% NaOCl for 30 s, followed by 17% EDTA for 30 s and 5.25% NaOCl for 30 s. Irrigation was performed in a flow rate of 15 mL/min per specimen.
CUI group. A P500-µm ultrasonic irrigation needle (ProUltra PiezoFlow; Dentsply Sirona Endodontics) connected to the P5 Newtron ultrasonic system, at a power setting of 5, was positioned 1 mm short of binding, no deeper than 2/3 of the WL, and activated in 1-2 mm up-and-down movements with 5.25% NaOCl for 30 s, followed by 17% EDTA for 30 s and 5.25% NaOCl for 30 s. Irrigation was performed in a flow rate of 15 mL/min per specimen.
ANP group. Firstly, macroirrigation was performed with 5.25% NaOCl for 30 s using the macrocannula. Then, 3 cycles of microirrigation were done. For the first cycle, the microcannula was inserted 1 mm short of the WL and 5.25% NaOCl was continuously restocked for 20 s. The two successors’ microirrigation cycles were similar but using 17% EDTA and lastly 5.25% NaOCl.
CIFO group. A conventional syringe irrigation was performed with a front outlet NaviTip needle (Ultradent Products Inc.; South Jordan, UT, USA) positioned at 1 mm short of the WL using 5.25% NaOCl for 30 s followed by 17% EDTA for 30 s and 5.25% NaOCl for 30 s.
CISV group. The same sequence of the CIFO group was used herein. The only difference was the use of a side-vented needle (Max-I-Probe).
A total of 20 mL of irrigant was used per canal in all groups, with a flow rate of 15 mL/min with the same irrigation and activation time in each group. After the final irrigant protocols, the root canals were dried with absorbent paper points (Dentsply Sirona Endodontics). The same experienced operator performed all chemical-mechanical procedures.
SEM preparation and evaluation
After the final irrigant protocols, the specimens were grooved at 4, 8 and 12 mm from the root apex, defining the coronal, middle and apical thirds, respectively. The most coronal 3 mm of each root was discarded. Then, a SEM was used to evaluate the smear layer on each root surface. Longitudinal grooves were made on the buccal and lingual surfaces of each root with a diamond disc, without penetrating the root canal, to facilitate its posterior fracture with a chisel. An F4 gutta-percha master cone (Dentsply Sirona Endodontics) was placed inside the root canal to prevent contamination during root fracture. Only the half of each root that conserved the most visible part of its root canal was used; the other half was discarded.
For sample fixation, the protocol described by Perdigão et al.  was used. Briefly, the specimens were immersed in 2.5% glutaraldehyde/2% paraformaldehyde in 0.1 M sodium cacodylate buffer at pH 7.4 for 12 h at 4°C. Then, they were rinsed with 10 mL 0.1 M sodium cacodylate buffer, pH 7.4, for 2 h and postfixed with 2% osmium tetroxide in 0.1 M sodium cacodylate buffer for 1 h, followed by washing in 0.1 M sodium cacodylate for 1 h. The specimens were rinsed four times with deionized water and dehydrated through ascending grades of ethanol. After sample fixation and dehydration with ethanol, each specimen was orificated for observation under a SEM (FE-SEM; Sigma, Carl Zeiss, Germany). Microphotographs were taken at X1000 magnification to evaluate the presence of smear layer at the root canal surface in the center of each root third.
Two independent examiners, trained in the scoring process and with concordance verified with the κ test, scored the samples following the criteria described by Torabinejad et al. : 0 = no smear layer (absence of smear layer on the surface of the root canal, all dentinal tubules clean and open), 1 = moderate smear layer (no smear layer on the surface of the root canal, but dentinal tubules contain debris) and 2 = heavy smear layer (smear layer covers the root canal surface and dentinal tubules). The final results of the smear layer removal analysis were obtained by statistical analysis of the scores for each root third in each of the five experimental groups.
Significant differences in the amount of smear layer removal achieved by the final irrigant protocols were sought using Pearson’s χ2 test. The level of statistical significance was set at P < 0.05. All statistical analyses were performed using the SPSS software (ver. 20.0; SPSS Inc., Chicago, IL, USA).