The study design was approved by the Institutional Ethics Committee of the Second Xiangya Hospital, Central South University (LYE No. 2021023). All methods were performed in accordance with the relevant guidelines and regulations. Twenty-seven intact premolars extracted from orthodontic reasons were collected after informed consent from the patients or their parents or guardians. All specimen were radiographically confirmed the presence of a single root straight canal with a mature apex and excluded teeth with cracks or root fractures. Following the removal of superficial soft tissues with a brush, and the teeth were kept in 0.1% thymol solution (Sigma-Aldrich, Missouri, USA) at 4 °C until used.
Root canal therapy
The root canal preparation followed recognized studies, the Guide to Clinical Endodontics, published by the American Association of Endodontists30,31. All samples were confirmed the working length (WL) by inserting No.10 K files (Dentsply Maillefer, Ballaigues, Switzerland) reaching the apical foramen and subtracting 1 mm from this point. Then, root canals were instrumented with nickel-titanium X-Taper Universal files (Easyinsmile, Staten Island, NY, USA) up to F3 size32 following the WL by the same operator. Following the completion of canals enlargement, canals were rinsed with 2 mL of 0.9% NaCl for 1 min through an in-and-out motion with a 5 ml needle (NaviTip, Ultradent, South Jordan, UT) and dried with absorbent paper points.
Final irrigation procedures
The teeth were randomly divided to 2 experimental groups (n = 9) according to the final irrigation treatment; 9 additional teeth selected as a control group. Final irrigation protocol of each group was as follows:
Group 1 (needle irrigation, NI)
A total of 1.5 ml of 3% NaOCl was applied for the final irrigation19,33-35 by a needle (NaviTip, Ultradent, South Jordan, UT). The tip of needle was placed 2 mm short of the WL.
Group 2 (passive ultrasonic irrigation, PUI)
The final irrigation was performed using 1.5 ml of 3% NaOCl for 45 s but with passive ultrasonic activation. It was performed using an ultrasonic tip (size 25) that was placed 2 mm from the WL.
Group 3 (EASYDO ACTIVATOR, EA)
Similarly, 1.5 ml of 3% NaOCl was delivered but with an EA system with a power setting of 2. The EA tip was applied 2 mm short of the working length and moved in 2 mm amplitudes from the WL.
SEM preparation and analysis
After the intracanal procedures, teeth were marked 2 longitudinal grooves at buccolingual direction with a diamond-coated high-speed bur (TF-14; SHARK, Boston, USA) as reported by Wu and Wesselink31. A mallet and a chisel were used to split root canals and care was noticed so that decreased the grooves penetrating into canals. To clearly and objectively distinguish the sections of each root canals, horizontal signs were made at the coronal, middle, and apical sections from apex using a sharp scalpel inside the canals. All sections of teeth were subjected to critical point drying and coated for later examined under SEM. The root canal surface of all sections was examined using a scanning electron microscopy (JSM-IT100; Jeol, Tokyo, Japan). Following screening of all canal walls, photomicrographs at 1000× and 2000× magnification were taken to visualize three portions at the levels previously marked by a scalpel scratch.
The smear layer was recognized as the surface film of debris produced by root canal instrumentation, which may become a barrier and increase bacterial invasion into dentinal tubules 32. Scoring of the presence of a smear layer was analyzed using a ×1000 magnification, and the scores were proposed by Hülsmann et al.17 on a grade from 1 to 5 as follows: score 1: no smear layer, all orifice of dentinal tubules open; score 2: slight smear layer, some dentinal tubules obstructed by debris; score 3: homogeneous amount of smear layer covering the canal wall, only a few dentinal tubules clean and open; score 4: whole root canal wall covered by heterogeneous smear layer, no patent orifice of dentinal tubules; score 5: the root canal wall obstructed by heavy, nonhomogeneous smear layer completely.
Debris was defined as inorganic dentine chips, pulp tissues residue, microorganisms and necrotic products and remnant attached to canal walls closely 17,32. × 2000 magnification images were analyzed for the presence of debris. The images were graded followed to the criteria reported by Hülsmann et al.: score 1: root canal wall clean and smooth, few small debris remnants present; Score 2: dense debris packing part of root canal wall; Score 3: less than 50% of the root canal wall packed by agglomerations of debris; Score 4: over 50% of the root canal wall covered by uneven debris; Score 5: whole root canal wall covered by a great number of debris nearly.
The smear layer and debris score data were analyzed using the Pearson chi square test by SPSS software (SPSS v. 23, IBM, Armonk, NY, USA) to verify the assumption of normality. The level of significance was set at p ≤ 0.05.