Efficacy of ultrasonic activation of NaOCl and orange oil in removing filling material from mesial canals of mandibular molars with and without isthmus

ABSTRACT Objectives The aim of this study was to evaluate the volume of remaining filling material after passive ultrasonic irrigation (PUI) of sodium hypochlorite (NaOCl) and orange oil in mesial canals of mandibular molars, with and without isthmus. Material and Methods Thirty mesial roots of mandibular molars were divided according to the presence or absence of isthmus. Canals were prepared and filled (Micro-CT #1). Filling was removed using rotary instruments, and specimens were sub-divided into three groups according to the irrigation procedures: Conventional – conventional irrigation with NaOCl, PUI/NaOCl – PUI of NaOCl (three activations, 20 seconds each), and PUI/orange oil – PUI of orange oil (Micro-CT#2). Specimens were enlarged using the X2 and X3 ProTaper Next instruments and submitted to the same irrigation protocols (Micro-CT #3). Results No differences were found between the experimental groups in each stage of assessment (P>0.05). The volume of residual filling material was similar to those in Micro-CT #2 and Micro-CT #3, but lower than those observed in Micro-CT #1 (P<0.05). When groups were pooled according to the presence or absence of an isthmus, volume of residual filling material was higher in specimens presenting isthmus (P<0.05). Conclusions PUI of NaOCl or orange oil did not improve filling removal. Isthmus consists in an anatomical obstacle that impairs the removal of filling material.


INTRODUCTION
When endodontic failure occurs, nonsurgical endodontic retreatment is often indicated. Mesial root canals of mandibular molars are usually isthmus, which communicates the buccal with the lingual canals 22 . The inaccessibility of instruments and chemical irrigants into the isthmus impairs root canal disinfection 31 . must be removed to enable re-preparation and disinfection of the root canal system 23 . The most commonly used techniques for filling removal 10 . These instruments can be used with chemical solvents to 25,28 . However, due the complexity of the root canal system, complete regardless of the employed technique 31 .
Organic solvents can be used during retreatment the tooth 26 . Chemical solvents present different proprieties that should be considered, such as efficacy in dissolving root filling material and cytotoxicity levels 3 . Orange oil is as effective as xylol 13,14 . Additionally, it has lower cytotoxicity when compared to eucalyptol, xylene, and chloroform 4 .

MATERIAL AND METHODS
This study was approved by the Ethics Committee of the Federal University of Rio Grande do Sul. Thirty mesial roots of mandibular molars were used in this study. An initial micro-CT scan was used to select with independent mesiobuccal and mesiolingual canals using a desktop X-ray microfocus CT scanner (SkyScan 1174v2; Bruker-microCT, Kontich, Belgium). The scanning procedures were performed using the following parameters: 50 kV scanned at a time. Scans with 1304x1024 pixels were obtained with acquisition intervals of 1° over a total of 360° rotation. Thus, data were recorded for tooth selection, and the sequences of scans were reconstructed using the NReconv1. 6 Paul, MN, USA) and the specimens were stored at 100% humidity and 37°C for one week to allow the sealer to set. Then, the specimens were scanned (Micro-CT #1) as described above. ProTaper Retreatment instruments (Dentsply Maillefer, Ballaigues, Switzerland) were used with 500 rpm and 2 N/cm of torque for removal of the canals were irrigated with 2 mL of 2.5% NaOCl. After using the ProTaper Retreatment instruments, specimens were allocated into six groups according or absence of an isthmus (n=5): -Conventional -Continuous irrigation with 10 mL of 2.5% NaOCl in each canal for 1 minute. 0.5 mL of 2.5% NaOCl. Next, the solution was activated using passive ultrasonic irrigation with a size 20 Paulo, SP, Brazil). The ultrasonic tip was passively inserted into root canals 2 mm short of the WL. PUI was performed at 40 kHz for 20 seconds in each canal. The PUI protocol was repeated twice with 2 mL of 2.5% NaOCl per canal each time. Finally, continuous irrigation was performed with 6 mL of 2.5% NaOCl. The total volume of NaOCl was 10 mL in each root canal.
-PUI/orange oil -0.5 mL of orange oil was used in each canal (Biodinâmica, Paraná, PR, Brazil). Next, the PUI protocol was performed as described above, using three activations of the solvent for 20 seconds in each root canal. Between activations, the solvent was refreshed. Finally, each canal was continuously irrigated with 6 mL of 2.5% NaOCl. Canals were dried with absorbent paper points and new micro-CT scans were performed (Micro-CT #2).
In groups that received PUI, the movements of ultrasonic tip were performed toward the isthmus.
Canals were re-prepared with X2 and X3 ProTaper Next instruments (Dentsply Maillefer, Ballaigues, Switzerland) at all WLs with 300 rpm and 2 N/cm of torque. After using these instruments, Finally, specimens were subjected to the same irrigation procedures previously described. Micro-CT #3 was performed to quantify the volume of the The amount of remaining filling material was volumetrically analyzed using the CTscan v1.11.10.0 (Brucker-microCT, Kontich, Belgium) software. The initial image was analyzed by limiting the region of interest (ROI) of the specimen with the CTscan software, and the new ROI data were saved in a separate folder. Then, the set of sample Figure 1-A, B and C represent specimens from the Conventional group; D, E and F from the PUI/NaOCl group; and data was opened and the binary value was adjusted according to the raw images. The value was recorded and used as a parameter for Micro-CT #2 and Micro-CT #3. Finally, a 3D plug-in analysis was performed to quantitatively measure the amount of overall volume (mm 3 ) from the 3D image of binary selected objects (white color). For each sample, the volume was calculated at three root levels: 1 to 3 mm, 3 to 5 mm, and 5 to 7 mm from the apex. All measurements and analyses were performed by the same operator who was blinded from the irrigating protocols.

Statistical analysis
Statistical analysis was performed using the SPSS 16.0 software (SPSS Co., Chicago, IL, USA). The Shapiro-Wilk test showed normal distribution of the data (P>0.05). Therefore, the volume of remaining filling material was analyzed using parametric tests. The repeated measures ANOVA and Tukey's post-hoc tests were used to compare The comparison of the irrigation protocols was performed using a one-factor ANOVA test. To assess if the presence or absence of an isthmus impairs t-test and the repeated measures ANOVA test were used. The level

RESULTS
experimental group was assessed. No differences were observed between Conventional, PUI/NaOCl and PUI/orange oil groups at any of the root levels analyzed (P>0.05). After Micro-CT #2, all groups volume (P<0.05). However, no differences between them were observed (P<0.05). When the specimens were re-prepared with ProTaper Next instruments and the irrigation protocols were repeated, the After Micro-CT #3, no differences were observed between the irrigating protocols (P>0.05) ( Table  1). Figure 1 shows representative images from Conventional, PUI/NaOCl and PUI/orange oil groups during the three experimental stages: Micro-CT irrigation procedures) and Micro-CT #3 (after repreparation and irrigation procedures).
in specimens that contained an isthmus when compared to those that contained two independent canals (P<0.05) (

DISCUSSION
The goal of root canal retreatment is to remove disinfection 11 . However, there is no technique capable of completely removing gutta-percha and endodontic sealer from root canal walls, especially 31 . Some studies used invasive, destructive and two-dimensional methodologies to assess the 24 . Só, et al. 24 (2008) sectioned the specimens longitudinally to using a dental operating microscope, which makes it impossible to evaluate one sample by different stages of retreatment. On the other hand, micro-CT is a quick, accurate and non-destructive method 9 . Micro-CT enables the visualization of three-dimensional images with a high level of detail. However, the main advantage of micro-CT over other methodologies is that it allows for the acquisition of images of the same sample during different experimental stages 18 .
The aim of this study was to compare the volume irrigation, PUI with NaOCl, and PUI with orange oil. Micro-CT #1 was performed to ensure that the

Conventional, PUI/NaOCl and PUI/orange oil
Cavenago, et al. 7 (2014) evaluated the volume different procedures performed sequentially (i.e., mechanical removal, post-xylene and post-PUI). In contrast to the results of the present study, the after PUI when compared to mechanical removal. It should be noted that those authors performed all retreatment procedures using an operating microscope with 5× magnification. Literature reports that using an operating microscope especially at the cervical and middle thirds of mesial roots of mandibular molars 20 . At the apical portion, such visualization may be impaired due to the degree of curvature. This study also found many endodontic retreatment studies 7,12 . was observed after using ProTaper retreatment instruments and applying the irrigation protocols (Micro-CT #2) (P<0.05). Nevertheless, the mechanical action of the instruments was the volume. On the other hand, neither PUI/NaOCl nor PUI/orange oil protocols were capable of reducing system after re-preparation using X2 and X3 ProTaper Next instruments (P>0.05). Even newly developed instruments are not able to contact all dentinal walls during a root canal preparation 30 . Additionally, Rödig, et al. 19 (2014) observed residual ProTaper Retreatment system, Reciproc instruments and Hedstroem hand files. The ProTaper Next system has a variable taper and rectangular crosssection, and uses an asymmetric rotary motion 17 . The combination of the rectangular cross-section with asymmetric rotary motion results in contact of only two cutting edges of the instrument with dentinal walls. The other two edges move freely, which reduces the torsion on the spirals and, thus, the risk of torsional fractures.
Several solvents have been evaluated for their 4,13,14 . Chloroform, xylene, eucalyptol and orange oil are the most commonly used solvents in endodontic retreatment. The main goal of combining solvents with endodontic instruments. Orange oil was chosen because of its low toxicity and for being able to dissolve gutta-percha, regardless of its concentration. According to Magalhães, et al. 13 (2007) orange oil, eucalyptol and chloroform can similarly dissolve gutta-percha, but less so than xylene. On the other hand, Martos, et al. 14 (2006) found that xylene and orange oil similarly dissolved gutta-percha and three endodontic sealers (i.e., Roekoseal, Endofill and Sealer 26). The main drawback reported concerning solvents is that they form a thin layer of softened gutta-percha and sealer that adheres to the canal walls and hinder its removal 8 .
In this study, PUI/NaOCl and PUI/orange oil were material (P>0.05). This result can be explained by the solvent contacting gutta-percha and forming a paste that adheres to the root canal walls by penetrating into canal irregularities. This impairs rotary instruments, and after re-preparation in the presence and absence of an isthmus. Specimens with an isthmus contained a larger amount of compared to specimens with two independent canals (P<0.05). The penetration of gutta-percha and sealer into the isthmus and irregular areas with such anatomical features. PUI appears to be effective for debris removal because of a phenomenon known as acoustic microstreaming and cavitation, which acts on root canals 1 . Castagna, et al. 6 (2013) found that PUI used with 17% EDTA promoted greater debris removal, especially at the cervical third. In addition, the apical third was the most critical area that required cleaning. Some studies have evaluated the effectiveness of PUI on root canal dressing removal 2,5 . According to Capar, et al. 5  to detach the endodontic sealer from the dentin wall when using PUI 27 . These factors may explain the presence of sealer in the canal walls and anfractuosities after endodontic retreatment.
of narrow and curved canals presents a challenge for clinicians 19 . When centering rotary instruments during re-preparation of the canal it is common to leave unprepared areas within the isthmus and 12 . Because of this, mesial roots of mandibular molars were included in this study. In addition, these roots have an isthmus in 55% of cases 15 . Therefore, 50% of the samples studied were mesial roots with an isthmus and 50% contained independent mesio-buccal and mesiolingual canals. Because no difference was observed among the irrigation protocols, data were pooled to This study model contains some limitations. All procedures were performed in a controlled environment with extracted and crownless teeth that may not be equivalent to an average clinic situation. Additionally, an operating clinical microscope was not used. According to Pécora and Andreana 16 (1993), the use of an operating clinical microscope enhances the visualization of root canal anatomy and improves sanitization and root canal preparation procedures.

CONCLUSIONS
Given the findings of this study and the limitations described, it can be concluded that passive ultrasonic activation of NaOCl and orange oil mesial roots of mandibular molars when compared to conventional irrigation with NaOCl. Additionally, the isthmus is an anatomical obstacle that impairs canals of mandibular molars.