Infl uence of retreatment in the formation of dentinal microcracks in mandibular molars fi lled with a calcium silicate based sealer

Some studies have evaluated, by micro-CT, the formation of microcracks after chemical-mechanical preparation with different instruments [4-10]. In most of these studies, the formation of new microcracks was not observed, regardless of the type or kinematics of the instrument used (continuous or reciprocating rotation) [4-6,8,10]. De Deus, et al. [5] using micro-CT and stereomicroscopy demonstrated that the defects identi ied in the stereomicroscopy analysis were already present before instrumentation or were absent at both times of the micro-CT analysis, indicating that the formation of defects resulted from the sectioning procedure performed prior to the stereomicroscopy. The studies that found microcracks did not report the use of a software to coregister the image stacks [7,9], which con irms whether the same slice was analyzed and if it was in the same position, whilst this could raise doubts about the real presence of new microcracks [6]. Abstract


Introduction
Vertical root fracture (VRF) is de ined as a severe crack longitudinally along the central axis of the root and may extend from the interior of the root canal to the external surface of the root [1]. In endodontically treated teeth, dentinal defects such as microcracks, may progress to a VRF which can lead to tooth loss [2,3].
Some studies have evaluated, by micro-CT, the formation of microcracks after chemical-mechanical preparation with different instruments [4][5][6][7][8][9][10]. In most of these studies, the formation of new microcracks was not observed, regardless of the type or kinematics of the instrument used (continuous or reciprocating rotation) [4][5][6]8,10]. De Deus, et al. [5] using micro-CT and stereomicroscopy demonstrated that the defects identi ied in the stereomicroscopy analysis were already present before instrumentation or were absent at both times of the micro-CT analysis, indicating that the formation of defects resulted from the sectioning procedure performed prior to the stereomicroscopy. The studies that found microcracks did not report the use of a software to coregister the image stacks [7,9], which con irms whether the same slice was analyzed and if it was in the same position, whilst this could raise doubts about the real presence of new microcracks [6].
Other studies have also evaluated the formation of microcracks after illing procedures [11] and after guttapercha removal during retreatment [12]. These studies did not observe new microcracks after different illing techniques with AH plus sealer [11] and after retreatment with AH26 sealer [12].
The complete removal of the illing material during retreatment is essential for adequate cleaning and disinfection of the root canal system, thus playing an important role in the success of endodontic therapy [13]. Filling material can be removed using heat and instruments with or without gutta-percha solvents [14]. The use of rotary nickel-titanium (NiTi) instruments is safer and faster than the use of manual instruments, reducing operator and patient fatigue [15].
Bioceramic sealers ill most of the properties desired for an ideal root canal sealer: antibacterial properties, biocompatibility and radiopacity [16]. Due to their chemical composition and interaction with dentin walls, some studies raised concern about the dif iculty of their removal during retreatment, owing to the possible formation of dentinal defects [17,18].
The chemical-mechanical preparation and root canal illing using different techniques do not seem to in luence the formation of dentinal microcracks. However, few studies have evaluated the in luence of retreatment on the formation of these defects, particularly in root canals obturated with bioceramic sealers. Therefore, the purpose of this study was to evaluate the formation of dentinal microcracks during illing removal in endodontic retreatment of root canals illed with gutta-percha and Total Fill BC bioceramic sealer.

Sample selection and image acquisition
The present study was approved by the ethics and research committee of University Hospital Clementino Fraga Filho under the protocol number 02235718.0.0000.5257. A total sample size of twelve was found using Wilcoxon signed rank test for one sample case with an α error of 0.05 and a power of 80% (G*Power 3.1). Twenty mandibular molars were atraumatically extracted for clinical reasons and stored in 0.1% thymol solution at 4ºC until use. Teeth with caries, calci ied canal, internal or external resorption were excluded from the study. The teeth were then analyzed by micro-CT to con irm similarity to the anatomy.
A custom-made mold of self-polymerizing resin was created for each tooth to standardize the mounting of the specimen [19]. The teeth were placed in a micro-CT scanner (SkyScan 1173; Bruker, Kontich, Belgium) on a custom aluminum attachment. This base ensured the standardization of the images obtained initially and after the canal obturation and retreatment, as it allowed precise re-placement of the sample inside the scanner. Scanning was performed through 360º rotation with a rotation step of 0.30 using a 1.0-mm thick aluminum ilter, 70-kV energy, 114-mA current, 14.8-mm pixel size, and 21.39-mm resolution.

Root canal treatment
The root surfaces were covered with a light-based silicone molding material to simulate the periodontal ligament and around the specimens were added a self-curing acrylic resin to act as a support. Mesial roots were instrumented with the NiTi K3XF ile system (SybronEndo, Orange, Canada) following the sequence: # 25/.08, # 25/.06 and # 25/.04. Files were used passively and apical enlargement was performed using iles # 25.06 and # 30.04. The pulp chamber was illed with 5.25% sodium hypochlorite (NaOCl) throughout the instrumentation. The canal was irrigated with 3 ml 5.25% NaOCl after each instrument. After instrumentation, all canals were irrigated with 3 ml 17% EDTA for 3 minutes (1 ml/min) and 3 ml 5.25% NaOCl, and dried with FM-size paper points (SybronEndo). Then, root canals were illed with Total Fill gutta-percha cones (FKG, La Chaux-de-Fonds, Switzerland Brasseler) # 30 or # 35 and Total Fill BC Sealer (FKG, La Chauxde-Fonds, Switzerland). The illing was performed according to the manufacturer's recommendations, using the Single Cone Technique. Then, the coronal sealing was performed with cotton and zinc oxide-based temporary material, and the teeth were stored for 7 days in an oven at 37ºC and 100% humidity for the total setting of the sealer. The teeth then underwent a new micro-CT image acquisition.
To remove illing material, a #3 Gates Glidden drill (Dentsply-Maillefer) was used in the cervical 3 mm. The nickeltitanium (NiTi) ProTaper Universal Retreatment (PTUR) (Dentsply-Maillefer) sequence was employed according to the manufacturer's instructions: D1, D2, and D3 were used in the cervical, middle, and apical regions, respectively, without solvents. The removal of the illing material was considered complete when no gutta-percha residue or sealer was observed in the instrument and the working length was reached. After this step, a NiTi ProTaper Next X4 ile (#40.06) (Dentsply-Maillefer ® ) was used up to the working length, followed by irrigation with 5 mL 5.25% NaOCl. The teeth then underwent a new micro-CT image acquisition.

Analysis of dentinal defects
The 3D Slicer v.4.6.2 software (available at http://www. slicer.org) was used to co-register the stacks of images by an automatic overlay process based on the external root contour. Then, cross-sectional images of the mesial roots, from furcation to apex (n = 24,444), were analyzed by 3 experienced endodontists who were trained to identify the presence of dentinal microcracks. To verify the formation of new cracks, the images obtained after the illing were analyzed and compared with the images obtained after the removal of the illing material in the Image J software. Defects were de ined either as visible cracks that extends from the dentinal wall inside the root canal but did not reach the external root surface or visible crack extending from the inside dentinal wall of the root canal to the external root surface.

Statistical analysis
A descriptive statistical analysis was performed using SPSS 25.0 software (IBM Corp. Chicago, IL). The results were expressed as the relative percentage of the number of sections with dentinal microcracks. To compare the relative percentages of dentinal microcracks in the initial, post-illing and after removal of material illing images, Chi-square test was used. The level of statistical signi icance was 5% (SPSS v20.0, Chicago, USA).

Results
A total of 24.444 slices were analyzed. From these slices, 5.67% (n = 1.386) showed some dentinal defects, with 0.51% (n = 125) in the initial images, 2.58% (n = 630) in the postilling images, and 2.58% (n = 630) in the images obtained after the removal of the illing material. There was no statistical difference (p > 0.05) in the relative percentages of dentinal microcracks in the initial, post-illing and retreatment images. When the initial and post-illing images were compared, it was observed that 1.65% (n = 403) of the slices showed new microcracks. All the dentinal defects identi ied in the analysis of the images obtained after the removal of the illing material were already present in the corresponding images obtained after the illing. Therefore, no new microcracks were observed after the removal of the illing material.

Discussion
This study evaluated the presence of dentinal microcracks after the retreatment of mesial roots of mandibular molars illed with gutta-percha and the bioceramic Total Fill BC sealer. The results showed that new microcracks were not formed after the removal of the illing material, and all the dentinal defects identi ied in the analysis of the images obtained after the removal of the illing material were already present in the corresponding images obtained after the illing.
The control for this study was the images obtained by micro-CT after root canal illing. These images were compared with the ones obtained after the retreatment of the canals, since the objective was to evaluate the incidence of dentinal microcracks generated during this procedure. The micro-CT analysis performed previously to the chemical-mechanical preparation of the canals had the objective of verifying the similarity of the root canals in terms of the anatomy. The analysis after the chemical-mechanical preparation was not performed since this analysis was not necessary for the evaluation proposed for the study. Moreover, most of the in vitro studies that evaluated the incidence of microcracks after the instrumentation failed to notice new microcracks related to this procedure. [4][5][6]8,10]. Furthermore, a recent in vivo study con irmed that root canal instrumentation with either rotary or hand instruments did not result in the formation of dentinal microcracks [20].
Rotary instruments are recommended for removing illing material during root canal retreatment because they are fast, safe and ef icient. In the present study, PTUR was selected for its proven effectiveness and safety in the apical extrusion of debris [21,22]. Furthermore, no gutta-percha solvent was used during retreatment, as its use is still controversial in the literature. Although the use of solvent favours the penetration of instruments in the obturation mass, it has been shown by MEV that the dentinal tubules of teeth re-treated with solvents can be obliterated by residues of gutta-percha and sealer, thus making root canal system cleaning dif icult [23].
The results reported herein are in accordance with others that did not observe production of microcracks in micro-CT analysis after chemical-mechanical preparation and root canal illing [4][5][6]8,10]. Our results are also in agreement with Yilmaz et al. [12], who did not observe new microcracks after retreatment of canals illed with gutta-percha and AH26 sealer.
Divergent results were reported by Jani, et al. [24] that found a signi icant increase in the formation of dentinal microcracks after root canal preparation, followed by obturation and retreatment [24]. They showed by micro-CT analysis that all the three root canal therapy procedures generate dentinal defects, with a maximum number registered during retreatment with PTUR ile system. The discrepancy in the results may be explained by the methodological differences, such as sample used, the type and resolution of the microtomograph device and the fact that the teeth were not mounted in a silicone base material to simulate the periodontal ligament. This is an important consideration and a limitation of the in vitro studies. The presence of periodontal ligament and the surrounding alveolar bone plays an essential role in distributing forces, therefore, it is important to either use a soft material to involve the teeth, like it was done in our study, or to perform the experiments using human mandibular or maxillary cadavers [25][26][27]. Furthermore, in vitro conditions without standardization of age and pre-extraction conditions may explain the divergent results. It has been reported that VRF is more common in patients older than 40 years of age, since root dentin in older individuals may exhibit a decrease in strength and resistance to fatigue because of the changes in the microstructure and chemical composition [20].

Conclusion
It is possible to conclude that no formation of dentinal defects was associated with retreatment of root canals illed with gutta-percha and Total Fill BC sealer.