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Efficacy of Combined Use of Different Nickel-Titanium Files on Removing Root Canal Filling Materials
Basic Research—Technology
Efficacy of Combined Use of Different Nickel-Titanium Files on Removing Root Canal Filling Materials € kkaya, DDS, PhD,† Sinan Y€ ur€ uker, MD, PhD,* Melahat G€ orduysus, DDS, PhD,†‡ Selen K€ u¸c u † § Emel Uzuno glu, DDS, PhD, Can Ilgın, MD,* Orhan G€ ulen, DDS, PhD, € Behram Tuncel, DDS, PhD,† and Mehmet Omer G€ orduysus, DDS, PhD†k Abstract Introduction: The aim of this study was to estimate the remaining amount of residual filling materials in root canals after retreatment using ProTaper Universal Retreatment (PTUR) files (Dentsply Maillefer, Ballaigues, Switzerland) alone or with the additional use of the Self-Adjusting File (SAF; ReDent-Nova, Ra’anana, Israel), Reciproc (VDW, Munich, Germany), or Hedstr€om-files (H-file; VDW, Antaeos, Munich, Germany) with volumetric estimation using the stereologic method via cone-beam computed tomographic images. Methods: Forty-eight mandibular premolars with single canals were used. The canals were instrumented with ProTaper rotary instruments up to F4 and filled with gutta-percha and AH26 sealer (Dentsply De Trey, Johnson City, TN). All the samples were placed into the silicone models. Samples were scanned with cone-beam computed tomographic imaging and assigned into 4 groups (n = 12) according to retreatment files: the PTUR system group, the PTUR system plus SAF group, the PTUR system plus Reciproc group, and the PTUR system plus H-file group. The specimens were rescanned after retreatment procedures, and the volume estimations of the remaining filling materials were performed using the stereologic method. Data were analyzed using Kruskal-Wallis and Dunn tests. Results: There was no significant difference among the groups regarding mean percentage volumes of the filling materials before retreatment procedures (P > .05). None of the retreatment procedures provided complete removal of the filling materials. The additional use of the SAF did not significantly improve the removal of filling materials when compared with the PTUR system alone (P > .05). However, the additional use of Reciproc or hand H-files significantly improved the removal of filling materials when compared with the PTUR system alone (P < .05). Conclusions: The additional use of files with different motion kinetics improved the removal of
root canal fillings; however, none of the systems completely removed the root canal filling material from the root canals. (J Endod 2016;-:1–6)
Key Words Reciprocation, root canal retreatment, rotation, self-adjusting file, stereology
N
onsurgical root canal retreatment is indicated in cases of post-treatment disease because intraradicular infection is the major cause of failure in initial endodontic treatment (1). The effective removal of the filling material is an essential part of retreatment procedures to achieve better cleaning and disinfection of the entire root canal system (2). There are several techniques for removing the filling materials from root canals including hand files, burs, and rotary systems (3). Furthermore, new rotary nickeltitanium systems that were specially designed for retreatment procedures have been introduced to the field of endodontics (4–6). The ProTaper Universal Retreatment (PTUR) system (Dentsply Maillefer, Ballaigues, Switzerland) is 1 of these systems and includes 3 retreatment instruments: D1 (30/.09), D2 (25/.08), and D3 (20/ .07). D1 has a cutting tip to facilitate initial penetration into the filling material. D2 and D3 both have noncutting tips and are used to remove material from the middle and apical thirds, respectively (4). Recently, new concepts were introduced in which root canal preparation is accomplished using different motions than rotation (7, 8). Reciproc (VDW, Munich, Germany) is a reciprocating instrumentation system to prepare and clean root canals with only 1 instrument. The reciprocating motion relieves the stress on the instrument by special counterclockwise (cutting action) and clockwise (release of the instrument) movements and, therefore, results in improved resistance to cyclic fatigue of the instrument (9). The system includes 3 sizes of instruments (R25, R40, and R50) to be used according to the initial canal diameter. This instrument is also advocated for retreatment purposes to remove any residual filling material (10). The Self-Adjusting File System (SAF; ReDent-Nova, Ra’anana, Israel) is another new concept in the design and operation motion of endodontic files (8). The SAF is a hollow and flexible file that adapts itself 3-dimensionally to the shape of the root canal, both longitudinally and along the cross section. Therefore, it offers a new potential approach to retreatment procedures because of its scraping motion with simultaneous irrigation and its ability to touch a higher percentage of root canal walls (11). Most of the previous studies used destructive and 2-dimensional methods to determine the amount of remaining filling materials after retreatment procedures (3, 9, 12–14). However, nondestructive analysis through 3-dimensional images
From the *Department of Histology and Embryology, Faculty of Medicine and †Department of Endodontics, Faculty of Dentistry, Hacettepe University, Ankara, Turkey; §Department of Dento-Maxillofacial Radiology, Ministry of Health, Ataturk Public Hospital, Ankara, Turkey; ‡RAK College of Dental Science, RAK Medical and Health Science University, Ras Al Khaimah, United Arab Emirates; and kDepartment of Endodontics, Dental College, Sharjah University, United Arab Emirates. Address requests for reprints to Dr Selen K€uc¸€ukkaya, Department of Endodontics, Faculty of Dentistry, Hacettepe University Sihhiye, Ankara, Turkey, 06100. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2016 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2015.11.019
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Combined Use of Different NiTi Files
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Basic Research—Technology can provide more precise results via volumetric evaluation of dental tissues as well as the root canal filling materials (15). Cone-beam computed tomographic (CBCT) imaging has become available for dental offices because of the reduced costs, dimension, and lower irradiation doses compared with conventional computed tomographic (CT) imaging. It has been shown that CBCT imaging is a reliable, noninvasive measuring tool that can be used to explore in detail the root canal system in all spatial planes (16). Stereology is a methodology for the estimation of cell number, volume, length, and surface area representing 3-dimensional structures from cut sections. Those sections can be physically cut tissue sections or sections from radiologic devices such as CT or magnetic resonance imaging (17). The Cavalieri method is a stereologic measurement of volume via the analysis of sections through 3-dimensional objects and provides a mathematically unbiased estimate of the volume of a structure of arbitrary shape and size. However, no studies to date used the Cavalieri method for the estimation of the remaining filling material in the root canal (18). The aim of this study was to compare the remaining filling material after instrumentation using the PTUR system alone or combined with Reciproc, SAF, and hand files using the Cavalieri method through CBCT images.
transferred to a computer for analysis of the volume of root canal filling of each specimen with stereologic measurement.
Stereologic Measurement The equipment for volume estimation consisted of stereologic software (Stereo Investigator; MBF Bioscience, Williston, VT) and a workstation (IBM Z Pro; IBM, Armonk, NY). The CBCT images were transferred to the stereologic software. Sagittal sections of each specimen were used for volumetric measurements. By using the Cavalieri method, the areas of the sections through the structure of interest were estimated by marker counting. Two markers with a size of 200 mm on both x and y directions were selected for marking empty and filled areas, and each section was marked according to the presence or absence of obturation material (Fig. 1A–D). Then, the software program calculated the total markings for both empty and filled areas and estimated the mathematical measurement of that section. After performing this procedure for all sections of each specimen, measurement was accomplished as total volumes for both empty and filled spaces of specimens according to the following formula (19): X pE taðpÞ Ve : Vf :
Materials and Methods Specimen Preparation After university ethics committee approval (ethics board no: GO13/274), 48 freshly extracted human mandibular premolar teeth with fully formed roots, single straight canals, patency, and no calcification as confirmed radiographically were selected for this study. Debris and soft tissue remnants were cleaned, and the teeth were kept in physiological saline solution at 4 C until experiments. The crowns were then removed with a diamond disc to leave a root length of 16 mm. After gaining access, a #10 K-file was placed into the canal until visible at the apical foramen. The working length (WL) was established 1 mm short of this length. All root canals were instrumented using ProTaper rotary instruments (Dentsply Maillefer) in a crown-down manner. Initially, the cervical and middle portions of the roots were prepared by using SX. Later, S1, S2, F1, F2, F3, and F4 files were sequentially used at the full WL. Between each instrument, apical patency was checked with a #15 K-file, and irrigation was performed with 2 mL 2.5% sodium hypochlorite (NaOCl) solution. After completion of instrumentation, final irrigation was performed with 2 mL 17% EDTA, 2 mL 2.5% NaOCl, and 5 mL distilled water, respectively. The root canals were then dried with paper points and obturated using lateral compaction with a size F4 ProTaper cone with accessory gutta-percha cones and AH 26 sealer (Dentsply DeTrey, Konstanz, Germany). Specimens were radiographed in both the buccolingual and mesiodistal directions to confirm the adequacy of the root canal filling. All specimens were then stored at 37 C and 100% humidity for 2 weeks to allow for full setting of the sealer. All specimens were mounted in silicone models to ensure subsequent and precise repositioning during the scanning procedure each time. A small metal ball with a diameter of 500 mm was also placed into each silicone block for calibration of the stereologic system measurements. A CBCT device (Carestream 9000, 3D; Carestream Dental LLC, Rochester, NY) was used to obtain the section images in the axial, coronal, and sagittal directions at 5 mA, 64 Kvp, and 76-mm thickness (field of view: 56 37 mm, voxel: 76 mm). The images were
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X
pF taðpÞ
P
pE isPthe total number of points that do not touch the root canal filling, pF is the total number of points that touch the root canal filling, t is the mean section thickness, and a(p) is the area related to the point. After the estimation of the total volume, the software also calculated the coefficient of error representing the accuracy of the estimation.
Retreatment Techniques The specimens were taken out from their holes in the silicone model and randomly divided into 4 groups according to the removal techniques of obturation materials from the root canals as follows. 1. The PTUR system group: The PTUR system was used to remove obturation materials from the root canals. The D1 file was used to remove the coronal filling material, the D2 file was used for the middle, and the D3 file was used for removal of the apical filling material with light apical pulses of pressure until the WL was reached and no further filling material could be removed from the root canals. All instruments were used with an endodontic motor (X-Smart, Dentsply Maillefer) with a constant speed of 500 rpm and a torque of 3 Ncm according to the manufacturer’s instructions. 2. The PTUR system plus SAF group: After the described procedure in the PTUR system group, the additional use of the SAF file was performed until reaching the WL to remove the filling residue left in the canal. A 2.0-mm-diameter SAF was operated in the canal using an RDT3-NX head (ReDent-Nova) at 5000 rpm with an amplitude of 0.4 mm. 3. The PTUR system plus Reciproc group: After the described procedure in the PTUR system group, the additional use of the R40 instrument of the Reciproc system (size 40 with a taper of 0.06 over the first apical millimeters) was performed until reaching the WL. The Reciproc file was operated with a torque-controlled endodontic motor (Silver Reciproc, VDW) in the preprogrammed ‘‘RECIPROC ALL’’ mode. The file was used in a reciprocating, slow in-and-out pecking motion with an amplitude of approximately 3 mm. Gentle apical
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Basic Research—Technology
Figure 1. Slices sectioned in the sagittal plane and point counting grid superimposed on the CBCT scans. (A and B) Before retreatment procedures and (C and D) after retreatment procedures. Red markers represent the filling material.
pressure was combined with a brushing motion against the lateral root canal walls. 4. The PTUR system plus H-file group: After the described procedure in PTUR system group, the additional use of #15, 20, 25, 30, 35, and 40 Hedstr€om files (H-file) (VDW, Munich, Germany), was performed until they reached the WL in a circumferential filing motion to remove the filling residue left in the canal. All specimens in each group were irrigated with 2 mL 2.5% NaOCl between each instrument during retreatment procedures. After the completion of instrumentation, final irrigation was performed with 2 mL 17% EDTA, 2 mL 2.5% NaOCl, and 5 mL distilled water, respectively. All the specimens were replaced in their holes in the silicone model, and their image scans were taken a second time in the same condition as mentioned previously. In this study, a single operator (K.S.) performed the initial treatment, another one (U.E.) the retreatment procedures, and a third one (I.C.) the evaluations to reduce interoperator variability. Each inJOE — Volume -, Number -, - 2016
strument was discarded after using 4 times, and no solvent was applied to facilitate material removal in all groups.
Evaluation of the Remaining Filling Material The volumetric amount of remaining root canal filling material in the canal after the retreatment procedures was evaluated using the Cavalieri method as described previously. Statistical Analysis Statistical analysis was performed using the Kruskal-Wallis and Dunn tests at 95% confidence level (P < .05).
Results No statistical difference was observed between the mean percentage volumes of the filling material in groups before the retreatment procedures (P > .05) (Table 1). None of the retreatment procedures used
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Basic Research—Technology TABLE 1. The Percentage Volume of the Filling Material in Groups before the Retreatment Procedures (mean standard deviation)* PTUR
PTUR-SAF
PTUR-Reciproc
PTUR-Hand H-file
90.5% 3.2%
90.1% 3.4%
89.7% 4%
92.1% 3.3%*
PTUR, ProTaper Universal Retreatment; SAF, Self-Adjusting File. *There was no statistical significant difference among groups regarding the volume of root canal filling materials, P > .05.
in this study completely removed the filling material from root canals. The additional use of the SAF did not significantly improve the removal of filling material when compared with the retreatment procedure with the PTUR system alone (P > .05). However, the additional use of Reciproc or hand H-files significantly improved the removal of filling material when compared with the PTUR system alone (P < .05 for both comparisons), (Fig. 2). The coefficient of error for volumetric estimation was between 0.1 and 0.01.
Discussion The main goal of root canal retreatment is to remove filling materials from the root canal because this promotes the effective action of instruments and irrigating solutions on debris and microorganisms responsible for apical periodontitis (20). Although it has not been proven that any remaining material will cause retreatment failure, removing as much filling material as possible would seem essential because infected remnants in the root canal may lead to persistent disease (1). Recently, it has been suggested that the combination of techniques might result in more efficient removal of material from the root canal system (21). New instruments with different motion kinetics designed for effective preparation of root canals have been introduced to the market and also recommended for retreatment purposes (9, 13). This study set out to evaluate the effectiveness of the combination of techniques in removing the filling material from the root canal system and to compare them with the PTUR system, which was specifically designed for retreatment procedures. In the present study, mandibular premolar
teeth with single straight roots and canals were selected, their crowns were removed partially to attain the same WL, and all root canal treatment procedures were the same in all samples to standardize the experimental procedures. According to the results of first scans, no statistical difference was observed between the mean percentage volumes of the filling materials in groups before the retreatment procedures. Thus, this allowed us to compare the amount of remaining filling material more precisely after the second scans because of standardized samples at the beginning of the retreatment procedures. During the retreatment procedures, no solvent was used for the purpose of eliminating a possible confounding factor. According to the results of the second scans, none of the techniques evaluated resulted in complete removal of filling materials from the root canals. This finding is consistent with previous studies (3, 9–11, 14, 21–23). The remaining filling material after the PTUR system was higher than the combination groups. This might be attributed to the fact that all canals were enlarged to a size F4 ProTaper file, which has a tip size of 40, whereas the D3 ProTaper retreatment file has a tip size of 20, which meant the D3 file tip did not engage with the canal walls. However, Marques de Silva et al (24) reported that the additional use of the F4 file after the PTUR system was not enough to obtain statistical difference regarding remaining root canal filling materials compared with the use of the PTUR system alone. The recent introduction of new systems with different motion kinetics has offered new potential approaches to the later stages of root canal filling removal (9, 14). Although there was no significance, the additional use of the SAF system showed better removal of filling material than the PTUR system alone in the present study. This
Figure 2. The mean percentage volume of the remaining filling materials. Different letters indicate statistical significance, P < .05.
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Basic Research—Technology finding is in agreement with a recent study that reported the additional use of the SAF improved the removal of filling material after the retreatment procedure with rotary instruments (11). One explanation may be the ability of the SAF to adapt to the cross section of the root canal and to have a scrubbing effect on the canal walls (14). Because the shape of the root canal system may render it difficult to clean properly, the different motion kinetics of the SAF may have contributed to the results of the present study. However, it must be noted that the SAF cannot be considered as an instrument capable of removing the bulk of root filling material because it is not a penetrating instrument and is too flexible to accomplish such a task (13). In the present study, the other combination group that included the additional use of Reciproc showed significant improvement in the removal of filling material when compared with the PTUR system. Reciprocating systems produce a broader motion in the counterclockwise direction yet shorter in the clockwise direction, keeping the file more centered in the canal (9). This factor may have created a greater contact area between the instrument and filling material, allowing better removal than continuous rotation. Similarly, the additional use of the hand H-file presented significantly better results than the PTUR system alone. The design of the flutes and circumferential movement of the hand H-file may have facilitated gutta-percha removal. Recently, it has been advocated that the use of rotary devices in endodontic retreatment should be followed by hand instrumentation to achieve optimal cleanliness of root canal walls (25). Most recent studies have used micro–computed tomographic (micro-CT) scanning to calculate the volume of residual filling material after retreatment procedures (10, 11, 15, 21, 22). Micro-CT imaging offers a highly accurate, noninvasive, and reproducible technique for 3-dimensional evaluation of the root canal system (10, 15, 21, 22). Despite its numerous advantages, micro-CT scanning requires a long time for analysis, it can be highly costly, and it cannot be used for human imaging in vivo (26). In the present study, the CBCT images were analyzed using a stereologic method named the Cavalieri principle, which provides numeric values expressing precise and unbiased quantitative volumetric measurements of biological structures (27, 28). In this principle, sections of an object that are parallel and of equal thickness are taken, and the volume of the object is calculated by the total number of section areas and thickness of the section. Briefly, the structure of interest is divided into exhaustive serial sections, and then the volume of each section is estimated by the section interval or section thickness. Area estimation on sectional surfaces is generally conducted by superimposing a point grid on the sectional profiles and counting all the points hitting the area of interest. Because every point in the grid represents a unit area, multiplying this area by the total point count yields the total area of the sectional surfaces. When section interval or section thickness is multiplied by sectional area estimation, an unbiased estimate of the volume of the structure is obtained (29). In this principle, analysis can be done using sections in the axial, sagittal, or coronal planes. In the present study, the aim was to calculate the overall remaining material in the root canal system. Therefore, the calculation of the residual material of each specimen was made by using all sections in the sagittal plane regardless of the root canal thirds. It was shown that the plane worked by stereologic methods has no adverse effect on quantitative measurements (29, 30). However, future studies could examine axial sections to calculate the amount of remaining filling material according to root canal thirds. In stereologic studies, it is also important to know the amount of error in calculation. The total amount of error arising from sampling estimation procedures in a stereologic study can be expressed as the coefficient error. Usually, a total JOE — Volume -, Number -, - 2016
coefficient error of less than 0.05 is considered to be adequate for most of the studies to provide accurate quantitative information (27). The coefficient of error in the present study was between 0.1 and 0.01, indicating 90%–99% accuracy for the calculations. The stereologic measurement using the Cavalieri method through CBCT images can be an alternative method to micro-CT analysis from a time and expense perspective. Furthermore, this novel technique has the potential to be used in clinical studies because CBCT imaging is available for human imaging in vivo (16). However, further research is warranted to assess the correlation between the stereologic measurement of CBCT images and micro-CT scanning analysis. Within the limitations of this in vitro study, it can be concluded that the combination techniques resulted in less filling material, but none of the techniques caused complete removal of material. Modern stereologic methodology seems to be a reliable, accurate, and promising method for estimating the remaining volume of residual root canal filling material after retreatment procedures.
Acknowledgments Supported by the Hacettepe University Fund (grant no. 013D05201002). The authors deny any conflicts of interest related to this study.
References 1. Nair PN. On the causes of persistent apical periodontitis: a review. Int Endod J 2006; 39:249–81. 2. Ruddle CJ. Nonsurgical retreatment. J Endod 2004;30:827–45. 3. Zuolo AS, Mello JE Jr, Cunha RS, et al. Efficacy of reciprocating and rotary techniques for removing filling material during root canal retreatment. Int Endod J 2013;46:947–53. 4. Gu LS, Ling JQ, Wei X, Huang XY. Efficacy of ProTaper Universal rotary retreatment system for gutta-percha removal from root canals. Int Endod J 2008;41:288–95. 5. Tasdemir T, Yildirim T, Celik D. Comparative study of removal of current endodontic fillings. J Endod 2008;34:326–9. 6. Takahashi CM, Cunha RS, de Martin AS, et al. In vitro evaluation of the effectiveness of ProTaper universal rotary retreatment system for gutta-percha removal with or without a solvent. J Endod 2009;35:1580–3. 7. Yared G. Canal preparation using only one Ni-Ti rotary instrument: preliminary observations. Int Endod J 2008;41:339–44. 8. Metzger Z, Teperovich E, Zary R, et al. The self-adjusting file (SAF). Part 1: respecting the root canal anatomy–a new concept of endodontic files and its implementation. J Endod 2010;36:679–90. 9. Rios Mde A, Villela AM, Cunha RS, et al. Efficacy of 2 reciprocating systems compared with a rotary retreatment system for gutta-percha removal. J Endod 2014;40:543–6. 10. Fruchi Lde C, Ordinola-Zapata R, Cavenago BC, et al. Efficacy of reciprocating instruments for removing filling material in curved canals obturated with a single-cone technique: a micro-computed tomographic analysis. J Endod 2014;40:1000–4. 11. Keles A, Alcin H, Kamalak A, Versiani MA. Oval-shaped canal retreatment with selfadjusting file: a micro-computed tomography study. Clin Oral Investig 2014;18: 1147–53. 12. Voet KC, Wu MK, Wesselink PR, Shemesh H. Removal of gutta-percha from root canals using the self-adjusting file. J Endod 2012;38:1004–6. 13. Abramovitz I, Relles-Bonar S, Baransi B, Kfir A. The effectiveness of a self-adjusting file to remove residual gutta-percha after retreatment with rotary files. Int Endod J 2012;45:386–92. 14. Keles A, Simsek N, Alcin H, et al. Retreatment of flat-oval root canals with a selfadjusting file: an SEM study. Dent Mater J 2014;33:786–91. 15. Solomonov M, Paque F, Kaya S, et al. Self-adjusting files in retreatment: a highresolution micro-computed tomography study. J Endod 2012;38:1283–7. 16. Michetti J, Maret D, Mallet JP, Diemer F. Validation of cone beam computed tomography as a tool to explore root canal anatomy. J Endod 2010;36:1187–90. 17. Garcia-Finana M, Cruz-Orive LM, Mackay CE, et al. Comparison of MR imaging against physical sectioning to estimate the volume of human cerebral compartments. Neuroimage 2003;18:505–16. 18. Roberts N, Puddephat MJ, McNulty V. The benefit of stereology for quantitative radiology. Br J Radiol 2000;73:679–97.
Combined Use of Different NiTi Files
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Basic Research—Technology 19. Scalize PH, de Sousa LG, Regalo SC, et al. Low-level laser therapy improves bone formation: stereology findings for osteoporosis in rat model. Lasers Med Sci 2015;30:1599–607. 20. Silva EJ, Orlowsky NB, Herrera DR, et al. Effectiveness of rotatory and reciprocating movements in root canal filling material removal. Braz Oral Res 2015;29:1–6. 21. Hammad M, Qualtrough A, Silikas N. Three-dimensional evaluation of effectiveness of hand and rotary instrumentation for retreatment of canals filled with different materials. J Endod 2008;34:1370–3. 22. Rodig T, Reicherts P, Konietschke F, et al. Efficacy of reciprocating and rotary NiTi instruments for retreatment of curved root canals assessed by micro-CT. Int Endod J 2014;47:942–8. 23. Colaco AS, Pai VA. Comparative evaluation of the efficiency of manual and rotary gutta-percha removal techniques. J Endod 2015;41:1871–4. 24. Marques da Silva B, Baratto-Filho F, Leonardi DP, et al. Effectiveness of ProTaper, DRaCe, and Mtwo retreatment files with and without supplementary instruments in the removal of root canal filling material. Int Endod J 2012;45:927–32.
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Y€ur€uker et al.
25. Bueno CE, Delboni MG, de Araujo RA, et al. Effectiveness of rotary and hand files in gutta-percha and sealer removal using chloroform or chlorhexidine gel. Braz Dent J 2006;17:139–43. 26. Nair MK, Nair UP. Digital and advanced imaging in endodontics: a review. J Endod 2007;33:1–6. 27. Gundersen HJ, Jensen EB. The efficiency of systematic sampling in stereology and its prediction. J Microsc 1987;147:229–63. 28. Mayhew TM, Gundersen HJ. If you assume, you can make an ass out of u and me’: a decade of the disector for stereological counting of particles in 3D space. J Anat 1996;188(Pt 1):1–15. 29. Sahin B, Mazonakis M, Akan H, et al. Dependence of computed tomography volume measurements upon section thickness: an application to human dry skulls. Clin Anat 2008;21:479–85. 30. Sahin B, Emirzeoglu M, Uzun A, et al. Unbiased estimation of the liver volume by the Cavalieri principle using magnetic resonance images. Eur J Radiol 2003;47: 164–70.
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Efficacy of Combined Use of Different Nickel-Titanium Files on Removing Root Canal Filling Materials € kkaya, DDS, PhD,† Sinan Y€ ur€ uker, MD, PhD,* Melahat G€ orduysus, DDS, PhD,†‡ Selen K€ u¸c u † § Emel Uzuno glu, DDS, PhD, Can Ilgın, MD,* Orhan G€ ulen, DDS, PhD, € Behram Tuncel, DDS, PhD,† and Mehmet Omer G€ orduysus, DDS, PhD†k Abstract Introduction: The aim of this study was to estimate the remaining amount of residual filling materials in root canals after retreatment using ProTaper Universal Retreatment (PTUR) files (Dentsply Maillefer, Ballaigues, Switzerland) alone or with the additional use of the Self-Adjusting File (SAF; ReDent-Nova, Ra’anana, Israel), Reciproc (VDW, Munich, Germany), or Hedstr€om-files (H-file; VDW, Antaeos, Munich, Germany) with volumetric estimation using the stereologic method via cone-beam computed tomographic images. Methods: Forty-eight mandibular premolars with single canals were used. The canals were instrumented with ProTaper rotary instruments up to F4 and filled with gutta-percha and AH26 sealer (Dentsply De Trey, Johnson City, TN). All the samples were placed into the silicone models. Samples were scanned with cone-beam computed tomographic imaging and assigned into 4 groups (n = 12) according to retreatment files: the PTUR system group, the PTUR system plus SAF group, the PTUR system plus Reciproc group, and the PTUR system plus H-file group. The specimens were rescanned after retreatment procedures, and the volume estimations of the remaining filling materials were performed using the stereologic method. Data were analyzed using Kruskal-Wallis and Dunn tests. Results: There was no significant difference among the groups regarding mean percentage volumes of the filling materials before retreatment procedures (P > .05). None of the retreatment procedures provided complete removal of the filling materials. The additional use of the SAF did not significantly improve the removal of filling materials when compared with the PTUR system alone (P > .05). However, the additional use of Reciproc or hand H-files significantly improved the removal of filling materials when compared with the PTUR system alone (P < .05). Conclusions: The additional use of files with different motion kinetics improved the removal of
root canal fillings; however, none of the systems completely removed the root canal filling material from the root canals. (J Endod 2016;-:1–6)
Key Words Reciprocation, root canal retreatment, rotation, self-adjusting file, stereology
N
onsurgical root canal retreatment is indicated in cases of post-treatment disease because intraradicular infection is the major cause of failure in initial endodontic treatment (1). The effective removal of the filling material is an essential part of retreatment procedures to achieve better cleaning and disinfection of the entire root canal system (2). There are several techniques for removing the filling materials from root canals including hand files, burs, and rotary systems (3). Furthermore, new rotary nickeltitanium systems that were specially designed for retreatment procedures have been introduced to the field of endodontics (4–6). The ProTaper Universal Retreatment (PTUR) system (Dentsply Maillefer, Ballaigues, Switzerland) is 1 of these systems and includes 3 retreatment instruments: D1 (30/.09), D2 (25/.08), and D3 (20/ .07). D1 has a cutting tip to facilitate initial penetration into the filling material. D2 and D3 both have noncutting tips and are used to remove material from the middle and apical thirds, respectively (4). Recently, new concepts were introduced in which root canal preparation is accomplished using different motions than rotation (7, 8). Reciproc (VDW, Munich, Germany) is a reciprocating instrumentation system to prepare and clean root canals with only 1 instrument. The reciprocating motion relieves the stress on the instrument by special counterclockwise (cutting action) and clockwise (release of the instrument) movements and, therefore, results in improved resistance to cyclic fatigue of the instrument (9). The system includes 3 sizes of instruments (R25, R40, and R50) to be used according to the initial canal diameter. This instrument is also advocated for retreatment purposes to remove any residual filling material (10). The Self-Adjusting File System (SAF; ReDent-Nova, Ra’anana, Israel) is another new concept in the design and operation motion of endodontic files (8). The SAF is a hollow and flexible file that adapts itself 3-dimensionally to the shape of the root canal, both longitudinally and along the cross section. Therefore, it offers a new potential approach to retreatment procedures because of its scraping motion with simultaneous irrigation and its ability to touch a higher percentage of root canal walls (11). Most of the previous studies used destructive and 2-dimensional methods to determine the amount of remaining filling materials after retreatment procedures (3, 9, 12–14). However, nondestructive analysis through 3-dimensional images
From the *Department of Histology and Embryology, Faculty of Medicine and †Department of Endodontics, Faculty of Dentistry, Hacettepe University, Ankara, Turkey; §Department of Dento-Maxillofacial Radiology, Ministry of Health, Ataturk Public Hospital, Ankara, Turkey; ‡RAK College of Dental Science, RAK Medical and Health Science University, Ras Al Khaimah, United Arab Emirates; and kDepartment of Endodontics, Dental College, Sharjah University, United Arab Emirates. Address requests for reprints to Dr Selen K€uc¸€ukkaya, Department of Endodontics, Faculty of Dentistry, Hacettepe University Sihhiye, Ankara, Turkey, 06100. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2016 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2015.11.019
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Basic Research—Technology can provide more precise results via volumetric evaluation of dental tissues as well as the root canal filling materials (15). Cone-beam computed tomographic (CBCT) imaging has become available for dental offices because of the reduced costs, dimension, and lower irradiation doses compared with conventional computed tomographic (CT) imaging. It has been shown that CBCT imaging is a reliable, noninvasive measuring tool that can be used to explore in detail the root canal system in all spatial planes (16). Stereology is a methodology for the estimation of cell number, volume, length, and surface area representing 3-dimensional structures from cut sections. Those sections can be physically cut tissue sections or sections from radiologic devices such as CT or magnetic resonance imaging (17). The Cavalieri method is a stereologic measurement of volume via the analysis of sections through 3-dimensional objects and provides a mathematically unbiased estimate of the volume of a structure of arbitrary shape and size. However, no studies to date used the Cavalieri method for the estimation of the remaining filling material in the root canal (18). The aim of this study was to compare the remaining filling material after instrumentation using the PTUR system alone or combined with Reciproc, SAF, and hand files using the Cavalieri method through CBCT images.
transferred to a computer for analysis of the volume of root canal filling of each specimen with stereologic measurement.
Stereologic Measurement The equipment for volume estimation consisted of stereologic software (Stereo Investigator; MBF Bioscience, Williston, VT) and a workstation (IBM Z Pro; IBM, Armonk, NY). The CBCT images were transferred to the stereologic software. Sagittal sections of each specimen were used for volumetric measurements. By using the Cavalieri method, the areas of the sections through the structure of interest were estimated by marker counting. Two markers with a size of 200 mm on both x and y directions were selected for marking empty and filled areas, and each section was marked according to the presence or absence of obturation material (Fig. 1A–D). Then, the software program calculated the total markings for both empty and filled areas and estimated the mathematical measurement of that section. After performing this procedure for all sections of each specimen, measurement was accomplished as total volumes for both empty and filled spaces of specimens according to the following formula (19): X pE taðpÞ Ve : Vf :
Materials and Methods Specimen Preparation After university ethics committee approval (ethics board no: GO13/274), 48 freshly extracted human mandibular premolar teeth with fully formed roots, single straight canals, patency, and no calcification as confirmed radiographically were selected for this study. Debris and soft tissue remnants were cleaned, and the teeth were kept in physiological saline solution at 4 C until experiments. The crowns were then removed with a diamond disc to leave a root length of 16 mm. After gaining access, a #10 K-file was placed into the canal until visible at the apical foramen. The working length (WL) was established 1 mm short of this length. All root canals were instrumented using ProTaper rotary instruments (Dentsply Maillefer) in a crown-down manner. Initially, the cervical and middle portions of the roots were prepared by using SX. Later, S1, S2, F1, F2, F3, and F4 files were sequentially used at the full WL. Between each instrument, apical patency was checked with a #15 K-file, and irrigation was performed with 2 mL 2.5% sodium hypochlorite (NaOCl) solution. After completion of instrumentation, final irrigation was performed with 2 mL 17% EDTA, 2 mL 2.5% NaOCl, and 5 mL distilled water, respectively. The root canals were then dried with paper points and obturated using lateral compaction with a size F4 ProTaper cone with accessory gutta-percha cones and AH 26 sealer (Dentsply DeTrey, Konstanz, Germany). Specimens were radiographed in both the buccolingual and mesiodistal directions to confirm the adequacy of the root canal filling. All specimens were then stored at 37 C and 100% humidity for 2 weeks to allow for full setting of the sealer. All specimens were mounted in silicone models to ensure subsequent and precise repositioning during the scanning procedure each time. A small metal ball with a diameter of 500 mm was also placed into each silicone block for calibration of the stereologic system measurements. A CBCT device (Carestream 9000, 3D; Carestream Dental LLC, Rochester, NY) was used to obtain the section images in the axial, coronal, and sagittal directions at 5 mA, 64 Kvp, and 76-mm thickness (field of view: 56 37 mm, voxel: 76 mm). The images were
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X
pF taðpÞ
P
pE isPthe total number of points that do not touch the root canal filling, pF is the total number of points that touch the root canal filling, t is the mean section thickness, and a(p) is the area related to the point. After the estimation of the total volume, the software also calculated the coefficient of error representing the accuracy of the estimation.
Retreatment Techniques The specimens were taken out from their holes in the silicone model and randomly divided into 4 groups according to the removal techniques of obturation materials from the root canals as follows. 1. The PTUR system group: The PTUR system was used to remove obturation materials from the root canals. The D1 file was used to remove the coronal filling material, the D2 file was used for the middle, and the D3 file was used for removal of the apical filling material with light apical pulses of pressure until the WL was reached and no further filling material could be removed from the root canals. All instruments were used with an endodontic motor (X-Smart, Dentsply Maillefer) with a constant speed of 500 rpm and a torque of 3 Ncm according to the manufacturer’s instructions. 2. The PTUR system plus SAF group: After the described procedure in the PTUR system group, the additional use of the SAF file was performed until reaching the WL to remove the filling residue left in the canal. A 2.0-mm-diameter SAF was operated in the canal using an RDT3-NX head (ReDent-Nova) at 5000 rpm with an amplitude of 0.4 mm. 3. The PTUR system plus Reciproc group: After the described procedure in the PTUR system group, the additional use of the R40 instrument of the Reciproc system (size 40 with a taper of 0.06 over the first apical millimeters) was performed until reaching the WL. The Reciproc file was operated with a torque-controlled endodontic motor (Silver Reciproc, VDW) in the preprogrammed ‘‘RECIPROC ALL’’ mode. The file was used in a reciprocating, slow in-and-out pecking motion with an amplitude of approximately 3 mm. Gentle apical
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Figure 1. Slices sectioned in the sagittal plane and point counting grid superimposed on the CBCT scans. (A and B) Before retreatment procedures and (C and D) after retreatment procedures. Red markers represent the filling material.
pressure was combined with a brushing motion against the lateral root canal walls. 4. The PTUR system plus H-file group: After the described procedure in PTUR system group, the additional use of #15, 20, 25, 30, 35, and 40 Hedstr€om files (H-file) (VDW, Munich, Germany), was performed until they reached the WL in a circumferential filing motion to remove the filling residue left in the canal. All specimens in each group were irrigated with 2 mL 2.5% NaOCl between each instrument during retreatment procedures. After the completion of instrumentation, final irrigation was performed with 2 mL 17% EDTA, 2 mL 2.5% NaOCl, and 5 mL distilled water, respectively. All the specimens were replaced in their holes in the silicone model, and their image scans were taken a second time in the same condition as mentioned previously. In this study, a single operator (K.S.) performed the initial treatment, another one (U.E.) the retreatment procedures, and a third one (I.C.) the evaluations to reduce interoperator variability. Each inJOE — Volume -, Number -, - 2016
strument was discarded after using 4 times, and no solvent was applied to facilitate material removal in all groups.
Evaluation of the Remaining Filling Material The volumetric amount of remaining root canal filling material in the canal after the retreatment procedures was evaluated using the Cavalieri method as described previously. Statistical Analysis Statistical analysis was performed using the Kruskal-Wallis and Dunn tests at 95% confidence level (P < .05).
Results No statistical difference was observed between the mean percentage volumes of the filling material in groups before the retreatment procedures (P > .05) (Table 1). None of the retreatment procedures used
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Basic Research—Technology TABLE 1. The Percentage Volume of the Filling Material in Groups before the Retreatment Procedures (mean standard deviation)* PTUR
PTUR-SAF
PTUR-Reciproc
PTUR-Hand H-file
90.5% 3.2%
90.1% 3.4%
89.7% 4%
92.1% 3.3%*
PTUR, ProTaper Universal Retreatment; SAF, Self-Adjusting File. *There was no statistical significant difference among groups regarding the volume of root canal filling materials, P > .05.
in this study completely removed the filling material from root canals. The additional use of the SAF did not significantly improve the removal of filling material when compared with the retreatment procedure with the PTUR system alone (P > .05). However, the additional use of Reciproc or hand H-files significantly improved the removal of filling material when compared with the PTUR system alone (P < .05 for both comparisons), (Fig. 2). The coefficient of error for volumetric estimation was between 0.1 and 0.01.
Discussion The main goal of root canal retreatment is to remove filling materials from the root canal because this promotes the effective action of instruments and irrigating solutions on debris and microorganisms responsible for apical periodontitis (20). Although it has not been proven that any remaining material will cause retreatment failure, removing as much filling material as possible would seem essential because infected remnants in the root canal may lead to persistent disease (1). Recently, it has been suggested that the combination of techniques might result in more efficient removal of material from the root canal system (21). New instruments with different motion kinetics designed for effective preparation of root canals have been introduced to the market and also recommended for retreatment purposes (9, 13). This study set out to evaluate the effectiveness of the combination of techniques in removing the filling material from the root canal system and to compare them with the PTUR system, which was specifically designed for retreatment procedures. In the present study, mandibular premolar
teeth with single straight roots and canals were selected, their crowns were removed partially to attain the same WL, and all root canal treatment procedures were the same in all samples to standardize the experimental procedures. According to the results of first scans, no statistical difference was observed between the mean percentage volumes of the filling materials in groups before the retreatment procedures. Thus, this allowed us to compare the amount of remaining filling material more precisely after the second scans because of standardized samples at the beginning of the retreatment procedures. During the retreatment procedures, no solvent was used for the purpose of eliminating a possible confounding factor. According to the results of the second scans, none of the techniques evaluated resulted in complete removal of filling materials from the root canals. This finding is consistent with previous studies (3, 9–11, 14, 21–23). The remaining filling material after the PTUR system was higher than the combination groups. This might be attributed to the fact that all canals were enlarged to a size F4 ProTaper file, which has a tip size of 40, whereas the D3 ProTaper retreatment file has a tip size of 20, which meant the D3 file tip did not engage with the canal walls. However, Marques de Silva et al (24) reported that the additional use of the F4 file after the PTUR system was not enough to obtain statistical difference regarding remaining root canal filling materials compared with the use of the PTUR system alone. The recent introduction of new systems with different motion kinetics has offered new potential approaches to the later stages of root canal filling removal (9, 14). Although there was no significance, the additional use of the SAF system showed better removal of filling material than the PTUR system alone in the present study. This
Figure 2. The mean percentage volume of the remaining filling materials. Different letters indicate statistical significance, P < .05.
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Basic Research—Technology finding is in agreement with a recent study that reported the additional use of the SAF improved the removal of filling material after the retreatment procedure with rotary instruments (11). One explanation may be the ability of the SAF to adapt to the cross section of the root canal and to have a scrubbing effect on the canal walls (14). Because the shape of the root canal system may render it difficult to clean properly, the different motion kinetics of the SAF may have contributed to the results of the present study. However, it must be noted that the SAF cannot be considered as an instrument capable of removing the bulk of root filling material because it is not a penetrating instrument and is too flexible to accomplish such a task (13). In the present study, the other combination group that included the additional use of Reciproc showed significant improvement in the removal of filling material when compared with the PTUR system. Reciprocating systems produce a broader motion in the counterclockwise direction yet shorter in the clockwise direction, keeping the file more centered in the canal (9). This factor may have created a greater contact area between the instrument and filling material, allowing better removal than continuous rotation. Similarly, the additional use of the hand H-file presented significantly better results than the PTUR system alone. The design of the flutes and circumferential movement of the hand H-file may have facilitated gutta-percha removal. Recently, it has been advocated that the use of rotary devices in endodontic retreatment should be followed by hand instrumentation to achieve optimal cleanliness of root canal walls (25). Most recent studies have used micro–computed tomographic (micro-CT) scanning to calculate the volume of residual filling material after retreatment procedures (10, 11, 15, 21, 22). Micro-CT imaging offers a highly accurate, noninvasive, and reproducible technique for 3-dimensional evaluation of the root canal system (10, 15, 21, 22). Despite its numerous advantages, micro-CT scanning requires a long time for analysis, it can be highly costly, and it cannot be used for human imaging in vivo (26). In the present study, the CBCT images were analyzed using a stereologic method named the Cavalieri principle, which provides numeric values expressing precise and unbiased quantitative volumetric measurements of biological structures (27, 28). In this principle, sections of an object that are parallel and of equal thickness are taken, and the volume of the object is calculated by the total number of section areas and thickness of the section. Briefly, the structure of interest is divided into exhaustive serial sections, and then the volume of each section is estimated by the section interval or section thickness. Area estimation on sectional surfaces is generally conducted by superimposing a point grid on the sectional profiles and counting all the points hitting the area of interest. Because every point in the grid represents a unit area, multiplying this area by the total point count yields the total area of the sectional surfaces. When section interval or section thickness is multiplied by sectional area estimation, an unbiased estimate of the volume of the structure is obtained (29). In this principle, analysis can be done using sections in the axial, sagittal, or coronal planes. In the present study, the aim was to calculate the overall remaining material in the root canal system. Therefore, the calculation of the residual material of each specimen was made by using all sections in the sagittal plane regardless of the root canal thirds. It was shown that the plane worked by stereologic methods has no adverse effect on quantitative measurements (29, 30). However, future studies could examine axial sections to calculate the amount of remaining filling material according to root canal thirds. In stereologic studies, it is also important to know the amount of error in calculation. The total amount of error arising from sampling estimation procedures in a stereologic study can be expressed as the coefficient error. Usually, a total JOE — Volume -, Number -, - 2016
coefficient error of less than 0.05 is considered to be adequate for most of the studies to provide accurate quantitative information (27). The coefficient of error in the present study was between 0.1 and 0.01, indicating 90%–99% accuracy for the calculations. The stereologic measurement using the Cavalieri method through CBCT images can be an alternative method to micro-CT analysis from a time and expense perspective. Furthermore, this novel technique has the potential to be used in clinical studies because CBCT imaging is available for human imaging in vivo (16). However, further research is warranted to assess the correlation between the stereologic measurement of CBCT images and micro-CT scanning analysis. Within the limitations of this in vitro study, it can be concluded that the combination techniques resulted in less filling material, but none of the techniques caused complete removal of material. Modern stereologic methodology seems to be a reliable, accurate, and promising method for estimating the remaining volume of residual root canal filling material after retreatment procedures.
Acknowledgments Supported by the Hacettepe University Fund (grant no. 013D05201002). The authors deny any conflicts of interest related to this study.
References 1. Nair PN. On the causes of persistent apical periodontitis: a review. Int Endod J 2006; 39:249–81. 2. Ruddle CJ. Nonsurgical retreatment. J Endod 2004;30:827–45. 3. Zuolo AS, Mello JE Jr, Cunha RS, et al. Efficacy of reciprocating and rotary techniques for removing filling material during root canal retreatment. Int Endod J 2013;46:947–53. 4. Gu LS, Ling JQ, Wei X, Huang XY. Efficacy of ProTaper Universal rotary retreatment system for gutta-percha removal from root canals. Int Endod J 2008;41:288–95. 5. Tasdemir T, Yildirim T, Celik D. Comparative study of removal of current endodontic fillings. J Endod 2008;34:326–9. 6. Takahashi CM, Cunha RS, de Martin AS, et al. In vitro evaluation of the effectiveness of ProTaper universal rotary retreatment system for gutta-percha removal with or without a solvent. J Endod 2009;35:1580–3. 7. Yared G. Canal preparation using only one Ni-Ti rotary instrument: preliminary observations. Int Endod J 2008;41:339–44. 8. Metzger Z, Teperovich E, Zary R, et al. The self-adjusting file (SAF). Part 1: respecting the root canal anatomy–a new concept of endodontic files and its implementation. J Endod 2010;36:679–90. 9. Rios Mde A, Villela AM, Cunha RS, et al. Efficacy of 2 reciprocating systems compared with a rotary retreatment system for gutta-percha removal. J Endod 2014;40:543–6. 10. Fruchi Lde C, Ordinola-Zapata R, Cavenago BC, et al. Efficacy of reciprocating instruments for removing filling material in curved canals obturated with a single-cone technique: a micro-computed tomographic analysis. J Endod 2014;40:1000–4. 11. Keles A, Alcin H, Kamalak A, Versiani MA. Oval-shaped canal retreatment with selfadjusting file: a micro-computed tomography study. Clin Oral Investig 2014;18: 1147–53. 12. Voet KC, Wu MK, Wesselink PR, Shemesh H. Removal of gutta-percha from root canals using the self-adjusting file. J Endod 2012;38:1004–6. 13. Abramovitz I, Relles-Bonar S, Baransi B, Kfir A. The effectiveness of a self-adjusting file to remove residual gutta-percha after retreatment with rotary files. Int Endod J 2012;45:386–92. 14. Keles A, Simsek N, Alcin H, et al. Retreatment of flat-oval root canals with a selfadjusting file: an SEM study. Dent Mater J 2014;33:786–91. 15. Solomonov M, Paque F, Kaya S, et al. Self-adjusting files in retreatment: a highresolution micro-computed tomography study. J Endod 2012;38:1283–7. 16. Michetti J, Maret D, Mallet JP, Diemer F. Validation of cone beam computed tomography as a tool to explore root canal anatomy. J Endod 2010;36:1187–90. 17. Garcia-Finana M, Cruz-Orive LM, Mackay CE, et al. Comparison of MR imaging against physical sectioning to estimate the volume of human cerebral compartments. Neuroimage 2003;18:505–16. 18. Roberts N, Puddephat MJ, McNulty V. The benefit of stereology for quantitative radiology. Br J Radiol 2000;73:679–97.
Combined Use of Different NiTi Files
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Basic Research—Technology 19. Scalize PH, de Sousa LG, Regalo SC, et al. Low-level laser therapy improves bone formation: stereology findings for osteoporosis in rat model. Lasers Med Sci 2015;30:1599–607. 20. Silva EJ, Orlowsky NB, Herrera DR, et al. Effectiveness of rotatory and reciprocating movements in root canal filling material removal. Braz Oral Res 2015;29:1–6. 21. Hammad M, Qualtrough A, Silikas N. Three-dimensional evaluation of effectiveness of hand and rotary instrumentation for retreatment of canals filled with different materials. J Endod 2008;34:1370–3. 22. Rodig T, Reicherts P, Konietschke F, et al. Efficacy of reciprocating and rotary NiTi instruments for retreatment of curved root canals assessed by micro-CT. Int Endod J 2014;47:942–8. 23. Colaco AS, Pai VA. Comparative evaluation of the efficiency of manual and rotary gutta-percha removal techniques. J Endod 2015;41:1871–4. 24. Marques da Silva B, Baratto-Filho F, Leonardi DP, et al. Effectiveness of ProTaper, DRaCe, and Mtwo retreatment files with and without supplementary instruments in the removal of root canal filling material. Int Endod J 2012;45:927–32.
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25. Bueno CE, Delboni MG, de Araujo RA, et al. Effectiveness of rotary and hand files in gutta-percha and sealer removal using chloroform or chlorhexidine gel. Braz Dent J 2006;17:139–43. 26. Nair MK, Nair UP. Digital and advanced imaging in endodontics: a review. J Endod 2007;33:1–6. 27. Gundersen HJ, Jensen EB. The efficiency of systematic sampling in stereology and its prediction. J Microsc 1987;147:229–63. 28. Mayhew TM, Gundersen HJ. If you assume, you can make an ass out of u and me’: a decade of the disector for stereological counting of particles in 3D space. J Anat 1996;188(Pt 1):1–15. 29. Sahin B, Mazonakis M, Akan H, et al. Dependence of computed tomography volume measurements upon section thickness: an application to human dry skulls. Clin Anat 2008;21:479–85. 30. Sahin B, Emirzeoglu M, Uzun A, et al. Unbiased estimation of the liver volume by the Cavalieri principle using magnetic resonance images. Eur J Radiol 2003;47: 164–70.
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