Basic Research—Technology

Supplementary Steps for Removing Hard TissueDebris from Isthmus-containing Canal Systems

Ali Keles, PhD,* Hatice Alcin, PhD,† Manoel D. Sousa-Neto, PhD,‡ and Marco A. Versiani, PhD‡

Abstract

Significance

In infected canals, hard tissue debris may containmicroorganisms and serve as a nidus for root canalreinfection. Itmayalso interferewithdisinfectionbypreventing irrigant flow. Therefore, irrigation ad-juncts for debris removal after root canal prepara-tion are of the utmost importance.

Introduction: The purpose of this ex vivo study was toevaluate the percentage reduction of accumulated hardtissue debris (AHTD) in the mesial root canal system ofmandibular molars under different final irrigation regi-mens by means of micro–computed tomographic imag-ing. Methods: Sixty curved mesial roots of mandibularmolars with 2 independent canals joint apically by anisthmus (Vertucci type II) were selected. Specimenswere scanned at a resolution of 12.5 mm, anatomicallymatched, and distributed into 3 groups (n = 20) accord-ing to the preparation protocol: Self-Adjusting File (SAF;ReDent Nova, Ra’anana, Israel), Reciproc (VDW GmbH,Munich, Germany), and Revo-S (Micro-Mega, Besancon,France) systems. Then, each group was subdivided into2 subgroups (n = 10) according to the final irrigationprotocol with the SAF or EndoVac system (Discus Dental,Culver City, CA). The percentage volume and percentagereduction of AHTD after root canal preparation and finalirrigation protocols were statistically compared using 1-way analysis of variance, the paired sample and the in-dependent Student’s t tests. The level of significancewas set at 5%. Results: Within groups, the mean per-centage volume was significantly reduced after the finalirrigation procedures in either the SAF (from 1.52%–1.78% to 1.01%–1.20%) or EndoVac (from 2.11%–2.23% to 1.31%–1.52%) subgroups (P < .05). In theexperimental groups, the mean percentage reductionof AHTD ranged from 29.15%–39.90% after the irriga-tion protocols, with no statistical difference betweengroups (P > .05). Conclusions: None of the irrigationapproaches succeeded in rendering the mesial rootcanal system free of AHTD. A similar percentage reduc-tion of AHTD was achieved after final irrigation proto-cols using either the SAF or EndoVac system. (J Endod2016;-:1–6)

Key WordsHard tissue debris, isthmus, mandibular molar, mesialcanal, micro–computed tomography, root canal irriga-tion

From the *Department of Endodontics, Faculty of Dentistry, OnRestorative Dentistry, Dental School of Ribeirao Preto, University of

Address requests for reprints to Dr Ali Keles, Ondokuz Mayıs Unalikeles29@yahoo.com0099-2399/$ - see front matter

Copyright ª 2016 American Association of Endodontists.http://dx.doi.org/10.1016/j.joen.2016.07.025

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Endodontic treatmentoutcome is dependent

on successful intracanalinfection control by meansof using effective chemo-mechanical protocols (1,2). With the advent ofnickel-titanium rotaryand reciprocating instru-

ments, several shortcomings of the conventional preparation procedures that couldadversely affect the endodontic therapy were overcome (3, 4). However, studiesusing high-resolution micro–computed tomographic (micro-CT) technology haveshown that debris created by the cutting action of instruments on dentin duringmechan-ical preparation may be packed into the anatomic complexities of the root canal systempreventing the hydrodynamic action of irrigant flow (5–13). Consequently, disinfectionprocedures could be compromised, and persistent microorganisms in these areas maydevelop or maintain the apical periodontitis (14).

In the last years, several efforts have been expended toward the development ofadjunctive approaches to enhance root canal disinfection (15). Among the supplemen-tary approaches mostly used to drive irrigants into the anatomic complexities of the rootcanal system are the sonic devices, ultrasonic techniques, and the EndoVac System(Discus Dental, Culver City, CA) (7, 13, 16). The Self-Adjusting File (SAF; ReDentNova, Ra’anana, Israel) has also been proven to be effective in the reduction of accu-mulated hard tissue debris (AHTD) during or after root canal preparation (8, 11, 17).Despite the fact that recent studies have shown the effectiveness of these approaches aspotential irrigation adjuncts for debris removal after root canal preparation (7, 17),comprehensive knowledge regarding the activation of irrigants in different finalirrigation protocols in order to remove hard tissue debris from the isthmus area isstill lacking. Therefore, this ex vivo study aimed to evaluate the percentagereduction of AHTD in isthmus-containing mesial root canals of mandibular molars un-der different final irrigation regimens by means of micro-CT imaging. The null hypoth-esis was that there is no difference in the reduction of AHTD among the tested irrigationprotocols.

Materials and MethodsSample Size Estimation

The sample size was calculated after the effect size estimation of the percentagevolume of AHTD as reported by Paqu�e et al (8). In that study, the percentage volumeof AHTD after SAF preparation was 1.7%. An a priori analysis of variance (ANOVA)

dokuz Mayıs University, Samsun, Turkey; †Private Practice, Ankara, Turkey; and ‡Department ofS~ao Paulo, Ribeir~ao Preto, S~ao Paulo, Brazil.iversity, Faculty of Dentistry, Department of Endodontics, Samsun 55139, Turkey. E-mail address:

Hard Tissue Removal after Irrigation 1

Basic Research—Technology

(fixed effects, omnibus, 1-way) was selected from the F test family inG*Power 3.1.7 software for Macintosh (Heinrich Heine, Universit€atD€usseldorf, Dusseldorf, Germany).

Tooth Specimen SelectionAfter local research ethics committee approval (protocol no.

2013/145), 250 mesial roots of mandibular molars were obtainedfrom a pool of teeth. Gender and age of the patients were unknown.In order to prevent the introduction of confounding variables, distalroots were sectioned, and teeth were decoronated �3 mm above thecementoenamel junction. Each mesial root was initially inspectedwith the aid of a stereomicroscope under 12�magnification and radio-graphed in both the buccolingual and mesiodistal directions to detectany possible root canal obstruction. The inclusion criteria comprisedonly teeth presenting mesial roots with moderate curvature (15�–20�) and a fully formed apex. Exclusion criteria were previous canaltreatment and the presence of dentinal defects at the external aspectof the roots. As a result, 160 mesial roots were selected and imagedseparately at an isotropic resolution of 12.5mmusing amicro-CT device(SkyScan 1172; Bruker-microCT, Kontich, Belgium). The scanner pa-rameters were set at 80 kV, 124 mA, 180� rotation around the verticalaxis, a rotation step of 0.48�, and a camera exposure time of 620 mil-liseconds using a 0.5-mm-thick aluminum filter. Then, 60 moderatelycurved and fully formed mesial roots of mandibular molars with 2 in-dependent canals joint apically by an isthmus (Vertucci type II config-uration) were selected.

The acquired projection images were reconstructed (NReconv.1.6.9 software, Bruker-microCT), resulting in the acquisition of1000 to 1200 transverse cross sections per root. The volume of interestwas selected extending from the furcation level to the apex, set by inte-gration of all cross sections. For the purpose of this study, the region ofinterest in each slice comprised the area of the mesial canals and theisthmus. Preoperative 3-dimensional models of the mesial root canalsystems were rendered (CTVol v.2.2.1, Bruker micro-CT), andmorphologic parameters of the isthmus area (length, volume, and sur-face area) and the entire root canal system (volume and surface area)were calculated (CTAn v.1.14.4 software, Bruker-microCT). Aiming toenhance the internal validity of the experiment, the mesial canals werematched to create 20 groups of 3 based on the morphologic aspects ofthe canal. Then, 1 root from each group was randomly assigned to 1 ofthe 3 experimental groups (n = 20) according to the preparation pro-tocol: SAF, Reciproc (VDW GmbH, Munich, Germany), and Revo-S (Mi-cro-Mega, Besancon, France) systems. After checking the normality

TABLE 1. Mean and Range Values of the Baseline Parameters (length, volume, surfaMandibular First Molars Evaluated before and after Preparation with Self-Adjusting

Baseline parameters Self-Adjusting File (n = 20)

Before preparationRoot canals

Volume (mm3) 3.68 � 1.71 (1.56–9.11)Surface area (mm2) 39.14 � 11.08 (24.31–69.23)

IsthmusLength (mm) 4.98 � 1.47 (2.94–8.04)Volume (mm3) 0.041 � 0.06 (0.001–0.25)Surface area (mm2) 7.14 � 8.83 (0.45–34.97)

After preparationRoot canals

Volume (mm3) 7.04 � 1.55 (4.41–10.92)Surface area (mm2) 50.95 � 8.94 (39.27–72.40)

Isthmus% Vol debris 1.86 � 0.96 (0.25–3.93)

2 Keles et al.

assumption (Shapiro-Wilk test) and the equality of variances (Levenetest), the degree of homogeneity (baseline) of the groups with respectto each one of the morphologic parameters was confirmed (1-way AN-OVA test, P > .05) (Table 1).

Root Canal PreparationAfter the apices of the mesial roots were sealed with fast-set epoxy

resin to create a closed-end system (18, 19), canals were accessed, andthe coronal third was sequentially flared with Gates Glidden burs sizes 2and 3 (Dentsply Maillefer, Baillagues, Switzerland) followed byirrigation with 3 mL 2.5% sodium hypochlorite (NaOCl). Patency wasconfirmed by inserting a size 10 K-file (Dentsply Maillefer) throughthe apical foramen before and after completion of canal preparation.For all groups, a glide path was created by scouting a stainless steelsize 15 K-file (Dentsply Maillefer) up to the working length (WL),which was established by deducting 1 mm from the canal length.Then, root canals in each group were prepared according to thefollowing protocols (Fig. 1):

1. The SAF system (n = 20): A 1.5-mm SAF instrument was oper-ated using an RDT3-NX head (ReDent Nova) adapted to a low-speed handpiece (NSK, Tokyo, Japan) operated at 5000 rpmfor 4 minutes. The SAF was gradually inserted into the canaland used to the WL with a pecking motion. Continuous irrigationat a flow rate of 5 mL/min with 2.5% NaOCl was appliedthroughout the procedure using a special irrigation apparatus(VATEA, ReDent-Nova).

2. The Reciproc system (n = 20): The R25 instrument (size 25, 0.08taper over the first 3 mm) was introduced into the canal until resis-tance was felt and then activated in reciprocating motion using theVDW Silver motor (VDW GmbH). The instrument was moved slowlyin the apical direction using a gentle in-and-out pecking motion ofabout 3 mm in amplitude. After 3 pecking motions, the instrumentwas removed from the canal and cleaned. Each time the Reciprocinstrument was removed, 2 mL 2.5% NaOCl was applied using a27-G closed-end tip needle adapted to a disposable plastic syringeand placed 1 mm short of the WL.

3. Revo-S system (n = 20): SC1 (size 25, 0.06 taper), SC2 (size 25,0.04 taper), and SU (size 25, 0.06 taper) Revo-S nickel-titanium ro-tary instruments were used at 300 rpm in a crown-down manner upto the WL using a gentle in-and-out pecking motion. Irrigation fol-lowed the same protocol as in the Reciproc group.

ce area, and percentage volume of debris) of 60 Mesial Root Canal Systems ofFile, Reciproc, and Revo-S Systems

Experimental groups

Reciproc (n = 20) Revo-S (n = 20)

4.01 � 1.29 (1.94–6.52) 3.89 � 1.55 (1.74–7.49)44.58 � 9.54 (25.99–59.41) 43.16 � 11.14 (38.41–81.50)

5.10 � 1.32 (2.66–7.01) 5.07 � 1.52 (2.25-7.80)0.052 � 0.08 (0.001–0.31) 0.047 � 0.07 (0.001–0.22)6.01 � 7.63 (0.62–26.56) 6.91 � 8.35 (0.65–27.67)

7.55 � 1.72 (4.18–10.52) 7.31 � 1.96 (4.87–11.72)56.04 � 10.29 (37.41–69.82) 53.04 � 11.16 (38.41–81.50)

2.01 � 1.31 (0.23–5.65) 1.92 � 1.15 (0.22–4.43)

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Figure 1. A flowchart of the experimental procedures.

Basic Research—Technology

In all groups, preparation was accomplished in 4 minutes using atotal of 20mL 2.5% NaOCl per canal. After preparation, a final rinse with5 mL 17% EDTA (pH = 7.7) delivered at 1 mL/min rate for 5 minutesfollowed by a 5-minute 5-mL rinse with bidistilled water were per-formed in each canal with the tip of the 27-G needle positioned1 mm short of the WL. Then, canals were slightly dried with absorbentpaper points and the roots submitted to a new scan, applying the param-eter settings previously mentioned (Fig. 1).

Postoperative scans were coregistered with their respective preop-erative data sets using the rigid registration module of the 3D Slicer4.3.1 software (available from http://www.slicer.org). Quantificationof AHTD was performed by the difference between the nonpreparedand prepared root canal space using postprocessing procedures withFiji software (Fiji v.1.47n; Fiji, Madison, WI). The sequence of imagesresulting from this operation was further used to identify the AHTDbymeans of morphologic operations (Fig. 2) as described in detail else-where (6). The presence of a material with density similar to dentin inregions previously occupied by air in the nonprepared root canal spacewas considered debris and quantified by the intersection betweenimages before and after canal instrumentation (6, 10). The totalvolume of AHTD was calculated in cubic millimeters (mm3) andexpressed as the percentage of the total canal system volume afterpreparation (%vol) (Table 1).

For the next experimental procedures, a plastic transparent auto-matrix band was adapted to the coronal part of the roots. Then, thespecimens were pair matched in each group with respect to the %vol

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of AHTD acquired in the postpreparation scan, and 1 root from eachpair was randomly assigned to 1 of 2 subgroups (n = 10) accordingto the following supplementary irrigation protocol (Fig. 1):

1. The SAF system (n = 10): The root canals were irrigated in 4 cy-cles of 60 seconds using a 1.5-mm SAF instrument connected tothe VATEA irrigation apparatus. In the first cycle, the SAF was in-serted to the WL and moved up and down with a continuous flowof 2.5% NaOCl irrigant at a flow rate of 5 mL/min. After that, theSAF was removed from the canal, and the irrigant was left undis-turbed for 60 seconds. This cycle was repeated. Then, in the thirdand fourth cycles, the SAF was used with continuous irrigation of5 mL/min 17% EDTA (pH = 7.7) and 5 mL/min 2.5% NaOCl,respectively.

2. The EndoVac system (n = 10): The root canals were irrigated in 4cycles of 60 seconds using the EndoVac System. In the first cycle,macroirrigation with 5 mL 2.5% NaOCl was accomplished using amacrocannula inserted into the canal and moved up and downfrom a point where it bounded at the canal walls. Then, the irrigantwas left undisturbed for 60 seconds. After that, 3 cycles of microir-rigation were followed. The microcannula was inserted 1 mm shortof the WL and held for 60 seconds, whereas 5 mL of the irrigant wasconstantly replenished. The microcannula was removed, and the ir-rigant was left undisturbed for 60 seconds. In the next 2 cycles ofmicroirrigation, 5 mL/min 17% EDTA (pH = 7.7) and 5 mL/min2.5% NaOCl were used, respectively.

Hard Tissue Removal after Irrigation 3

Figure 2. Cross-sectional images of the mesial root of a mandibular molar before and after canal preparation showing the morphologic operations used foridentification of the AHTD. (A) Mesial root canals connected by an isthmus before preparation. (B) Segmentation of the root canal system in A. (C) Mesialroot canals after preparation showing the presence of hard tissue debris in the isthmus area. (D) Segmentation of the areas without hard tissue debris after canalpreparation. (E) Superimposition of the segmented root canal before preparation over the prepared root canal. (F) Segmentation of the isthmus area. (G) Su-perimposition of the segmented area in F over the isthmus. (H) Segmentation of the hard tissue debris in the isthmus area.

Basic Research—Technology

After the supplementary irrigation procedures, the canals wereslightly dried with absorbent paper points, and a final scan was per-formed (Fig. 1). Data sets were registered with their respective counter-parts, and the %vol of AHTD in each canal was calculated. Then, thepercentage reduction (%red) of the AHTD was obtained according tothe following formula: 100 � ([VAF � 100]/VBF), where VBF and VAFare the volume of AHTD before and after irrigation protocols, respec-tively. Color-coded root canal models (green and red colors indicatingpre- and postoperative canal surfaces, respectively) and debris (inblack color) enabled qualitative comparison of the matched root canalsbefore and after the experimental procedures.

Statistical AnalysisData were normally (Shapiro-Wilk test) and homoscedastically

(Levene test) distributed. Therefore, results were expressed as themean and standard deviation and compared between groups using1-way ANOVA and within group by paired sample and independent Stu-dent t tests, with a significance level set at 5% (SPSS v17.0; SPSS Inc,Chicago, IL).

ResultsSample size estimation showed that 9 samples per group were

indicated as the ideal sample size (alpha-type error <0.05 and powerbeta of 99%). Pre- and postoperatively, the degree of homogeneity(baseline) of the groups and subgroups was confirmed regarding theanalyzed morphologic parameters of the root canals as well as the %vol of AHTD after preparation (P > .05) (Table 1).

TABLE 2. Mean Percentage Volume of Accumulated Hard Tissue Debris (%vol) bEndoVac System after Preparation of 60 Mesial Root Canal Systems of Mandibular

Supplementary final irrigation protocol Self-Adjusting File (n

Self-Adjusting File (n = 10)Before irrigation 1.52 � 0.76 (0.25–2After irrigation 1.01 � 0.44 (0.20–1

EndoVac (n = 10)Before irrigation 2.19 � 1.05 (0.80–3After irrigation 1.43 � 0.95 (0.41–3

No statistical difference was observed between the experimental groups before or after supplementary irr

4 Keles et al.

The mean %vol and %red of AHTD evaluated before and after thesupplementary irrigation procedures are detailed in Tables 2 and 3,respectively. Within groups, the mean %vol of AHTD was significantlyreduced after the final irrigation procedures with either SAF (from1.52%–1.78% to 1.01%–1.20%) or EndoVac (from 2.11%–2.23%to 1.31%–1.52%) systems (P < .05) (Table 2). Overall, the mean %red of AHTD ranged from 29.15%–39.90% with no statistical differencebetween the experimental groups (P > .05) (Table 3). Therefore, thenull hypothesis was confirmed. Three-dimensional models of represen-tative mesial root canals in each experimental group show the distribu-tion of the AHTD after chemomechanical preparation andsupplementary irrigation protocols (Fig. 3). Overall, residual debris af-ter final irrigation protocols were mostly observed at the apical thirdand in the isthmus area.

DiscussionIn general, root canal debridement has been evaluated bymeans of

conventional methods such as root sectioning, scanning electron mi-croscopy, and histology (20). More recently, nondestructive micro-CT technology was used as a precise tool for 3-dimensional quantitativeevaluation of hard tissue debris packed into recesses during canal prep-aration (7–10, 12, 13, 17). Evidence from these studies indicates thatdentin particles cut from the canal walls by the endodontic instrumentscan be actively packed into the anatomic complexities of the canalsystem, becoming more resistant to removal (8, 10). Despite the factthat it is difficult to draw reliable conclusions from the literatureregarding the most effective irrigation protocol to overcome this

efore and after Supplementary Irrigation Protocol with Self-Adjusting File andFirst Molars with Self-Adjusting File, Reciproc, and Revo-S Systems

Experimental groups

= 20) Reciproc (n = 20) Revo-S (n = 20)

.54) 1.78 � 0.90 (0.23–3.47) 1.73 � 1.35 (0.22–4.43)

.62) 1.16 � 0.77 (0.04–2.48) 1.20 � 0.96 (0.18–3.22)

.93) 2.23 � 1.66 (0.41–5.65) 2.11 � 0.95 (0.50–3.81)

.59) 1.31 � 1.32 (0.29–4.60) 1.52 � 0.86 (0.31–3.25)

igation protocols (1-way analysis of variance, P > .05).

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TABLE 3. Mean (range) Percentage Reduction of AHTD (%red) after Supplementary Irrigation Protocols with Self-Adjusting File and EndoVac System afterPreparation of 60 Mesial Root Canal Systems of Mandibular Molars with Self-Adjusting File, Reciproc, and Revo-S Systems

Supplementary irrigation protocols

Experimental groups

Self-Adjusting File (n = 20) Reciproc (n = 20) Revo-S (n = 20)

Self-Adjusting File (n = 10) 29.15 � 15.51 (5.56–55.05) 39.90 � 25.30 (11.06–83.33) 27.27 � 21.76 (4.27–75.01)EndoVac (n = 10) 36.99 � 20.56 (8.71–68.18) 36.98 � 27.56 (8.01–88.46) 29.49 � 19.16 (6.77–62.84)

Basic Research—Technology

problem (20, 21), a general agreement exists about the benefits ofactivating the irrigant during and after preparation procedures (22).Overall, previous studies have shown that the use of supplementary irri-gation approaches after canal preparation results in less debris withinthe complexities of the root canal system (7–9, 12, 13, 17), as shown inthe present study.

The SAF is a hollow nickel-titanium latticelike instrument thatscrubs the canal walls by vertical vibrations and allows simultaneousand continuous irrigation throughout the mechanical preparation ofthe root canal (23). To date, only 2 studies attempted to quantifythe volume of hard tissue debris accumulated in the isthmus areaof the mesial roots of mandibular molars after using the SAF system(8, 17). In 1 of them, the SAF was used as a finishing instrumentafter root canal preparation and left 4.3% of the canal volumefilled with AHTD (17). This result was more than twice that re-ported by Paqu�e et al (8) and the results found in the present study,but it may be explained because SAF’s protocol was modified and

Figure 3. Distal views of 6 representative reconstructions of anatomically matchedSAF, Reciproc, and Revo-S systems and after supplementary irrigation protocols w

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applied for only 1 minute in each canal to equalize the experimentalgroups in terms of preparation time and amount of irrigant (17).Therefore, it may be advised that studies using SAF as a supplemen-tary step for root canal cleaning should follow its full protocol, asused herein. However, despite the fact that SAF has been proven effi-cient as a potential irrigation adjunct for debris removal, it must behighlighted that this instrument is unable to remove all AHTD con-tent from the canal because during its use it continues to scrub thecanal walls, producing more dentinal debris. Besides, although SAFconforms to the canal shape, it cannot enter into the isthmus area.Consequently, its efficacy in the present study may be attributed tothe continuous flow with a fresh and fully active irrigantreplacement (23).

The EndoVac system comprises a different irrigation regimen thatinvolves apical negative pressure and uses a master tip to deliver andevacuate the irrigant concomitantly at the pulp chamber level, whereasmicrocannulas are used deeply in the canal (21). In the literature, the

mesial root canal systems before (in green) and after (in red) preparation withith SAF and Endovac. AHTD is depicted in black.

Hard Tissue Removal after Irrigation 5

Basic Research—Technology

comparison of the cleaning and disinfecting effects of EndoVac and con-ventional irrigation reached inconclusive results. Although some studiesshowed superior bacterial elimination and better cleaning using the En-doVac system, other authors found no significant differences betweenthese techniques (20, 21). Recently, 2 studies evaluated the efficacyof EndoVac to remove hard tissue debris accumulated in isthmus-containing mesial root canals of mandibular molars. It was reportedthat the median percentage volume of AHTD was reduced to 2.12%(7) and 3.4% (13) after using the EndoVac system as a final irrigationprotocol, which is in agreement with the present results. However, it isimportant to point out that the root canal configuration of the samplesselected herein and in those studies (Vertucci type II) may favor the ef-ficacy of the EndoVac system when compared with other irrigation ap-proaches. In this type of configuration in which root canals areconnected by an isthmus, the mechanical flushing action of EndoVacproduces a current flow of the irrigant toward the apical third (21)in both root canals simultaneously. Consequently, this constant irrigantflow may displace the hard tissue debris from within the isthmus areafavoring its removal, which may explain these results.

Although a final irrigation protocol with SAF and EndoVac resultedin a significant reduction in the debris content, which can be translatedinto improved cleanliness of the root canal system, its clinical relevanceremains unclear, and further studies are necessary to evaluate its impacton the success rate of the root canal treatment (17). It is also worthmentioning that the experimental groups in this study differed notonly in the mode of irrigant delivery but also in the delivery protocol;the EndoVac system delivers the irrigant into the pulp chamber andnot in the root canal system (16). However, these differences did notseem to negatively influence the results, probably because of the sampledistribution approach used herein, which was based on the morpho-logic aspects of both root canals and the isthmus area (Table 1).This methodological step is paramount to reduce the potential signifi-cant anatomic biases that could interfere with the results, enhancingthe internal validity of the experiment (13, 17, 24).

Despite the findings of this study revealing that the adjunctive irri-gation protocols with SAF or EndoVac systems did not succeed inrendering the isthmus-containing mesial root canal system of mandib-ular molar teeth free of AHTD, their efficacy in reducing the debriscreated by the instruments during the cleaning and shaping procedureswas shown.

AcknowledgmentsSupported by the Scientific and Technological Research Coun-

cil of Turkey-TUBITAK (grant #114S002).The authors deny any conflicts of interest related to this study.

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JOE — Volume -, Number -, - 2016