DR.

ÖZGÜR İLKE ATASOY ULUSOY (Orcid ID : 0000-0001-8779-770X)

PROF. TAYFUN ALACAM (Orcid ID : 0000-0002-1456-0223)

Accepted Article
Article type : Original Scientific Article

The effectiveness of various irrigation protocols on organic tissue removal from simulated

internal resorption defects

Ö. İ. Ulusoy1, İ. G. Savur1, T. Alaçam1, B. Çelik2

1
Department of Endodontics, Faculty of Dentistry, and 2Department of Statistics, Faculty of Science,

Gazi University, Ankara, Turkey

Running title: Dissolution capacity of irrigants

Key words: Etidronic acid, sodium hypochlorite, tissue dissolution

Corresponding author:

Özgür İlke Ulusoy

Department of Endodontics, Faculty of Dentistry, Gazi University, 82.Street, 06510 Emek, Ankara,

Turkey

business tel: +90 312 203 41 07

fax: +90 312 223 92 26

e-mail: ilkeatasoy@yahoo.com

This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/iej.12919
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 ABSTRACT

Aim To evaluate the effectiveness of NaOCl, NaOCl-EDTA, and NaOCl+HEBP activated by

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ultrasonics and XP-endo Finisher on organic tissue removal from simulated internal root resorption

cavities.

Methodology The root canals of 144 single-rooted teeth were instrumented. The teeth were split

longitudinally and semicircular cavities were prepared in the canals on each half of the roots. Samples

obtained from ground bovine muscle tissue were weighed and adapted into the semicircular cavities.

The root fragments were reassembled and cemented to create a circular simulated resorption cavity

within the canal. Teeth were divided into twelve groups (n=12) according to the irrigation protocols:

Group1: NaOCl, no activation; Group2: NaOCl, Passive ultrasonic irrigation (PUI); Group3: NaOCl,

XP-endo Finisher; Group4: NaOCl-EDTA, no activation; Group5: NaOCl-EDTA, PUI; Group6:

NaOCl-EDTA, XP-endo Finisher; Group7: NaOCl+HEBP, no activation; Group8: NaOCl+HEBP,

PUI; Group9: NaOCl+HEBP, XP-endo Finisher; Group 10: Distilled water, no activation; Group11:

Distilled water, PUI; Group 12: Distilled water, XP-endo Finisher. Teeth were disassembled and the

tissue samples inside the resorption cavities were weighed. The data was analysed statistically using

two-way ANOVA and Fisher’s LSD tests with a significance level of 0.05.

Results The use of XP-endo Finisher with the experimental solutions resulted in the highest tissue

weight loss compared to the other activation protocols (p<0.001). There was no significant difference

between NaOCl and NaOCl+HEBP in terms of tissue removal.

Conclusion The use of a NaOCl+HEBP mixture activated with XP-endo Finisher was an effective

irrigation regimen for removing simulated organic tissues from artificial internal root resorption

cavities in the root canals of single-rooted teeth.

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 Introduction

Internal root resorption is a chronic inflammatory process leading to a progressive destruction of

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dentine (Patel et al. 2010). The irregular areas of internally resorbed teeth complicate the

chemomechanical preparation and filling of root canals during root canal treatment. Removal of

inflamed pulpal and granulation tissue filling the resorption lacuna is essential in the management of

the internal root resorption cases (Lyroudia et al. 2002)

Sodium hypochlorite (NaOCl) is one of the most commonly used irrigation solution due to its

enhanced antimicrobial activity, and organic tissue dissolution capacity for the elimination of necrotic

and granulation tissues from internal resorption cavities (Cobankara et al. 2010, Haapasalo et al.

2014). However, when applied with a conventional irrigation technique, NaOCl may not reach the

necrotic tissue and debris within the irregularities of the root canal system. Several agitation methods

including ultrasonics, sonics, and lasers have been suggested to increase tissue dissolution and

antimicrobial activity of irrigants particularly within inaccessible areas of the root canal system

(Neelakantan et al. 2015, Conde et al. 2016).

XP-endo Finisher (FKG, La Chaux-de-Fonds, Switzerland) is manufactured to improve the

effectiveness of final irrigation after root canal instrumentation (Bao et al. 2017, FKG 2015). NiTi

Max-Wire (Martensite-Austenite- electropolish-fleX), a special type of alloy used in its fabrication,

increases the flexibility of XP-endo Finisher (FKG 2015). As it can adapt to the root canal walls three

dimensionally, the manufacturer recommends its use during the chemomechanical preparation of root

canals with internal resorption cavities (FKG 2015).

Use of 17% ethylenediaminetetraacetic acid (EDTA) after NaOCl irrigation is the most commonly

applied irrigation protocol during root canal treatment to provide smear layer removal, antimicrobial

disinfection, and necrotic tissue dissolution (Lottanti et al. 2009). However, the combined use of

NaOCl and EDTA may decrease the dissolution capacity and antimicrobial property of NaOCl due to

reduced available chlorine (Grawehr et al. 2003). A1-hydroxyethylidene-1, 1-bisphosphonate

(HEBP), etidronic acid, has previously been shown to cause only a slight dose-dependent decrease in

the available chlorine of NaOCl (Girard et al. 2005). Due to the lack of interaction between these two

irrigants, HEBP and NaOCl have been proposed for use as a single irrigant in the biomechanical

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 preparation of root canals (Zehnder et al. 2005, Tartari et al. 2015). Tartari et al. (2015) reported that

a mixture of etidronate and sodium hypochlorite was able to dissolve organic tissue in a similar way
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to that of NaOCl alone. Furthermore, this combination has been reported to achieve smear layer and

hard tissue debris removal in the root canals (Lottanti et al. 2009, Paque et al. 2012).

The complete removal of inflamed pulp and granulation tissue from internal resorption cavities during

chemomechanical treatment can be difficult because of irregular shape of the root canal. The aim of

this laboratory study was to investigate the effectiveness of NaOCl, NaOCl-EDTA, and

NaOCl+HEBP activated with XP-endo Finisher and ultrasonics on simulated organic tissue removal

from the artificial internal resorption cavities in root canals. The null hypothesis tested was that there

is no difference between NaOCl, NaOCl-EDTA, and NaOCl+HEBP activated using XP-endo Finisher

or ultrasonics regarding organic tissue removal in the internal root resorption cavities.

Materials and Methods

Freshly extracted maxillary anterior teeth with similar dimensions and straight root canals were

selected. Teeth with caries, root cracks, resorptions, immature apex, and more than one root canal

were excluded. Presence of a single root canal was confirmed using radiographic examination. One

hundred and forty-four teeth meeting the criteria were cleaned from calculus and soft tissue remnants,

then stored in 0.1% thymol solution at 4°C prior to the experiment. All teeth were shortened coronally

to a standardized length of 20 mm using diamond fissure burs under water spray. After preparation of

a conventional access cavity, working length was measured 1 mm short of the apical foramen using a

size 15 K-file (Dentsply Sirona Endodontics, Ballaigues, Switzerland). All the root canals were

instrumented using ProTaper Universal rotary instruments (Dentsply Sirona Endodontics) to a master

apical size of F3, then irrigated with 2 mL 2.5% NaOCl after each instrument use. Final irrigation

were performed using 17% EDTA (Wizard, Rehber Chemistry, Istanbul, Turkey) for 1 min. The root

canals were finally washed using distilled water and dried with paper points.

Two longitudinal grooves were created on the buccal and lingual surfaces of the teeth using a

diamond disc. The teeth were split into two halves along their long axis with a chisel and hammer.

Two semicircular cavities with a diameter of 2 mm were prepared in the halves of each root canal

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 using round burs (Figure 1). The diameter of the cavities were measured using digital calipers with an

accuracy of 0.01 mm. The location of the cavities were 8 mm from the apex. One hundred and forty-
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four samples were obtained from bovine muscle tissue that was ground using a meat grinder (PKM

32, Arı Makina, İstanbul, Turkey) used by the food industry. The tissue samples were adapted into the

two semicircular cavities of each root (Figure 2). Then they were replaced and weighed 3 times using

a precision balance with an accuracy of 0.0001 g in an air-tight container (Precisa XB 220A, Dietikon,

Switzerland). Arithmetic mean of three measurements were calculated and recorded as the baseline

value. Then the preweighed tissue samples were again placed in the semicircular cavities. The root

sections including tissue samples were reassembled and cemented together using a glue (Pattex Super

Glue, Henkel Co, Düsseldorf, Germany). Thus, a circular cavity representing an internal resorption

defect was formed. The apices of all roots were sealed with sticky wax to simulate a closed-end canal

model.

The reassembled roots were randomly divided into twelve (n=12) groups according to the irrigation

protocols:

Group 1 (NaOCl, no activation): The root canals were irrigated manually with 6 mL 2.5% NaOCl

using a syringe and 30-G irrigation needle (NaviTip, Ultradent, South Jordan, UT) at a flow rate of

3mL/min without activation. The tip of the needle was placed 1 mm short of the apex and the total

irrigation time was 2 min.

Group 2 (NaOCl, Passive ultrasonic irrigation (PUI)): The root canals were irrigated with 3 mL 2.5%

NaOCl, which was activated for 1 minute using an ultrasonic tip (NSK Various E4D, Nakanishi,

Tochigi, Japan) mounted on an ultrasonic unit (NSK Varios 750) at a power setting of 4. The

ultrasonic tip was placed 2 mm short of the apex. After 1 min, 3 mL freshly prepared irrigation

solution was introduced into the root canals, and activated for 1 min using the same procedure.

Group 3 (NaOCl, XP-endo Finisher): The root canals were flushed with 3 mL 2.5% NaOCl. The

irrigant was activated for 1 min using an XP-endo Finisher file (FKG, La Chaux-de-Fonds,

Switzerland) that was mounted in a torque-controlled endodontic motor (Sybron-Endo, Orange, Ca,

USA) operated at 800 rpm and 1Ncm torque. Briefly, the file inside its plastic tube was cooled using a

cold spray (Endo-Ice, Whaledent, Mahwah, NJ, USA), then it was removed from the tube in rotation

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 mode. The XP-endo Finisher file was inserted into the root canals filled with the irrigant and activated

for 1 min using slow and gentle 7-8 mm lengthwise movements to contact the full length of the canal
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(FKG 2015). A freshly prepared solution was then applied into the root canals and activated for 1 min

using the same procedure.

Group 4 (NaOCl-EDTA, no activation): The root canals were irrigated manually with 3 mL 2.5%

NaOCl for 1 min in the same manner as Group 1. Then 3 mL 17% EDTA (Wizard) was applied into

the root canals without any activation for 1 min.

Group 5 (NaOCl-EDTA, PUI): The root canals were irrigated with 3 mL 2.5% NaOCl, which then

was activated for 1 min using ultrasonics as described in Group 2. Then 3 mL 17% EDTA (Wizard)

was applied into the root canals, and activated using ultrasonic tip for 1 min.

Group 6 (NaOCl-EDTA, XP-endo Finisher): The root canals were filled with 3 mL 2.5% NaOCl and

then activated with XP-endo Finisher for 1 min using the same protocol as in the Group 3. After that,

the root canals were irrigated with 17% EDTA, which was activated by XP-endo Finisher for 1 min.

Group 7 (NaOCl+HEBP, no activation): Sixty percent aqueous HEBP solution, which was obtained

from a commercial source (SigmaAldrich, St Louis, MO, USA), was mixed with ultrapure water to a

weight/volume ratio of 18%. The solution was stored in a glass-bottle at room temperature before use.

3 mL of 18% HEBP solution and 3 mL of 5% NaOCl were mixed to produce a single solution with a

volume of 6 mL. Thus, the resultant concentrations of the solutions in the mixture were 9%, and 2.5%

respectively. The root canals were passively irrigated with this solution for 2 min as mentioned in

Group 1.

Group 8 (NaOCl+HEBP, PUI): A single solution containing the mixture of 3 mL 9% HEBP and

2.5% NaOCl was prepared as described in Group 7. The root canals were irrigated with this solution

that was activated using ultrasonics for 1 min. Then, a freshly prepared solution was applied to the

root canals, and activated using ultrasonics for another 1 min.

Group 9 (NaOCl+HEBP, XP-endo Finisher): The mixture of 3 mL 9% HEBP and 2.5% NaOCl was

applied to the root canals, then activated using XP-endo Finisher for 1 min using the same protocol in

Group 3. Then the activation was stopped and the root canals were irrigated with a freshly prepared

solution which then was activated using XP-endo Finisher for another 1 min.

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 Group 10 (Distilled water, no activation): The root canals were irrigated with 6 mL distilled water for

2 min without any activation using the same procedure as in Group 1.

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Group 11 (Distilled water, PUI): The root canals were irrigated with 3 mL distilled water, which then

was activated for 1 min using ultrasonics as described in Group 2. After 1 min, a freshly prepared 3

mL distilled water was introduced into the root canals, and activated for 1 min using the same

procedure.

Group 12 (Distilled water, XP-endo Finisher): 3 mL distilled water was introduced into the root

canals and activated using XP-endo Finisher for 1 min. Then the activation was stopped and the

procedure was repeated for 1 min.

The total activation time was 2 minutes in all experimental groups. All the root canals were finally

flushed with distilled water to prevent the prolonged effect of the irrigants, and dried with paper

points. The teeth were disassembled and the tissue samples inside the resorption cavities were blotted

dry (Figure 3). They were weighed three times and the arithmetic mean of three measurements were

calculated and recorded.

The estimated sample size was calculated using F-test family and ANOVA with PASS 2008 software,

according to the parameters which were determined from a pilot study. Minimum detectable

difference between means=0.001, coefficient of variation=0.001, probability level  = 0.05, statistical

power=0.80. Thus, the minimum sample size was determined as eight for each group.

Data analysis was performed using SPSS 15.0 statistical package (SPSS Inc., Chicago, IL, USA). The

data were examined for normal distribution using the Shapiro-Wilk test and homogenity of variance

with Levene’s test. Two-way ANOVA was performed to analyse the influence of the two factors

(irrigant and activation method) and their interactions on organic tissue removal. The independent

variables were the irrigation solutions and the activation method. The dependent variable was tissue

weight reduction after final irrigation. Post-hoc multiple comparisons were performed by using the

Fisher’s Least Significant Difference (LSD) test. All statistical analyses were two-sided, and a P-value

< 0.05 was considered to be significant.

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 Results

The percentage reduction in weight of the tissue specimens are summarized in Table 1. There was no
Accepted Article
significant difference between the baseline weight of the specimens (p>0.05). Two-way ANOVA

revealed that tissue weight reduction was significantly affected by the irrigation solution and the

activation method (p<0.001). There was also a significant interaction between the irrigation solution

and the activation method (p<0.001). The use of XP-endo Finisher with the experimental solutions

(NaOCl, NaOCl-EDTA, NaOCl+HEBP) resulted in the greatest tissue weight loss compared to the

other activation protocols (p<0.001). Lower reduction values in tissue weight were obtained from the

NaOCl-EDTA and distilled water groups without agitation. The tissue weight changes of the NaOCl

and NaOCl+HEBP groups with no activation or activated using two methods, were not significantly

different from each other (p>0.05).

Discussion

Irregularities of the root canal system create difficulties for clinicians during root canal treatment. The

manufacturer of XP-endo Finisher recommends its use for the chemomechanical preparation of root

canals with highly complex morphologies such as internal root resorption cavities (FKG 2015). The

file has also been proposed to improve the benefits of irrigant (Keskin et al. 2017), which is of utmost

importance when managing irregular shaped root canals. However, there is limited information

regarding the efficiency of XP-endo Finisher in the treatment of internal root resorption cases (Keskin

et al. 2017). The elimination of the inflamed pulp and granulation tissue inside the resorption cavity is

fundamental for a successful outcome, as they not only complicate chemomechanical canal

preparation due to the bleeding impairing visibility, but also provide a substrate for the growth of

microorganisms (Patel et al. 2010).

The results of the present study revealed that the use of XP-endo Finisher was more effective than

PUI in eliminating simulated tissue samples from artificial internal resorption cavities in straight root

canals of extracted teeth. This can be attributed to the differences between the action mechanisms of

XP-endo Finisher and PUI. XP-endo Finisher was designed primarily to dislodge tissues

mechanically. This file can adapt to the root canals three dimensionally and has an expansion capacity

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 that can reach an area with a diameter of 6 mm (FKG 2015, Trope & Debelian 2015). Furthermore, a

7-8 mm vertical movement was applied in an attempt to contact the full length of the canal. Therefore,
Accepted Article
in addition to the tissue dissolution ability of the irrigants activated with XP-endo Finisher, the

physical removal of the organic tissues from simulated resorption cavities could have increased the

tissue weight loss of the groups, in which XP-endo Finisher was used. However, the mechanism of

PUI includes acceleration of dissolution reaction (Munley & Goodell 2007). The files activated with

ultrasonic energy produce acoustic streaming, which enhances the activity of irrigants by

hydrodynamic shear stress (Munley & Goodell 2007). Additionally in the present study, the ultrasonic

tips were statically placed “2 mm” from the apex without any vertical movement. As the shear stress

is highest around the tip (Chen et al. 2014), if it was placed at the same level on the resorption

cavities, the dissolution values obtained from PUI groups could be different. The results are in conflict

with those of Keskin et al. (2017) who showed a similar efficacy of XP-endo Finisher and PUI in

removal of calcium hydroxide from simulated internal resorption cavities. This contradiction may be

related to the possible differences between the dissolution mechanisms of the calcium hydroxide and

bovine muscle tissue.

The present findings also support previous research, which revealed that HEBP did not interfere with

NaOCl by decreasing its dissolution ability (Zehnder et al. 2005, Tartari et al. 2015). The use of the

HEBP+NaOCl mixture, when inactivated or agitated with ultrasonics, resulted in more tissue weight

reduction compared to the sequential use of NaOCl and EDTA. Although EDTA and NaOCl were not

mixed, the remnants of NaOCl in the irregular areas could have interacted with the subsequent use of

EDTA, reducing the dissolution rate of the tissue samples in the resorption cavities.

The contact time and volume of the irrigation solutions are important parameters that can influence

their dissolution capacity. In the present study, the same exposure time (2 min) was used in all groups.

However, to standardize the total irrigant volume (6 mL) in the groups, the NaOCl volume had to be

adjusted to 3 mL in the EDTA and HEBP groups. In the groups where NaOCl was used as the sole

irrigant, its volume was 6 mL. This variation could have influenced the present results and can be

considered as a limitation.

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 Artificial internal root resorption cavities were created for evaluating the removal ability of the

irrigants activated with the various methods. However, in a clinical setting, there would already be
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some considerable dissolution of dental pulp tissue during cleaning and shaping prior to the final

irrigation protocol. In the present study, for standardization purposes, the organic tissues that were

placed in the simulated cavities were directly treated with the irrigation protocols. The main root canal

was prepared before resorption cavity preparation, therefore, the organic tissues inside the cavities

were not subjected to the cleaning and shaping procedures as in the clinical conditions. This may be

another limitation of this study.

Conclusion

Use of XP-endo Finisher was more effective compared to ultrasonics in removing simulated organic

tissue from artificial internal resorption cavities in straight root canals in extracted teeth. The

combined use of NaOCl and HEBP in a single irrigant did not inhibit the dissolution of organic tissues

within the irregularities of the root canal system.

Conflict of Interest statement

The authors have stated explicitly that there are no conflicts of interest in connection with this article.

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 References

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 FIGURE LEGENDS

Figure 1 Prepared internal root resorption cavities

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Figure 2 Adaptation of organic tissues inside the resorption cavities

Figure 3 The remaining tissue samples after final irrigation protocols

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ccepted Articl
Table 1 Means and standard deviations of % reduction in tissue weight values

Activation method P-value of two-way ANOVA

Irrigation solution No activation PUI XP-endo Finisher Irrigation Activation Irrigation solution x

Mean (SD) Mean (SD) Mean (SD) solution method Activation method

NaOCl 28.5 (7.5) ABa 52.3 (16.0) Ab 85.0 (8.2) Ac

NaOCl-EDTA 20.9 (13.5) Ba 26.5 (9.7) Ba 80.4 (12.8) Ab

< 0.001 < 0.001 < 0.001
NaOCl+HEBP 30.8 (11.5) Aa 47.8 (14.5) Ab 89.4 (6.9) Ac

Distilled water 21.2 (5.2) Ba 28.5 (7.4) Ba 60.5 (12.8) Bb

SD, standard deviation; NaOCl, sodium hypochlorite; EDTA, ethylenediaminetetraacetic acid; HEBP, etidronic acid; PUI, passive ultrasonic irrigation.

The different letters indicate statistically significant differences between the groups (p<0.05). Capital letters were used to compare groups in rows (irrigation solution), and

lower-case letters were used to compare groups in the columns (activation method) separately.

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