Invest Clin 62(2): 132 - 139, 2021 https://doi.org/10.22209/IC.v62n2a04

Corresponding author: Giampiero Rossi-Fedele. Adelaide Dental School. The University of Adelaide. Floor 10. Ad-

elaide Health and Medical Sciences Building. Adelaide, South Australia, Australia. E-mail:giampiero.rossi-fedele@

adelaide.edu.au

The use of chelating agents with passive

ultrasonic irrigation enhances root canal

filling sealing ability.

Gisele Jung Franciscatto

1, 2, 3

, Giampiero Rossi-Fedele

, Manuela Favarin Santini

Mariana de Carlo Bello

, Minéia Weber Blattes

and Carlos Alexandre Souza Bier

Graduate Program in Dental Science, Federal University of Santa Maria, Santa Maria,

Rio Grande do Sul, Brazil.

Graduate Program in Dentistry, School of Health and Life Sciences, Pontifical Catholic

University of Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil.

Medical and Dental Centre of the Military Police of Rio Grande do Sul, Porto Alegre,

Rio Grande do Sul, Brazil.

Adelaide Dental School, University of Adelaide, Australia.

School of Dentistry, Franciscan University, Santa Maria, Rio Grande do Sul, Brazil.

School of Dentistry, Lutheran University of Brazil. Canoas, Rio Grande do Sul, Brazil.

School of Pharmacy, Franciscan University, Santa Maria, Rio Grande do Sul, Brazil.

Key words: ethylenediaminetetraacetic acid; citric acid; root canal irrigants; root canal

obturation; ultrasonics.

Abstract. The aim of this study was to compare the influence of a final rinse

with and without passive ultrasonic irrigation (PUI), using 17% ethylenediamine-

tetraacetic acid (EDTA) or 10% citric acid (CA), with 2.5% sodium hypochlorite

(NaOCl) with PUI, on the sealing ability of root canal fillings. Root canal instru-

mentation was performed in single-rooted premolars using NaOCl as the irrigant.

Subsequently, the roots were randomly allocated into four experimental groups

(n=23): EDTA+PUI, EDTA, CA+PUI, CA and control group: NaOCl+PUI. The spec-

imens were root-filled using gutta-percha with a resin-based sealer and inserted into

a device to measure glucose leakage at 7 and 30 days. Kruskal-Wallis and Mann-

Whitney tests were used to evaluate the differences between the solutions and with

or without PUI. Friedman’s ANOVA was used to compare time points (P<0.05).

PUI groups showed lower infiltration values (EDTA+PUI 0.74±0.77 and CA+PUI

5.32±1.45) when compared to the groups on which PUI was not performed (EDTA

7.20±2.18 and CA 20.73±4.70), this difference was significant for EDTA (P<0.01)

and CA (P=0.000). Also, EDTA showed less glucose infiltration than CA, with or

without PUI (P<0.005). NaOCl+PUI showed a higher cumulative glucose infiltra-

tion (22.92±9.71). A final rinse using EDTA or CA with PUI enhances the sealing of

root canal fillings. EDTA showed less infiltration.

Chelators with PUI and root-lling sealing ability 133

Vol. 62(2): 132 - 139, 2021

El uso de agentes quelantes con irrigación ultrasónica pasiva

mejora la capacidad de sellado de la obturación de conducto

radicular.

Invest Clin 2021; 62 (2): 132-139

Palabras clave: ácido etilendiaminotetraacético; ácido cítrico; irrigantes de canales

radiculares, obturación de canales radiculares; ultrasonido.

Resumen. El objetivo de este estudio fue comparar la influencia de una

irrigación final con o sin activación ultrasónica pasiva (PUI), utilizando ácido

etilendiaminotetraacético (EDTA) al 17% o ácido cítrico (CA) al 10%, con hipo-

clorito de sodio (NaOCl) al 2,5% con PUI, sobre el sellado de la obturación de

conductos radiculares. Después de la instrumentación utilizando NaOCl como

irrigante, premolares de raíz única se asignaron aleatoriamente a cuatro grupos

experimentales (n = 23): EDTA+PUI, EDTA, CA+PUI, CA y grupo de control:

NaOCl+PUI. Los conductos se obturaron utilizando gutapercha con un sellador

a base de resina y se insertaron en un dispositivo para medir la infiltración de

glucosa a los 7 y 30 días. Se utilizaron las pruebas de Kruskal-Wallis y Mann-Whit-

ney para evaluar las diferencias entre las soluciones y con o sin PUI. Se utilizó el

ANOVA de Friedman para comparar los puntos temporales (P <0,05). Los gru-

pos de PUI mostraron menores valores de infiltración (EDTA+PUI 0,74±0,77

y CA+PUI 5,32±1,45) en comparación con los grupos en los que no se realizó

PUI (EDTA 7,20±2,18 y CA 20,73±4,70), esta diferencia fue significativa para

EDTA (P <0,01) y CA (P = 0,000). Además, EDTA mostró menos infiltración

de glucosa que CA, con o sin PUI (P <0,005). NaOCl+PUI mostró una mayor

infiltración acumulada de glucosa (22,92±9,71). Una irrigación final con EDTA

o CA con PUI mejora el sellado de la obturación de conducto radicular. EDTA

mostró menos infiltración.

Received: 12-11-2020 Accepted: 22-03-2021

INTRODUCTION

The endodontic literature suggests

that different irrigant solutions, with or

without agitation, are used globally, with

the choice influenced by the diagnosis, the

training of the operator and their eagerness

to remove the smear layer (1-4). Despite

the controversy of whether or not to remove

the smear layer during root canal prepara-

tion, its reduction enhances sealing ability

by promoting a closer contact between the

root filling material and dentinal tubules

(5, 6). This contact also reduces the forma-

tion of gaps between the root canal wall and

the filling, which could facilitate the pene-

tration of microorganisms from the coronal

portion (6).

Therefore, one of the aims of root canal

irrigation is the removal of the smear layer.

Studies have shown that a sodium hypochlo-

rite solution (NaOCl) does not promote an

effective final cleansing of the dentine walls

when used alone (7). Therefore, the use of

chelating agent solutions, such as 17% ethyl-

enediaminetetraacetic acid (EDTA), comple-

ments this process by being active against the

inorganic matter (7). Combining this chela-

tor with NaOCl enhances root canal cleansing

and improves the sealing of the root canal fill-

134 Jung Franciscatto et al.

Investigación Clínica 62(2): 2021

ing (8). Citric acid (CA) is a chelating agent

that also reacts with metals to form a soluble

non-ionic chelate. Moreover, it has antibac-

terial activity and has a root canal cleansing

ability comparable to other chelating solu-

tions, including EDTA (9, 10).

Despite the use of NaOCl in association

with chelating solutions, dentine debris and

smear layer may remain on the canal walls

(5), especially in irregularly shaped canals,

which are difficult to access for cleaning.

Thus, passive ultrasonic irrigation (PUI) is

a suggested strategy, which involves activat-

ing a file inside a root canal containing an

irrigating solution. When used with NaOCl,

PUI generates acoustic streaming in the so-

lution producing a more effective cleansing

(11). Using EDTA with PUI for one minute

as the final irrigation method is effective in

removing the smear layer and the dentinal

debris in the apical third of root canals (12).

Although the literature demonstrates the

superiority of PUI in removing debris and

cleaning isthmuses between canals (13, 14),

there is scarce evidence regarding the effect

of ultrasonic activation of chelating solu-

tions in regards to the root fillings sealing

ability (14, 15).

Thus, the aim of this study was to assess

the effect of PUI when EDTA or CA are used

as final irrigant solutions on the sealing of

root canals filled with gutta-percha and AH-

Plus sealer. The tested null hypothesis was

that PUI and these chelating solutions do

not influence the sealing ability of root ca-

nal fillings.

MATERIALS AND METHODS

The study was approved by the Insti-

tutional Health Sciences Research Ethics

Board (n

0234.0.243.000-11) and was car-

ried out in accordance with the Helsinki

Declaration of 1975, as revised in 2000.

Teeth selection and specimen preparation

One hundred and fifteen human premo-

lars with a single canal, having slight or no

curvature <10° for the supplementary angle

(16), were selected from the teeth bank of

the university. Radiographs of each premo-

lar were exposed from two angles to verify

a single canal and an oval shape, character-

ised by a bucco-lingual diameter twice the

mesio-distal diameter (17). Root-filled teeth

or those with calcified canals or other mor-

phologies and curvatures were excluded. The

teeth were kept in distilled water until use.

All procedures were performed by a special-

ist endodontist (GJF).

The coronal portion of the included

teeth was removed with a diamond bur un-

der refrigeration, standardising the root

lengths to 12 mm. The final working length

was determined by inserting a size 15 K-file

(Dentsply Maillefer, Ballaigues, Switzerland)

until the tip was observed at the apical fora-

men and subtracting 1 mm from this mea-

sure. The canals were instrumented with the

ProTaper system (Dentsply Maillefer), up to

the instrument F3. Each set of ProTaper in-

struments was used for 15 canals consecu-

tively. Final apical instrumentation was per-

formed with a size 35 K-file. Irrigation was

performed with 1 mL of 2.5% NaOCl after

each instrument change by using a dispos-

able syringe and Endo Eze tips (Ultradent

Products Inc., Indaiatuba, SP, Brazil) placed

at 3 mm short of the working length.

Root canal irrigation

The teeth were randomly assigned

(www.random.org) to four experimental

groups (n=23), according to their final ir-

rigation protocols: EDTA; EDTA+PUI; CA;

CA+PUI and one control group NaOCl+PUI.

The irrigant solutions used in the assays

were as follows: 17% EDTA (Novaderme, San-

ta Maria, RS, Brazil); 10% CA (Novaderme);

2.5 % NaOCl (Novaderme).

Irrigant solutions were delivered us-

ing a disposable syringe and Endo Eze tips

with the needled tip positioned at 3 mm

from the working length. PUI was performed

with an ultrasonic adapter and coupled to a

piezoelectric unit (Sonic Laxis – Schuster,

Chelators with PUI and root-lling sealing ability 135

Vol. 62(2): 132 - 139, 2021

Santa Maria, RS, Brazil) by inserting a size

15 K-file, at a frequency of 32.000 Hz, 1 mm

short of the working length. Three cycles of

1-minute agitation each were carried out per

sample for the PUI groups. At each cycle, the

root was irrigated with 1 mL of the solution,

thus all specimens were irrigated, in total,

with 3 mL of the solution.

A rinse with 1 mL of distilled water us-

ing syringe and needle as described above

was carried out, and the canals were dried

using paper points (Dentsply Maillefer).

Root canal obturation

The operator filling the teeth was un-

aware of the irrigation protocol used previ-

ously. An epoxy resin-based sealer (AH Plus,

Dentsply Maillefer) was mixed according to

the manufacturer’s instructions and a stan-

dardised amount was placed into the root

canal with a size 35 lentulo spiral. A size 35

gutta-percha point and size FF accessory

points (Dentsply Maillefer) were passively

inserted in the root canals without using a

spreader to the depth at which resistance

was met. The excess gutta-percha was sev-

ered at the level of the coronal section, using

a hot condenser.

Preparation and measurement

of the penetration model

The infiltration model used in the pres-

ent study was proposed by Xu et al. (18). All

the roots were covered with sticky wax (Asfer

Indústria Química, São Caetano do Sul, SP,

Brazil) and nail varnish (Risqué, São Paulo,

SP, Brazil), except for the coronal access, ca-

nal entrance, and apical foramen. The cor-

onal third was covered with sticky wax and

cyanoacrylate (Loctite, Itapevi, SP, Brazil) to

enable a leak-free surface between the Ep-

pendorf microtube (Sarstedt AG & Co. KG,

Nümbrecht, NRW, Germany) and the root.

This junction was also covered with nail var-

nish. A hole was created at the cap of the

microtube, and a 14 cm length with a 5 mL

capacity pipette was inserted. This assembly

was positioned into a sterile 5 mL glass bot-

tle. This interface and the interface between

the pipette and the microtube were sealed

with cyanoacrylate, sticky wax, and nail var-

nish. The glass bottle contained 2 mL of a

0.2% NaN

solution in which the apical third

of the roots were immersed.

A 1 mol/L glucose solution (pH = 7.0)

was used as the tracer. Five mL of this so-

lution containing 0.2% NaN

were injected

into the pipette until the top of the solution

was at 14 cm from the filling level in the

root canal, creating a hydrostatic pressure of

1.5 kPa (15 cm H

O). The glucose solution

that infiltrated through the root canal was

collected in the NaN

solution in the glass

bottle. All of the specimens were kept in an

incubator at 37°C.

Using a micropipette, 20 μL of the so-

lution were drawn from the glass bottle at

the 7

and 30

days. The same aliquot of

0.2% NaN

was inserted into the glass bottle

to maintain a constant volume. The sample

was analysed with a Glucose Kit (Glicose PAP

Liquiform, Labstest Diagnóstica S.A, Lagoa

Santa, MG, Brazil). In this test, glucose was

oxidised by glucose oxidase in the presence

of oxygen to yield gluconic acid and hydro-

gen peroxide. The hydrogen peroxide that

was formed reacted with 4-amino antipyrine

and phenol, and this reaction was catalysed

by peroxidase to form a red product in which

the intensity of the colour is proportional to

the amount of glucose in the sample. The

concentration of glucose in the samples

was determined via a spectrophotometer

(UV- 1100 Spectrophotometer, Pró-análise

Química e Diagnóstica Ltda, Porto Alegre,

RS, Brazil) at a wavelength of 505 nm, and

recorded by a blinded evaluator.

Statistical analysis

The statistical analysis was performed

using SPSS Statistics for Windows, Version

17.0. (SPSS Inc., Chicago, USA). Data are

presented as mg/dL. As the data were not

normally distributed, Kruskal-Wallis and

Mann-Whitney tests were used to evaluate

the differences among the solutions and with

136 Jung Franciscatto et al.

Investigación Clínica 62(2): 2021

or without PUI. Friedman’s ANOVA was used

for comparison between the time points (7

and 30

days). The significance level was set

at P<0.05.

RESULTS

Results are presented as mean values

± standard deviations. Glucose infiltration

(mean and SD) for 7 and 30 days are present-

ed in Figures 1 and 2. Kruskal-Wallis results

showed the groups were different at both

evaluated periods (P=0.000). NaOCl+PUI

showed a higher cumulative glucose infil-

tration on 30 days (29.79 ± 9.71). Accord-

ing to the Mann-Whitney test, PUI groups

showed lower infiltration values on both

periods (7 days: EDTA+PUI 0.19±0.44 and

CA+PUI 4.30±1.45; 30 days: EDTA+PUI

1.28±0.77 and CA+PUI 6.35±1.45) when

compared to the groups on which PUI was

not performed (7 days: EDTA 5.66±1.44 and

CA 17.40±3.15; 30 days: EDTA 8.74±2.18

and CA 24.05±4.70), these differences

were significant for EDTA (P<0.01) and CA

(P=0.000). Also, EDTA showed less glucose

infiltration than CA, with or without PUI

(P<0.005).

Fig. 1. Cumulative mean of glucose infiltration and SD after 7 days. Different superscript letters indicate

significant difference at the Mann-Whitney test (level of significance set at <0.05). EDTA: ethylenedi-

aminetetraacetic acid; PUI: passive ultrasonic irrigation; CA: citric acid; NaOCl: sodium hypochlorite.

Fig. 2. Cumulative mean of glucose infiltration and SD after 30 days. Different superscript letters indicate

significant difference at Mann-Whitney test (level of significance set at <0.05). EDTA: ethylenedia-

minetetraacetic acid; PUI: passive ultrasonic irrigation; CA: citric acid; NaOCl: sodium hypochlorite.

Chelators with PUI and root-lling sealing ability 137

Vol. 62(2): 132 - 139, 2021

According to Friedman’s ANOVA re-

sults, the glucose infiltration increased sig-

nificantly from day 7 to day 30 (P<0.05).

DISCUSSION

As a final rinse following chemo-me-

chanical instrumentation using NaOCl, PUI

with the chelating solutions tested promot-

ing a better sealing when gutta-percha and a

resin-based sealer were used, when compared

with NaOCl, thus rejecting the null hypoth-

esis. This finding may be related to the syn-

ergistic relationship between the ultrasound

and chelating solutions on the removal of the

inorganic part of the smear layer and the pos-

itive effect of removing this layer on the seal-

ing ability of the root canal filling (6, 11, 12).

The influence of the smear layer on

the sealing ability of the root canal fill-

ing has been studied with various method-

ologies. In the present study, glucose was

used as the tracer due to its low molecular

weight and because it is a bacterial sub-

strate (18). Thus, it is considered of clini-

cal relevance (18). Moreover, the glucose

tracer provides a quantitative analysis

of the process, also allowing the evalua-

tion of the infiltration over time (18-21).

Glucose may react with some endodontic

materials, which can significantly alter

experimental results (22). However, this

is not the case for an epoxy resin-based

sealer and gutta-percha (22), which were

used for the present study. Furthermore,

the glucose solution penetrates solely

through the dentine-filling interface (19).

The removal of the smear layer is recom-

mended to enhance the disinfection of the

root canal system and, subsequently, to en-

hance the sealing of the filling material (5,

6). This layer is composed of necrotic debris,

residual pulp, remaining dentine, and even-

tually, microorganisms. It results from root

canal instrumentation and remains slightly

adhered to the root canal walls (23, 24). The

smear layer may act as a substrate for micro-

organisms, prevent the penetration of irrigant

solutions and medicaments into the dentinal

tubules, and interfere with the penetration

and adhesion of sealers (6, 23, 24).

Sodium hypochlorite is the most used

irrigating solution in root canal treatment

due to its biocidal effect and its ability to

dissolve organic tissues, though it is not

fully effective in smear layer removal (25).

This ineffectiveness can explain the differ-

ences between the control group and the

other groups in the present study and it

is in agreement with a previous one (25).

Chelating agents enhance smear layer re-

moval because they can act on the inor-

ganic particles (24). Chelation involves

the reaction of the solutions with a metal

to form a non-ionic soluble chelate (10).

In other words, the solvent promotes the

sequestration of calcium ions (inorganic

particles) from the smear layer and facili-

tates their removal with the flushing ef-

fect of the root canal irrigants solution.

This is likely enhanced by the irrigation

dynamics associated with PUI.

The most widely used solution for

smear layer removal is EDTA (1-4). Its maxi-

mum cleansing effect is reached after pne

minute (26), which may limit secondary ef-

fects, such as decalcification and erosion

of the dentinal walls (27). Kuah et al. (12)

observed a better smear layer removal when

EDTA was activated ultrasonically for one

minute only. In the present study, to stan-

dardize with the NaOCl control group, this

procedure was repeated twice, and the solu-

tion in question was renewed (11). When

EDTA is used in association with NaOCl

(27), or ultrasonically activated, its erosive

effect is accentuated (28). Though, in our

experimental set-up, this purported dam-

age appears not to interfere with the seal-

ing ability of the root filling.

Citric acid is a weak organic acid that

also acts through chelation. In this study, CA

presented with increased leakage compared

to EDTA, regardless of the use of PUI. When

comparing smear layer removal with 17%

EDTA and MTAD, a mixture of 3% doxycycline,

138 Jung Franciscatto et al.

Investigación Clínica 62(2): 2021

4.25% citric acid, and 0.5% Tween-80, the lat-

ter was more efficient (29). EDTA tends to be

more effective than the CA for smear layer

removal when there is a longer contact time

(11), which may be associated with the ten-

dency of the demineralisation capacity of CA

to saturate after 60 seconds (30).

The use of PUI to potentiate the irri-

gation solution’s effect has been previously

recommended, especially in oval-shaped

root canals or in canals with irregular mor-

phology, such as isthmuses, which are hard-

er to reach (11, 12). PUI agitation of Na-

OCl without the use of chelating solutions

generates cleaner root canal walls, which

reflects in better root canal sealing (11).

In the present study, when used as a final

rinse following chemo-mechanical instru-

mentation with NaOCl, the use of PUI with

chelating solutions was more effective than

NaOCl alone agitated, suggesting a possi-

ble synergism between ultrasonic activation

and chelating solutions. Limitations of the

study included that the root canal morphol-

ogy of the samples was assessed using two-

dimensional rather than three-dimensional

imaging. This is important as the anatomy

of the root canal system is considered a

common confounding factor in leakage

studies (31).

Similarly, the root filling was

carried out using a matching gutta-percha

point and no condensation, and our results

may not be translatable to other obturation

materials and/or techniques.

Based on the findings of this study, it

can be concluded that a final rinse that in-

cludes EDTA or CA, agitated with PUI, is as-

sociated with enhanced sealing ability for

gutta-percha and sealer when compared with

the use of the same chelators in the absence

of agitation or the use of NaOCl with PUI.

EDTA performed better than CA. These re-

sults indicate that during non-surgical root

canal treatment, PUI with chelating solu-

tions helps to promote a better sealing for

root canal fillings.

REFERENCES

1. de Gregorio C, Arias A, Navarrete N, Cis-

neros R, Cohenca N. Differences in disin-

fection protocols for root canal treatment

between general dentist and endodontists.

J Am Dent Assoc 2015;146:536-543.

2. Savani GM, Sabbah W, Sedgley CM, Whit-

ten B. Current trends in endodontic treat-

ment by general dental practitioners: re-

port of a United States national survey. J

Endod 2014;40:618–624.

3. Dutner J, Mines P, Anderson A. Irrigation

trends among American Association of En-

dodontists members: a Web-based survey. J

Endod 2012;38:37-40.

4. Willerhausen I, Wolf TG, Schmidtmann I,

Berger C, Ehlers V, Willerhausen B, Brise-

ño B. Survey of root canal irrigating solu-

tions used in dental practices within Ger-

many. Int Endod J 2015;48:654-660.

5. Economides N, Liolios E, Kolokuris I.

Long-term evaluation of the influence of

smear layer removal on the sealing ability

of different sealers. J Endod 1999;25:123–

125.

6. Shahravan A, Haghdoost A-A, Alireza A,

Hessam R, Fahimeh S. Effect of smear layer

on sealing ability of canal obturation: a sys-

tematic review and meta-analysis. J Endod

2007;33:96–105.

7. Goldman M, Goldman LB, Cavaleri R, Bo-

gis J, Lin PS. The efficacy of several end-

odontic irrigating solutions: a scanning

electron microscopic study: Part 2. J Endod

1982;8:487–492.

8. Vivacqua-Gomes N, Ferraz CCR, Gomes

BPFA, Zaia AA, Teixeira FB, Souza-Filho FJ.

Influence of irrigants on the coronal micro-

leakage of laterally condensed gutta-percha

root fillings. Int Endod J 2002;35:791–795.

9. Yamaguchi M, Yoshida K, Suzuki R, Naka-

mura H. Root canal irrigation with citric

acid solution. J Endod 1996;22:27–29.

10. Kaushai R, Bansai R, Malhan S. A compara-

tive evaluation of smear layer removal by us-

ing ethylenediamine tetraacetic acid, citic

acid, and maleic acid as root canal irrigants:

an in vitro scanning electron microscopic

study. J Conserv Dent 2020;23:71-78.

Chelators with PUI and root-lling sealing ability 139

Vol. 62(2): 132 - 139, 2021

11. van der Sluis LWM, Shemesh H, Wu MK,

Wesselink PR. An evaluation of the influ-

ence of passive ultrasonic irrigation on

the seal of root canal fillings. Int Endod J

2007;40:356–361.

12. Kuah H-G, Lui J-N, Tseng PSK, Chen N-N.

The effect of EDTA with and without ultra-

sonics on removal of the smear layer. J En-

dod 2009;35:393–396.

13. van der Sluis LWM, Versluis M, Wu M-K,

Wesselink PR. Passive ultrasonic irrigation

of the root canal: a review of the literature.

Int Endod J 2007;40:415–426.

14. Burleson A, Nusstein J, Reader A, Beck M.

The in vivo evaluation of hand/rotary/ultra-

sound instrumentation in necrotic, human

mandibular molars. J Endod 2007;33:782–

787.

15. Singh CV, Rao SA, Chandrashekar V. An

in vitro comparison of penetration depth

of two root canal sealers: An SEM study. J

Conserv Dent 2012;15:261–264.

16. Hartmann RC, Fensterseifer M, Peters OA,

Figueredo JAP, Gomes MS, Rossi-Fedele G.

Methods for the measurement of root canal

curvature: a systematic and critical review.

Int Endo J 2019;52:169-180.

17. Wu MK, R’oris A, Barkis D, Wesselink PR.

Prevalence and extent of long oval canals in

the apical third. Oral Surg Oral Med Oral

Pathol Oral Radiol Endod 2000;89:739-743.

18. Xu Q, Fan M, Fan B, Cheung GSP, Hu H. A

new quantitative method using glucose for

analysis of endodontic leakage. Oral Surg

Oral Med Oral Pathol Oral Radiol Endod

2005;99:107–111.

19. Shemesh H, van den Bos M, Wu M-K,

Wesselink PR. Glucose penetration and

fluid transport through coronal root struc-

ture and filled root canals. Int Endod J

2007;40:866–172.

20. De-Deus G, Soares J, Leal F, Luna AS, Fi-

del S, Fidel RAS. Similar glucose leakage

pattern on smear-covered, EDTA-treated

and BioPure MTAD-treated dentin. J Endod

2008;34:459–462.

21. El Sayed MA, Taleb AA, Balbahaith MS.

Sealing ability of three single-cone obtura-

tion systems: an in-vitro glucose leakage

system. J Conserv Dent 2013;16:489-493.

22. Shemesh H, Souza EM, Wu M-K, Wesselink

PR. Glucose reactivity with filling materials

as a limitation for using the glucose leak-

age model. Int Endod J 2008;41:869–872.

23. Torabinejad M, Handysides R, Khademi

AA, Bakland LK. Clinical implications of

the smear layer in endodontics: a review.

Oral Surg Oral Med Oral Pathol Oral Radiol

Endod 2002;94:658–666.

24. Violich DR, Chandler NP. The smear lay-

er in endodontics - a review. Int Endod J

2010;43:2–15.

25. Wu L, Mu Y, Deng X, Zhang S, Zhou D.

Comparison of the effect of four decalcify-

ing agents combined with 60°C 3% sodium

hypochlorite on smear layer removal. J En-

dod 2012;38:381–384.

26. Calt S, Serper A. Time-dependent effects

of EDTA on dentin structures. J Endod

2002;28:17–19.

27. Ghisi AC, Kopper PM, Baldasso FE, Stürm-

er CP, Rossi-Fedele G, Steier L, Figueiredo

JA, Morgental RD, Vier-Pelisser FV. Effect

of superoxidized water and sodium hypo-

chlorite, associated or not with EDTA, on

organic and inorganic component of bovine

root dentin. J Endo 2015;41:925-930.

28. Hartmann R, Neuvald L, Barth V, de

Figueiredo JAP, Dias de Oliveira S, Rossi-

Fedele G. Antimicrobial efficacy of 0.5%

peracetic acid and EDTA with passive ultra-

sonic or manual agitation in an Enterococ-

cus faecalis biofilm model. Aust Endod J

2019;45:57-63.

29. Lotfi M, Vosoughhosseini S, Saghiri MA,

Zand V, Ranjkesh B, Ghasemi N. Effect of

MTAD as a final rinse on removal of smear

layer in ten-minute preparation time. J En-

dod 2012;38:1391–1394.

30. Arslan H, Capar ID, Saygili G, Uysal B,

Gok T, Ertas H, Topcuoglu HS. Efficacy

of various irrigation protocols on the re-

moval of triple antibiotic paste. Int Endod J

2014;47:594–599.

31. De-Deus G. Research that matters - root ca-

nal filling and leakage studies. Int Endod J

2012;45:1063–1064.