Antibacterial effectiveness of several irrigating

solutions and the Endox Plus system an ex vivo

study
A. R. Aranda-Garcia, J. M. Guerreiro-Tanomaru, N. B. Faria-Ju nior, G. M. Chavez-Andrade,
R. T. Leonardo, M. Tanomaru-Filho & I. Bonetti-Filho
Department of Restorative Dentistry, Araraquara Dental School, UNESP, Univ Estadual Paulista, Araraquara, Brazil
Abstract
Aranda-Garcia AR, Guerreiro-Tanomaru JM, Faria-
Ju nior NB, Chavez-Andrade GM, Leonardo RT, Tanomaru-
Filho M, Bonetti-Filho I. Antibacterial effectiveness of several
irrigating solutions and the Endox Plus system an ex vivo
study. International Endodontic Journal,
Aim To compare the ex vivo antibacterial effectiveness
of the Endox Plus system and sodium hypochlorite
(NaOCl) in combination with BioPure MTAD (Tulsa
Dental, Tulsa, OK, USA) or with EDTA in Enterococcus
faecaliscontaminated root canals.
Methodology After initial preparation, the root
canals of 70 single-rooted human teeth were inocu-
lated with E. faecalis (ATCC 29212) and incubated
for 21 days. Specimens were divided into ve groups:
Endox Plus/saline; 2.5% NaOCl/MTAD; 2.5% NaOCl/
EDTA; saline (positive control); negative control
(root canals not prepared, nor irrigated). Samples
were collected using paper points. Microbiological
analysis evaluated the number of CFUs. Data
were analysed by anova and Tukey tests at 0.05
signicance.
Results All specimens had bacterial growth after the
incubation period, with similar CFU per mL counts
(P > 0.05). After chemo-mechanical preparation, the
number of bacteria in all groups reduced, except for the
negative control. No signicant differences were
observed between 2.5% NaOCl/MTAD and 2.5% NaO-
Cl/EDTA, but these groups had lower CFU counts than
the other groups (P < 0.05). In the nal samples, an
increase in the bacterial counts was observed for Endox
Plus/saline, 2.5% NaOCl/MTAD, 2.5% NaOCl/EDTA
and saline (P < 0.05) with no signicant differences
between these groups.
Conclusions This ex vivo study revealed that the
Endox Plus system was associated with a reduced
antibacterial effectiveness compared with conventional
irrigation using 2.5% NaOCl/MTAD and 2.5% NaOCl/
EDTA. All irrigation procedures allowed recovery of
bacteria 7 days after treatment, demonstrating persis-
tence of contamination within the root canal system.
Keywords: Endox Plus, Enterococcus faecalis, irri-
gating solution, MTAD.
Received 13 December 2011; accepted 11 April 2012
Introduction
Chemo-mechanical canal preparation is capable of
reducing the number of microorganisms in the root
canal system, but achievement of complete disinfection
is extremely difcult owing to the complex anatomy of
the canal system (Bystrom & Sundqvist 1981, Siqueira
et al. 1999). Several solutions have been used as
irrigants, aiming to reduce the endodontic microbiota.
Amongst these solutions, the mostly widely used is
sodium hypochlorite (NaOCl), whose antibacterial
action has been demonstrated against planktonic
bacteria as well as against biolm (Arias-Moliz et al.
2009). Use of NaOCl in conjunction with EDTA has
shown superior antibacterial action and ability to
remove the smear layer (Berutti et al. 1997, Kishen
et al. 2008).
MTAD BioPure (Tulsa Dental, Tulsa, OK, USA) is an
irrigant introduced in the market in 2003 (Torabinejad
Correspondence: Mario Tanomaru-Filho, Rua Humaita, 1680,
Caixa Postal 331, Centro, 14801-903 Araraquara, SP, Brazil
(Tel.: +55 16 3301 6390; fax: +55 16 3301 6392; e-mail:
tanomaru@uol.com.br).
doi:10.1111/j.1365-2591.2012.02069.x
2012 International Endodontic Journal International Endodontic Journal 1
et al. 2003a), which is composed of 3% doxycycline,
4.25% citric acid and a detergent (Tween 80)
(Torabinejad et al. 2003b, Singla et al. 2011). It has
proven clinical effectiveness on post-operative discom-
fort and biocompatibility (Zhang et al. 2003, Torabine-
jad et al. 2005). Several studies have demonstrated its
antibacterial action against Enterococcus faecalis both in
biolm and in planktonic phase, when used as a nal
irrigant. These studies consisted of an immediate
analyses after the nal irrigation or agar diffusion tests
(Torabinejad et al. 2003b, Davis et al. 2007, Newberry
et al. 2007, Shabahang et al. 2008, Prabhakar et al.
2010).
The Endox Plus system (Anfratron Technologies
GmbH, Wasserburg, Germany) is a newer version of
the Endox system (Lysis Srl, Nova Milanese, Italy),
developed to promote disinfection of the root canal
system. Similarly to its original version, this device
included a ne surgical stainless steel needle that acted
as an active electrode, transmitting electrical impulses
within the root canal (Cassanelli et al. 2008). Its
operating system is based on a high-frequency alter-
nating current (HFAC). The differences between the
previous and the current version (Endox Plus system)
in an attempt to improve its performance are the higher
frequency: from 312.5 to 315 Khz and the higher
potency: from 110 W/140 ms to 180 W/120 ms.
Several studies have evaluated the Endox system,
with controversial results. Analysis of this system by
SEM demonstrated efcacy in dentine debris and smear
layer removal after biomechanical preparation (Lendini
et al. 2005). Other studies, however, did not show
greater antimicrobial action for this device in compar-
ison with conventional irrigation protocols (Virtej et al.
2007, Karale et al. 2011).
The aim of the present study was to compare the
antibacterial effectiveness of the Endox Plus system,
2.5% NaOCl in association with MTAD and 2.5%
NaOCl followed by EDTA during biomechanical prep-
aration of ex vivo E. faecaliscontaminated root canals.
The null hypothesis is that the Endox Plus system has
the same antibacterial effectiveness as other methods.
Material and methods
This study was approved by the Committee of Ethics in
Research of the Araraquara School of Dentistry,
UNESP, Brazil. A total of 70 roots of extracted single-
rooted human teeth were standardized to a length of
15 mm. Root canal anatomy was standardized using
round/oval root canals without the presence of
isthmuses and ramications as observed after radio-
graphic evaluation. The working length (WL) was
established 1 mm short of the apex, and root canals
were instrumented up to a size 35 K-le (Dentsply
Maillefer, Ballaigues, Switzerland). At each instrument
change, root canals were irrigated with 2 mL saline
using a 5-mL syringe (Ultradent Products, South
Jordan, UT, USA) and a 29-G (17-mm-long) needle
(NaviTips, Ultradent Products) with simultaneous
suction. After preparation, root canals were lled with
17% EDTA (Biodinamica, Ibipora, PR, Brazil) for 3 min,
irrigated with 5 mL saline and dried with absorbent
paper points. Subsequently, the apex of each specimen
was sealed with light-cured composite resin (Z100, 3M
ESPE, St. Paul, MN, USA), and the external root surface
was made impermeable with two layers of epoxy
adhesive (Araldite, Brascola Ltda., Taboao da Serra,
SP, Brazil).
Specimens were divided randomly into four 24-well
microplates (Corning Incorporated, Corning, NY, USA).
Each microplate received 15 roots (experimental groups
and negative control), and one microplate received 10
roots (negative control), which were attached to the
wells with self-curing acrylic resin (Classico Artigos
Odontologicos, Sao Paulo, SP, Brazil). A copper wire
was attached to the root apices with epoxy adhesive to
allow transmission of the HFAC from the Endox Plus
system. One of the ends of the wire was exposed, to
form a closed circuit. The application of a copper wire
directly in contact with the root apex neglected the
impedance. The microplates containing the specimens
were wrapped and sterilized by ethylene oxide.
Contamination of the root canals
These procedures were carried out in a laminar ow
chamber (VecoFlow Ltda., Campinas, SP, Brazil). Stan-
dard E. faecalis strains (ATCC 29212) were cultivated in
Tryptic Soy Broth TSB (Difco, Detroit, MI, USA) for
24 h. Bacteria were seeded onto Tryptic Soy Agar
TSA plates (Difco) and incubated in a microaerophilic
environment at 37C for 48 h. A bacterial suspension
was prepared in sterile saline, at a concentration of
1.5 10
8
CFU per mL. The optical density of the
suspension was adjusted using a spectrophotometer
(Model 600 Plus, Femto, Sao Paulo, SP, Brazil). The
culture medium (TSB) was mixed with the bacterial
suspension at a ratio of, 1 : 1 and root canals were
inoculated with 20 lL of this mixture. The microplates
were covered and kept in a microaerophilic environ-
ment at 37 C for an incubation period of 21 days.
Antibacterial effectiveness of Endox Plus Aranda-Garcia et al.
International Endodontic Journal 2012 International Endodontic Journal 2
Sterile TSB was added to each canal on alternate days,
as described in previous studies (Soares et al. 2010,
Dornelles-Morgental et al. 2011). After the incubation
period, initial samples were collected to conrm con-
tamination by E. faecalis. Two sterile paper points of size
35 (Miltex Inc., Plainsboro, NJ, USA) were used in
sequence in each root. The paper points remained in the
canal for 1 min, were subsequently transferred to tubes
containing 1 mL sterile saline and shaken for 1 min.
Irrigation and decontamination procedures
The microplates containing the specimens were ran-
domly divided into three experimental groups and two
controls, as shown in Table 1. All root canals in the
experimental and positive control groups were instru-
mented with a manual size 45 K-le to the WL, and
step-back preparation was performed up to a size
60 K-le.
In group I, the solution used was 2 mL of saline at
each le change during instrumentation, and a green
tip (length of 23 mm placed to the WL) of the Endox
Plus system was used during nal irrigation. The
system was used according to manufacturers instruc-
tions, by applying one pulse to each canal third
(coronal, middle and apical) through a closed circuit
connecting the neutral electrode and the copper wire
attached to each root apex. Final irrigation was
performed with 2 mL of saline solution.
Specimens in group II were irrigated with 2.5%
NaOCl (Ciclo Farma indu stria Qu mica Ltda. EPP
Serrana, SP, Brazil), and nal irrigation was performed
with 2 mL of BioPure MTAD (Dentsply Tulsa Dental,
Tulsa, OK, USA). Group III was treated similarly, but in
the nal irrigation, the root canal was lled with 2 mL
of EDTA for 3 min, which was agitated with the size
45 K-le and then ushed with sterile saline. Root
canals in group IV received 2 mL of sterile saline
applied as previously described, and in group V
(negative control), root canals were not instrumented
nor irrigated.
At each instrument change, root canals were
irrigated with 2mL saline using a 5-mL syringe
(Ultradent Products) and a 29-G (17-mm-long) needle
(NaviTips, Ultradent Products), using the solution to be
evaluated in its respective group. Immediately after
these procedures, root canals in groups II and III were
lled with 1% sodium thiosulfate for 2 min to neutral-
ize the NaOCl. The other groups received saline for the
same amount of time. After that, a second sample
collection was conducted similarly to the initial collec-
tion, using two absorbent paper points (Miltex, Inc.,
York, PA, USA) per specimen. The root canals were
then lled with sterile saline, and the covered micro-
plates were once again incubated in a microaerophilic
environment at 37 C for 7 days. After the nal
incubation, samples were collected following the same
steps as in the initial and post-instrumentation proce-
dures.
Microbiological analysis
Microbiological analysis of the samples collected imme-
diately after initial incubation, post-instrumentation
and 7 days later was performed by determining the
CFU per mL counts of E. faecalis. Tenfold serial dilutions
were made, and 20 lL aliquots were seeded in triplicate
onto Petri dishes containing TSA. Following that, the
plates were incubated in microaerophilic environment
at 37 C for 48 h.
Statistical analysis
Data obtained were subjected to base-10 logarithmic
transformation and analysed by the GraphPad Prism
3.0 software (San Diego, CA, USA). The data showed a
normal distribution. Comparison between the groups
was carried out by anova and Tukey tests at 0.05
signicance. Microbiological samples within the same
group were compared by repeated measures anova
(5%).
Results
Results are presented in Fig. 1. All specimens had
bacterial growth after the initial incubation period.
Standardization of the samples was conrmed by their
similar bacterial counts, with no statistical difference
between the groups (P > 0.05). After instrumentation,
irrigation and disinfection, the bacterial counts were
signicantly reduced in all groups, except in the
negative control (P < 0.05). No statistically signicant
Table 1 Experimental and control groups
Groups n
GI Endox Plus + SS 15
GII NaOCl + MTAD 15
GIII NaOCl + EDTA 15
GIV Positive control (SS) 15
GV Negative control 10
G, group; SS, saline solution; NaOCl, sodium hypochlorite
solution. Total volume used (12 mL).
Aranda-Garcia et al. Antibacterial effectiveness of Endox Plus
2012 International Endodontic Journal International Endodontic Journal 3
differences were observed between GI and GIV
(P > 0.05). In the post-instrumentation samples, GII
and GIII did not show bacterial growth, signicantly
differing from the other groups (P < 0.05). At the nal
sample collection 7 days later, bacterial counts in-
creased in groups I, II, III and IV, with no signicant
differences amongst the groups, but differing signi-
cantly from the previous (post-instrumentation) sam-
ples. Comparison between samples collected within
each group at different periods showed that in the
negative control group (GV), all samples had similar
bacterial counts, conrming that E. faecalis remained
viable throughout the entire experimental period.
Discussion
The methodology used in the present study aimed to
simulate a clinical case of contamination of the root
canal system by incubating the root canals with
E. faecalis for 21 days, as previously described (Bhuva
et al. 2010, Harrison et al. 2010). Enterococcus faecalis
has a proven ability to invade dentine tubules after
21 days of incubation (Harrison et al. 2010). Entero-
coccus faecalis was used as a microorganism that is easy
to grow under laboratory conditions and difcult to
eradicate from the root canal system, in other words,
simulating a challenging case for the irrigants/devices
(Stuart et al. 2006).
It has been reported that MTAD, used in conjunction
with 1.3% NaOCl, is able to eradicate E. faecalis from
the root canals (Newberry et al. 2007, Shabahang et al.
2008). Other studies, however, have shown that MTAD
does not eliminate this microorganism from the root
canal system and that irrigation with NaOCl followed
by EDTA is equally effective or even more effective than
MTAD (Kho & Baumgartner 2006, Johal et al. 2007).
Dunavant et al. (2006) demonstrated, by a direct
contact test, that 1% NaOCl was signicantly more
efcient for the removal of E. faecalis biolm than
MTAD.
After root canal instrumentation, the microbiota
from the root canal lumen is eliminated, but recolon-
ization may occur because of the persistence of micro-
organisms within the dentine tubules, which are not
reached by chemo-mechanical preparation (Siqueira
et al. 2002, Molander et al. 2007). This may explain
why the nal samples in the present study (collected
7 days after instrumentation) were associated with an
increase in the E. faecalis counts, even after being
irrigated with NaOCl followed by EDTA. These results
are in agreement with previous studies (Oliveira et al.
2007, Dornelles-Morgental et al. 2011).
The present study is the rst to evaluate the Endox
Plus system. Few studies have compared its previous
version (Endox system) with commonly used irrigation
protocols. Karale et al. (2011) compared ex vivo the
antibacterial efcacy of Endox, 3% NaOCl and 2%
chlorhexidine in human root canals contaminated with
E. faecalis. The incubation period was 24 h, and
samples were collected immediately after irrigation.
The results showed that although the three methods
were efcacious, NaOCl had the best results. Contras-
tingly, in the present study, the Endox Plus system
demonstrated low effectiveness. The incubation period
in the present study was longer, and the samples were
also collected 7 days after treatment, allowing the
Figure 1 Comparison between groups in the initial, post-instrumentation and nal samples (columns in each group) and between
samples within the same group (mean CFU per mL in log10). Equal uppercase letters in the same row and equal lowercase letters
in the same column indicate statistically similar mean values (P > 0.05). Group I Endox Plus/saline, Group II 2.5% NaOCl/
MTAD, Group III 2.5% NaOCl/EDTA, Group IV saline (positive control), Group V negative control.
Antibacterial effectiveness of Endox Plus Aranda-Garcia et al.
International Endodontic Journal 2012 International Endodontic Journal 4
recovery of microorganisms that had survived in the
dentine tubules after instrumentation.
Virtej et al. (2007) compared the antibacterial effec-
tiveness of Endox, MTAD, 3% NaOCl and HealOzone in
root canals contaminated with mixed microbiota by
analysing samples collected at three different time-
points: 1 week after inoculation, immediately after
disinfection procedures and 1 week later. The second
samples did not show signicant differences between
NaOCl, MTAD and HealOzone, rather, all showed
complete elimination of the microbiota. However, the
Endox group was signicantly less efcacious. The nal
samples, collected 1 week later, also revealed that the
Endox system had the worse antibacterial effect, which
is in agreement with the results from the present study.
Conclusions
This ex vivo study revealed that the Endox Plus system
had the weakest antimicrobial effectiveness compared
with irrigation with 2.5% NaOCl in conjunction with
MTAD and 2.5% NaOCl followed by EDTA during
chemo-mechanical preparation. The Endox System had
no additional antibacterial effect following saline irri-
gation. All groups allowed recovery of bacteria 7 days
after treatment, demonstrating persistence of contam-
ination in the RCS after the irrigation protocols.
References
Arias-Moliz MT, Ferrer-Luque CM, Espigares-Garcia M, Baca P
(2009) Enterococcus faecalis biolms eradication by root
canal irrigants. Journal of Endodontics 35, 7114.
Berutti E, Marini R, Angeretti A (1997) Penetration ability of
different irrigants into dentinal tubules. Journal of Endodon-
tics 23, 7257.
Bhuva B, Patel S, Wilson R, Niazi S, Beighton D, Mannocci F
(2010) The effectiveness of passive ultrasonic irrigation on
intraradicular Enterococcus faecalis biolms in extracted
single-rooted human teeth. International Endododontic Journal
43, 24150.
Bystrom A, Sundqvist G (1981) Bacteriologic evaluation of the
efcacy of mechanical root canal instrumentation in end-
odontic therapy. Scandinavian Journal of Dental Research 89,
3218.
Cassanelli C, Marchese A, Cagnacci S, Debbia EA (2008)
Alteration of membrane permeability of bacteria and yeast
by high frequency alternating current (HFAC). The Open
Microbiology Journal 2, 327.
Davis JM, Maki J, Bahcall JK (2007) An in vitro comparison
of the antimicrobial effects of various endodontic medica-
ments on Enterococcus faecalis. Journal of Endodontics 33,
5679.
Dornelles-Morgental R, Guerreiro-Tanomaru JM, de Faria-
Junior NB, Hungaro-Duarte MA, Kuga MC, Tanomaru-Filho
M (2011) Antibacterial efcacy of endodontic irrigating
solutions and their combinations in root canals contami-
nated with Enterococcus faecalis. Oral Surgery Oral Medicine
Oral Pathology Oral Radiology and Endodontics 112, 396
400.
Dunavant TR, Regan JD, Glickman GN, Solomon ES, Honey-
man AL (2006) Comparative evaluation of endodontic
irrigants against Enterococcus faecalis biolms. Journal of
Endodontics 32, 52731.
Harrison AJ, Chivatxaranukul P, Parashos P, Messer HH
(2010) The effect of ultrasonically activated irrigation on
reduction of Enterococcus faecalis in experimentally infected
root canals. International Endodontic Journal 43, 96877.
Johal S, Baumgartner JC, Marshall JG (2007) Comparison of
the antimicrobial efcacy of 1.3% NaOCl/BioPure MTAD to
5.25% NaOCl/15% EDTA for root canal irrigation. Journal of
Endodontics 33, 4851.
Karale R, Thakore A, Shetty V (2011) An evaluation of
antibacterial efcacy of 3% sodium hypochlorite, high-
frequency alternating current and 2% chlorhexidine on
Enterococcus faecalis: an in vitro study. Journal of Conser-
vative Dentistry 14, 25.
Kho P, Baumgartner JC (2006) A comparison of the antimi-
crobial efcacy of NaOCl/BioPure MTAD versus NaOCl/
EDTA against Enterococcus faecalis. Journal of Endodontics 32,
6525.
Kishen A, Sum CP, Mathew S, Lim CT (2008) Inuence of
irrigation regimens on the adherence of Enterococcus
faecalis to root canal dentin. Journal of Endodontics 34,
8504.
Lendini M, Alemanno E, Migliaretti G, Berutti E (2005) The
effect of high-frequency electrical pulses on organic tissue in
root canals. International Endododontic Journal 38, 5318.
Molander A, Warfvinge J, Reit C, Kvist T (2007) Clinical and
radiographic evaluation of one- and two-visit endodontic
treatment of asymptomatic necrotic teeth with apical
periodontitis: a randomized clinical trial. Journal of Endodon-
tics 33, 11458.
Newberry BM, Shabahang S, Johnson N, Aprecio RM, Tora-
binejad M (2007) The antimicrobial effect of BioPure MTAD
on eight strains of Enterococcus faecalis: an in vitro investi-
gation. Journal of Endodontics 33, 13524.
Oliveira DP, Barbizam JV, Trope M, Teixeira FB (2007) In vitro
antibacterial efcacy of endodontic irrigants against Entero-
coccus faecalis. Oral Surgery Oral Medicine Oral Pathology Oral
Radiology and Endodontics 103, 7026.
Prabhakar J, Senthilkumar M, Priya MS, Mahalakshmi K,
Sehgal PK, Sukumaran VG (2010) Evaluation of antimi-
crobial efcacy of herbal alternatives (Triphala and green
tea polyphenols), MTAD, and 5% sodium hypochlorite
against Enterococcus faecalis biolm formed on tooth
substrate: an in vitro study. Journal of Endodontics 36,
836.
Aranda-Garcia et al. Antibacterial effectiveness of Endox Plus
2012 International Endodontic Journal International Endodontic Journal 5
Shabahang S, Aslanyan J, Torabinejad M (2008) The substi-
tution of chlorhexidine for doxycycline in MTAD: the
antibacterial efcacy against a strain of Enterococcus faecalis.
Journal of Endodontics 34, 28890.
Singla MG, Garg A, Gupta S (2011) MTAD in endodontics: an
update review. Oral Surgery Oral Medicine Oral Pathology
Oral Radiology and Endodontics 112, e706.
Siqueira JF Jr, Lima KC, Magalhaes FA, Lopes HP, de Uzeda M
(1999) Mechanical reduction of the bacterial population in
the root canal by three instrumentation techniques. Journal
of Endodontics 25, 3325.
Siqueira JF Jr, Rocas IN, Santos SR, Lima KC, Magalhaes FA,
de Uzeda M (2002) Efcacy of instrumentation techniques
and irrigation regimens in reducing the bacterial population
within root canals. Journal of Endodontics 28, 1814.
Soares JA, Roque de Carvalho MA, Cunha Santos SM et al.
(2010) Effectiveness of chemomechanical preparation with
alternating use of sodium hypochlorite and EDTA in
eliminating intracanal Enterococcus faecalis biolm. Journal
of Endodontics 36, 8948.
Stuart CH, Schwartz SA, Beeson TJ, Owatz CB (2006)
Enterococcus faecalis: its role in root canal treatment failure
and current concepts in retreatment. Journal of Endodontics
32, 938.
Torabinejad M, Khademi AA, Babagoli J et al. (2003a) A new
solution for the removal of the smear layer. Journal of
Endodontics 29, 1705.
Torabinejad M, Shabahang S, Aprecio RM, Kettering JD
(2003b) The antimicrobial effect of MTAD: an in vitro
investigation. Journal of Endodontics 29, 4003.
Torabinejad M, Shabahang S, Bahjri K (2005) Effect of MTAD
on postoperative discomfort: a randomized clinical trial.
Journal of Endodontics 31, 1716.
Virtej A, MacKenzie CR, Raab WH, Pfeffer K, Barthel CR
(2007) Determination of the performance of various root
canal disinfection methods after in situ carriage. Journal of
Endodontics 33, 9269.
Zhang W, Torabinejad M, Li Y (2003) Evaluation of cytotox-
icity of MTAD using the MTT-tetrazolium method. Journal of
Endodontics 29, 6547.
Antibacterial effectiveness of Endox Plus Aranda-Garcia et al.
International Endodontic Journal 2012 International Endodontic Journal 6