doi:10.1111/j.1365-2591.2012.02069.

Antibacterial effectiveness of several irrigating solutions and the Endox Plus system an ex vivo study

nior, G. M. Chavez-Andrade, A. R. Aranda-Garcia, J. M. Guerreiro-Tanomaru, N. B. Faria-Ju 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 nior NB, Chavez-Andrade GM, Leonardo RT, TanomaruJu Filho M, Bonetti-Filho I. Antibacterial effectiveness of several
irrigating solutions and the Endox Plus system an ex vivo study. International Endodontic Journal, 45, 10911096, 2012.

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 inoculated 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% NaOCl/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 persistence of contamination within the root canal system. Keywords: Endox Plus, Enterococcus faecalis, irrigating 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 (Bystro m & Sundqvist 1981, Siqueira

rio Tanomaru-Filho, Rua Humaita , 1680, Correspondence: Ma 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).

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

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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 discomfort and biocompatibility (Zhang et al. 2003, Torabinejad 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 alternating 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 comparison 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 preparation of ex vivo E. faecaliscontaminated root canals. The null hypothesis is that the Endox Plus system has the same antibacterial effectiveness as other methods.

isthmuses and ramications as observed after radiographic 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 mica, Ibipora 17% EDTA (Biodina , 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., Taboa o 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 ssico Artigos wells with self-curing acrylic resin (Cla gicos, Sa Odontolo o 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). Standard 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 1.5 108 CFU per mL. The optical density of the suspension was adjusted using a spectrophotometer (Model 600 Plus, Femto, Sa o 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 environment at 37 C for an incubation period of 21 days.

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 singlerooted human teeth were standardized to a length of 15 mm. Root canal anatomy was standardized using round/oval root canals without the presence of

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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 contamination 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 randomly divided into three experimental groups and two controls, as shown in Table 1. All root canals in the experimental and positive control groups were instrumented 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 instructions, 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.
Table 1 Experimental and control groups
Groups GI Endox Plus + SS GII NaOCl + MTAD GIII NaOCl + EDTA GIV Positive control (SS) GV Negative control n 15 15 15 15 10

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 neutralize 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 collection, using two absorbent paper points (Miltex, Inc., York, PA, USA) per specimen. The root canals were then lled with sterile saline, and the covered microplates 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 procedures.

Microbiological analysis
Microbiological analysis of the samples collected immediately 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

G, group; SS, saline solution; NaOCl, sodium hypochlorite solution. Total volume used (12 mL).

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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.

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 increased in groups I, II, III and IV, with no signicant differences amongst the groups, but differing signicantly from the previous (post-instrumentation) samples. 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). Enterococcus 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 recolonization may occur because of the persistence of microorganisms 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. Contrastingly, 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

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recovery of microorganisms that had survived in the dentine tubules after instrumentation. Virtej et al. (2007) compared the antibacterial effectiveness of Endox, MTAD, 3% NaOCl and HealOzone in root canals contaminated with mixed microbiota by analysing samples collected at three different timepoints: 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 irrigation. All groups allowed recovery of bacteria 7 days after treatment, demonstrating persistence of contamination in the RCS after the irrigation protocols.

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