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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 15  |  Issue : 4  |  Page : 234-240

Antibacterial efficacy of newly suggested root canal irrigants in differently prepared root canals


1 Department of Restorative Dental Sciences, College of Dentistry, King Khalid University, Abha, KSA
2 Department of Restorative Dental Sciences, College of Dentistry, King Khalid University, Abha, KSA; Department of Endodontics, Faculty of Dentistry, Tanta University, Tanta, Egypt
3 Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Tanta University, Tanta, Egypt
4 Department of Endodontics, Faculty of Dentistry, Tanta University, Tanta, Egypt

Date of Submission07-Jul-2018
Date of Acceptance15-Aug-2018
Date of Web Publication21-Jan-2019

Correspondence Address:
Mohamed I Elshinawy
Department of Restorative Dental Sciences, College of Dentistry, King Khalid University, Abha 61471, PO Box 3263, KSA

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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/tdj.tdj_27_18

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  Abstract 

Aim
To evaluate the efficacy of trisodium phosphate (Na3PO4) and sodium bicarbonate (NaHCO3) irrigations against Enterococcus faecalis in differently prepared root canals.
Materials and methods
Ninety laboratory-infected root canals were endodontically prepared using K-file (group 1), Mtwo rotary (group 2) and OneShape rotary (group 3) file systems. Canals in each group were irrigated with the minimum bactericidal concentrations of sodium hypochlorite (subgroup 1), Na3PO4(subgroup 2) and NaHCO3(subgroup 3) solutions. Another 10 infected unprepared canals served as control group. Samples obtained from lumens and dentin linings of root canals were used to count the residual bacteria in each subgroup. Both two-way analysis of variance and Mann–Whitney comparisons at P value less than or equal to 0.05 were used to statistically analyze the results.
Results
All tested subgroups showed lower no of colony-forming units than the control (P < 0.05). Regardless the utilized irrigation, rotary-prepared root canals in group 2 and group 3 exhibited lower colony-forming units in comparison to hand-prepared ones in group 1 (P < 0.05). Both sodium hypochlorite and NaHCO3 showed the highest antibacterial efficacy in group 1 and group 3 (P < 0.05), while no difference was recorded between the utilized irrigation solutions in group 2 (P > 0.05).
Conclusion
Although none of the tested irrigation solutions are capable of completely eradicating E. faecalis, both Na3PO4 and NaHCO3 seem effective when used together with the nominated file systems.

Keywords: antibacterial efficacy, endodontic irrigation solutions, Enterococcus faecalis, rotary file system


How to cite this article:
Abdelaziz KM, Elshinawy MI, Al-Madboly LA, Ghoneim WM. Antibacterial efficacy of newly suggested root canal irrigants in differently prepared root canals. Tanta Dent J 2018;15:234-40

How to cite this URL:
Abdelaziz KM, Elshinawy MI, Al-Madboly LA, Ghoneim WM. Antibacterial efficacy of newly suggested root canal irrigants in differently prepared root canals. Tanta Dent J [serial online] 2018 [cited 2019 May 26];15:234-40. Available from: http://www.tmj.eg.net/text.asp?2018/15/4/234/250456


  Introduction Top


Complete debridement and elimination of microbial irritants is a fundamental prerequisite for successful endodontic therapy. Because of the complexity of root canal anatomy, these tasks cannot be completely achieved by only mechanical instrumentation. Therefore, many reports recommended the use of the chemo-mechanical preparation to achieve the most favorable cleaning effect [1],[2].

Although the time saving advantage of nickel–titanium rotary file systems has been proved in preparing root canals, the cleansing outcomes could be affected by the design features of these instruments [3]. As a result, many rotary file systems had been introduced to dental market. Mtwo rotary system had been introduced with a noncutting tip and S-shaped cross-section. This design is claimed to shape the entire length of the canal with minimum transportation of debris towards the apex [4]. In addition, OneShape single rotary file systemhas recently been introduced with different cross-sectional designs through the entire length of the working part. These features simplified the instrumentation protocols, and helped reduce the risk of cross-contamination [5].

Enterococcus faecalis is a virulent Gram positive facultative anaerobic bacterium that could adhere to root canal dentin, resist the intracanal medicaments and dominates in persistent endodontic infections [6]. In spite of this challenging situation, some root canal irrigants seemed effective against this kind of microbes [7]. The antimicrobial potency of sodium hypochlorite (NaOCl), the most commonly used root canal irrigant, is normally increased in higher concentrations that probably threaten the periapical tissue [8]. Some researchers [9],[10], at the same time, reported unacceptable antimicrobial efficacy of this irrigant in lower concentrations.

Sodium bicarbonate (NaHCO3) is widely used in dentifrices and mouth rinses industries. This agent is known by its safety, water solubility and acid buffering properties in addition to its antibacterial characteristics [11]. Trisodium phosphate (Na3 PO4) is a biocompatible chemical, naturally present in bones, and its antimicrobial effect was documented against many microbial strains including; Staphylococcus aureus,  Escherichia More Details coli,  Salmonella More Details spp., Campylobacter spp., and Pseudomonads spp. [12],[13]. However, the effectiveness of both NaHCO3 and Na3 PO4 against E. faecalis has not hitherto been studied.

Based on the aforementioned information, this in-vitro study aimed to investigate the antimicrobial efficacy of the aqueous solutions of NaHCO3 and Na3 PO4 when used in combination with either Mtwo or OneShape rotary file system to chemo-mechanically debride root canals. The null hypothesis, accordingly, was that none of the suggested chemo-mechanical combinations is able to completely eradicate the root canal microbes.


  Materials and Methods Top


One hundred freshly extracted human single-canalled premolars free from cracks or any developmental defects were collected from orthodontic private clinics after approval of the study protocol by The Research Ethics Committee at the Faculty of Dentistry, Tanta University, Egypt. The teeth were cleaned of both hard and soft deposits, disinfected and stored in distilled water in an incubator (45-EchoTherm Bench Top Incubator; Torrey Scientific Inc., West Carlsbad, California, USA) at 37 ± 1°C till the time of use.

Aqueous solutions for both of NaHCO3(Lombardi, Lucca, Italy), Na3PO4(Pandora Industries Ltd, New Delhi, Delhi, India) and NaOCl (Clorox Co., 10th of Ramadan, Egypt) were prepared and nominated as root canal irrigants in the current study.

Susceptibility of the test strains to different root canal irrigants

Resazurin assay was used to determine both the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the nominated irrigation solutions against E. faecalis (ATCC 29212; Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Tanta University, Tanta, Egypt). An equimolar volume of each of the nominated irrigation solutions was poured into individual wells in microtitration well plate each containing 100 μl of tryptic soy broth, shaked and then transferred to the next wells in a two-fold serial dilution manner. A 10 μl of 107 colony-forming units/ml of the selected bacterium was prepared and transferred to each well followed by the addition of 10 μl of 7.5 mg/ml Resazurin solution. The wells were then incubated for 24 h at 37 ± 1°C before checking for the color change from violet to pink that indicates the microbial growth. The same procedures were repeated three times before calculating the means and SDs for both MIC and MBC of each solution [14]. The collected data were statistically analyzed using both one-way analysis of variance (ANOVA) and Tukey's tests on Past software (version 2.17; Natural History Museum, University of Oslo, Oslo, Norway) to indicate the significance of differences detected between the nominated irrigation solutions.

Root specimens' preparation and sterilization

The collected teeth were decoronated using water-cooled diamond disc (Edenta AG, AU/SG, Switzerland) leaving root specimens of standardized 12 mm length. Canal lumen of each tooth was initially cleaned and standardized using size 20 initial apical K-Files (Dentsply Maillefer) at a working length of 11 mm under irrigation with distilled water. Root canals of all teeth were then conditioned with 10 ml of 17% EDTA (Pulpdent Corp., Watertown, Massachusetts, USA) for 3 min to remove the smear layer and washed with 5 ml of distilled water before their drying with size 20 paper points (Dentsply Maillefer, Ballaigues, VD, Switzerland) [15],[16].

After steam sterilization (Tomy ES-315; Tomy Tech Inc., Fremont, California, USA) for 20 min at 121 ± 1°C, apices of all roots were covered with resin composite (Z350; 3M ESPE, St. Paul, Minnesota, USA) and the external root surfaces were sealed with epoxy resin (Araldite, Brascola, Joinvile, SC, Brazil) under complete aseptic conditions inside laminar air flow chamber (BAVnp-01-Laminar-S-1.2; Lamsystems GmbH, Berlin, Germany) [17]. Each specimen was transferred to brain heart infusion (BHI) broth (Oxoid Limited, Basingstoke, Hampshire, UK) and incubated for 24 h at 37 ± 1°C to check their sterility up. These teeth were transferred to 2 ml sterile physiologic saline in individual tubes to wash out the BHI and reincubated for another 24 h [16].

Inoculation of roots with E. faecalis biofilm

Root canals of all teeth were carefully dried with sterile paper points under aseptic conditions. A single colony from the microbial overnight cultures was suspended in BHI to prepare 0.5 McFarland (1 × 108 cells/ml). Equal portions of the microbial suspension were mixed in Falcon tubes containing the premolar teeth then incubated under anaerobic conditions at 37 ± 1°C for 1 week in order to ensure a mature biofilm formation [18]. The culture broth was replaced with fresh one every second day to ensure bacterial viability and to remove dead cells. By the end of the incubation period, specimens were picked up from the tubes under strict aseptic conditions and washed with sterile phosphate-buffered saline solution to remove unbound microbes as well as the culture media [19].

Chemo-mechanical debridement of root canals

Ninety of the infected root canals were then chemo-mechanically debrided using three different file systems (groups 1–3, n = 30 each), while the remaining 10 root canals were left with no chemo-mechanical debridement to serve as control group. The debridement procedures took place in laminar air flow chamber to ensure complete aseptic conditions. Root canals in group 1 (G1) were prepared using hand stainless steel K-files (Dentsply Maillefer) in step-back technique up to (#35/0.02) master apical file.

In group 2 (G2), instrumentation was carried out up to #35/0.04 master apical file using Mtwo nickel–titanium rotary system (VDW, Munich, Germany) in gear reduction handpiece powered with a torque limited electric motor (Endo-mate TC, NSK, Tokyo, Japan) while in group 3 (G3), OneShape single rotary file (#25/0.06) (Micro-Mega, Besanc, on Cedex, France) was used to prepare the root canals. Instruments in all groups were used according to their manufacturers' instructions. Canals' irrigation in each group was performed in three subgroups (SG1–3, n = 10) using a total volume of 10 ml of the prepared MBC of each assigned irrigating solution [Table 1] and [Table 2].
Table 1: Mean minimum inhibitory concentration and minimum bactericidal concentration values (molar concentration) of the tested irrigation solutions for Enterococcus faecalis

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Table 2: Root canal chemo-mechanical preparation grouping

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Quantitative determination of the viable microbes in the biofilm

To determine the bacterial count (colony-forming units/ml) immediately after instrumentation, 5 ml of distilled water was introduced into each canal as a final irrigation step. Microbiological samples were collected from lumens of the canals by introducing three sterilized #15 paper points into root canal and left undisturbed for 1 min. In addition, samples from canal dentin walls were collected using sterile #25 K-files introduced into the debrided canals to the proper working length with circumferential pulling strokes for 30 s. The cutting blades of these files were separated and dropped together with the paper points for each root canal into an Eppendorf tube containing 0.5 ml of BHI broth and vortexed for 30 s. Each sample was subjected to five times serial two-fold dilutions before getting 10 μl of each planted onto BHI agar plates. All plates were incubated at 37 ± 1°C for 24 h. The viable microbial counts in all subgroups were then collected and their mean values were recorded to the log of 10 [16]. The collected data was analyzed using two-way ANOVA and Mann–Whitney's tests on Past software (Natural History Museum) version 2.17 to determine the significance of differences detected between subgroups.


  Results Top


The mean MIC and MBC of the utilized irrigation solutions against E. faecalis are listed in [Table 1] The one-way ANOVA test indicated significant differences between the tested irrigation solutions in terms of MIC (P = 1.12E − 09) and MBC (P = 2.47E–10). The lowest and highest MIC and MBC were recorded for NaOCl and Na3 PO4 respectively (Tukey's, P ≤ 0.05).

The mean bacterial counts in all groups are presented in [Table 3]. Two-way ANOVA test indicated significant differences between groups (canal preparation techniques, P < 0.0001) and between subgroups (irrigation solutions, P < 0.0001) in addition, a significant interaction between both variables was detected (P < 0.0001). Mann–Whitney comparisons indicated lower bacterial count in the prepared root canals of all test groups in comparison to the control (P < 0.05). At the same time, a significant differences were declared among all test groups (P < 0.05), where the highest bacterial count was recorded in G1 and the lowest was recorded in G2. The exception was between G2 and G3 (P = 0.6774), where no difference in the bacterial counts was detected in canals irrigated with NaHCO3(SG3).
Table 3: Mean bacterial count obtained from root canals of different subgroups

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The difference in the effect of the utilized irrigation solutions on the bacterial count was significant when each was used in conjunction with hand files to prepare root canals of G1 (P < 0.05), while no difference at all (P > 0.05) was noticed between the same irrigation solutions when combined with Mtwo rotary system in the preparation of root canals in group 2. In comparison to other irrigation solutions, NaHCO3(SG3) showed more potent effect on the bacterial count than both NaOCl and Na3 PO4(P = 0.0001827 and 0.0001817) when used, respectively, with OneShape rotary system to prepare root canals of G3.


  Discussion Top


The success of endodontic treatment is greatly dependent on getting rid of root canal microbes those reported to be responsible for post endodontic infections [20]. E. faecalis was usually isolated from cases of failed endodontic treatment and persistent periapical inflammation [21],[22]. This virulent microbe has the ability to deeply penetrate into dentinal tubules, forming biofilms on dentin walls and survive in both high and low pH situations. These features render this microbe resistant and remain vegetative even after root canal treatment [23],[24]. In spite of the popularity of NaOCl, its efficiency is noticed in higher concentrations those could harm the periapical tissues [25]. Therefore, both Na3 PO4 and NaHCO3 solutions were suggested in this study to irrigate root canals based on their safety and proved antimicrobial properties [11],[12],[13] though none of them had been used for this purpose before.

In this study, EDTA was used during the initial preparation of the tested root canals to help remove smear layer that could harbor many types of microbes [26]. This procedure together with steam sterilization of the initially prepared roots canals were supposed to offer microbe-free dentin surfaces before contaminating root canals with E. faecalis. This maneuver will help avoid the effect of any residual microbes on the outcomes of the suggested root canal chemo-mechanical debridement procedures.

Based on the fact that, cleaning efficiency of rotary files are affected by their design and working techniques [3], both Mtwo and OneShape rotary file systems were recruited to this study to be compared to the standard stainless steel K-files.

The MIC and MBC evaluation indicated clear differences between the tested irrigation solutions. The lowest recorded MIC and MBC of NaOCl against E. faecalis might be referred to its high alkalinity and hypertonicity that osmotically draw out cell fluids in addition to its oxidative and cell-protein hydrolyzing effects [27]. However, some previous studies revealed a necessity of using either a high concentration or repeated copious irrigation with a low concentration of NaOCl to obtain the desired bactericidal effect [9],[10]. The noticed antimicrobial effect of Na3 PO4 against E. faecalis could be referred to its high alkalinity and chelation of divalent cations in the outer bacterial cell membrane with subsequent adverse effects on cell components [28]. These mechanisms were supported by Sampathkumar et al. [13], who declared the role of the high pH of Na3 PO4 solution in damaging the bacterial cells. On the other hand, NaHCO3 showed lower MIC than Na3 PO4. This result might be related to its alkalinity, water solubility and acid buffering property that neutralizes bacterial-produced acids in dental biofilm[11],[29]. Moreover, the hypertonicity of NaHCO3 solution increases its bactericidal abilities as it derives water out of microbial cells causing their dehydration and eventual death [30]. Previous reports [29],[31],[32] indicated suppression of bacteria in dental biofilm in contact with NaHCO3, although its full antibacterial efficacy might need a relatively longer contact time to show up.

The recorded results indicated lower bacterial counts in all chemo-mechanically prepared root canals in comparison to the control (undebrided root canals). This finding could be supported by previous postulation that mechanical instrumentation dramatically reduces the bacterial counts of the infected root canals [33]. Several researchers [1],[2] also recommended the chemo-mechanical debridement of root canals aiming to achieve considerable canal preparation. This procedures help detach the bacterial biofilm and the infected dentin layer exposing them to the bactericidal and washing actions of the utilized irrigations.

At the same time, there was a significant difference between the three test groups (canal preparation techniques) regardless the utilized irrigation solutions. Generally, rotary-prepared root canals in G2 and G3 exhibited lower bacterial counts in comparison to those prepared with hand files in G1. These finding could be related to the expected higher cleaning and shaping efficiency of rotary-driven files that may accordingly help deeper cutting into the infected root canal dentin. This explanation came in agreement with the report of Guelzow et al. [34], that declared higher efficiency of six different rotary file systems in preparing root canals than hand files. Other studies [35],[36],[37] also proved the failure of traditional hand instruments in achieving adequate cleaning and shaping particularly in curved canals. On contrary, others [38],[39] indicated no difference in root canal debridement efficiency between rotary and hand files. This contradiction could be a result of using different file systems and irrigation solutions.

Using Mtwo system in preparing root canals in G2 showed lower bacterial counts than those obtained from canals prepared with OneShape system in G3. This difference might be related to different designs of the utilized instruments [3]. Mtwo file series having italic S-shaped cross-sections with two cutting blades with positive rake angles those maximize their dentin cutting effectiveness. The presence of variable helical angles also reduces intracanal file threading tendency and packing of dentin debris towards the apex [4]. On the other hand, OneShape is a single file system having three different cross-sectional designs over its entire cutting length together [5]. One study [40] revealed better cutting efficiency of Mtwo file system in comparison to other rotary systems with different designs and working actions. Another study [41] revealed superior shaping ability and cleaning effectiveness of the Mtwo file system to WaveOne single file systems that proved in a different study to have better cleaning performance than OneShape single file system[42].

In spite of using the predetermined MBC of each irrigation solution, clear differences were recorded between the total bacterial counts in the three tested subgroups of G1. The highest antibacterial efficacy was noticed with NaOCl in SG1 and the lowest with Na3 PO4 in SG2. The noticed difference could be related on one hand to the inability of hand filing to completely detach E. faecalis biofilms and the outer infected dentin layers and on the other hand to the difference in the organic dissolving potentials of the utilized chemical irrigants [9],[29],[35],[36],[37]. Many studies demonstrated higher alkalinity and organic dissolving effect of NaOCl in comparison to NaHCO3 and Na3 PO4, respectively [9],[10],[27]. Although the alkalinity of Na3 PO4 is higher than NaHCO3, the noticed higher antibacterial efficacy of NaHCO3 in SG3 could be referred to its ability to generate CO2 on heating that results from files' mechanical movement during canal preparation [13],[43],[44],[45].

On the other hand, no difference was noticed between the same irrigation solutions when combined with the use of Mtwo rotary file system to prepare root canals of G2. This finding reflected the higher cleaning efficacy of the utilized system in debriding root canals [5],[34]. Mtwo file system was reported to follow the original root canal path and to produce less amount of minimally contaminated smear layer at the time of canal preparation. These features helped minimize the existing bacterial biofilms and allowed proper contact of the utilized irrigation solutions with the residual bacterial population [37],[45],[46]. Unexpectedly, NaHCO3 solution showed the most potent effect on the E. faecalis in comparison to other irrigation solutions when used with OneShape rotary file system to prepare root canals in G3. This finding reflected a lower canal debriding capability of the OneShape in comparison to Mtwo rotary file system that could be related to the difference in size and taper of the utilized master apical files of both systems. Previous reports [47],[48] indicated marked reduction in the residual intracanal bacterial population as a result of increasing the size of apical preparation. The encountered results might also be related not only to the difference in the design and manufacturing properties of the utilized file systems, but also to the better antimicrobial effect of NaHCO3 with its high solubility and CO2 releasing capability [11],[45].

Although our results indicated significant interaction between canal preparation techniques and the types of irrigation solutions, none of the utilized chemo-mechanical debridement protocols was capable of complete microbial eradication. This finding not only directed toward the acceptance of the drawn null hypothesis, but also came in agreement with many of the previous studies that denied the ability of either hand or rotary files to reach all root canal topographical details [49],[50]. Others also elaborated reduced outcomes of chemo-mechanical debridement from the coronal to the apical parts of root canals [49],[51]. Therefore, some authors [39],[52] recommended the continuing search for more effective irrigation solutions and canal debridement techniques for better outcomes of endodontic treatment. In respect to that recommendation, the current study deduced a noticeable efficacy of both Na3 PO4 and NaHCO3 against E. faecalis when used to irrigate rotary-prepared root canals. However, the biocompatibility of the utilized MBCs of the suggested irrigation solutions together with their effect on dentin surface properties and bonding of contemporary endodontic sealers should be addressed before recommending these solutions for routine use.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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