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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 32  |  Issue : 3  |  Page : 112-117

Antimicrobial efficacy of intracanal and extracanal heated sodium hypochlorite against Enterococcus faecalis: An in vitro study


Department of Conservative Dentistry and Endodontics, Yogita Dental College and Hospital, Khed, Maharashtra, India

Date of Submission14-Feb-2020
Date of Decision14-Mar-2020
Date of Acceptance14-Jun-2020
Date of Web Publication28-Oct-2020

Correspondence Address:
Dr. Deepali Samel
Department of Conservative Dentistry and Endodontics, Yogita Dental College and Hospital, Near Narangi River Side, Khed, Ratnagiri - 415 709, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/endo.endo_21_20

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  Abstract 


Introduction: Enterococcus faecalis is the most common bacterial species in persistent root canal infections. Sodium hypochlorite (NaOCl) has shown antibacterial activity against E. faecalis at room temperature. It has been proven that heating enhances the properties of NaOCl. Thus, the aim of the present study is to compare the effectiveness of intracanal and extracanal heated NaOCl against E. faecalis.
Methodology: The specimens were decoronated up to cementoenamel junction and prepared up to size F3 and were inoculated with E. faecalis. Then, teeth were divided into four groups and irrigation was performed. Group A - 5% NaOCl (Prime Dental Products Pvt. Ltd., India) at room temperature, Group B - 5% NaOCl (Prime Dental Products Pvt. Ltd., India) heated extracanal at 600C in a water bath, Group C - 5% NaOCl (Prime Dental Products Pvt. Ltd., India) intracanal heated using 980 Nm diode laser, and Group D - 5% NaOCl (Prime Dental Products Pvt. Ltd., India) intracanal heated using Elements System-B. The antimicrobial efficacy was tested by collecting transfer fluid and counting the colony forming units of viable E. faecalis on agar plates.
Results: Following the disinfection protocol significant reduction in E. faecalis count was seen in the groups irrigated using intracanal heated and extracanal heated NaOCl with that of NaOCl used at room temperature with a significant value of P ≤ 0.001*.
Conclusion: Within the limitations of the present study, it can be concluded that intracanal heating using Elements System-B Heat Source and Diode Laser has better antimicrobial efficacy as compared to that of NaOCl extracanal heated and at room temperature.

Keywords: Diode laser, elements system B, Enterococcus faecalis, sodium hypochlorite


How to cite this article:
Rathore V, Samel D, Moogi P, Bandekar S, Kshirsagar S, Vyas C. Antimicrobial efficacy of intracanal and extracanal heated sodium hypochlorite against Enterococcus faecalis: An in vitro study. Endodontology 2020;32:112-7

How to cite this URL:
Rathore V, Samel D, Moogi P, Bandekar S, Kshirsagar S, Vyas C. Antimicrobial efficacy of intracanal and extracanal heated sodium hypochlorite against Enterococcus faecalis: An in vitro study. Endodontology [serial online] 2020 [cited 2020 Nov 27];32:112-7. Available from: https://www.endodontologyonweb.org/text.asp?2020/32/3/112/299283




  Introduction Top


The main purpose of root canal treatment is to eliminate the microorganisms and their byproducts from the root canal and its tubular system, and also to prevent the re-entry of the microorganisms into the root canal system.[1] Using only irrigating solutions or mechanical methods is incapable of thoroughly cleaning this complex tubular system.[2],[3] Thus, chemicomechanical methods are used in conjunction to enhance the cleaning efficacy in the infected root canals. When the bacterial infection occurs in the root canal system and the bacteria penetrate into the deep dentinal layers, cleaning of the dentinal tubules of the root canal becomes more difficult.[4]

Enterococcus faecalis is the most common bacterial species in resistant or recurrent infections leading to endodontic treatment failures.[5],[6] These cocci can resist the antibacterial agents such as sodium hypochlorite (NaOCl) through different mechanisms.[7],[8] In vitro studies showed that E. faecalis can form biofilm and invade dentinal tubules even under stressed conditions.[9] It is also resistant to alkaline pH and consequently to Ca (OH)2 pastes, that normally inhibits other bacteria.[9] The related mechanism may be related to the existence of a working active proton pump in the cellular membrane of this bacteria.[9]

Many irrigants have been utilized with the mechanical methods to thoroughly clean the root canal system. The existing irrigants remove microorganisms through direct contact with them and are thus unable to penetrate deep into the dentinal tubules and clean them.[9] NaOCl is the most commonly used endodontic irrigant.[10],[11] Many investigations have demonstrated the efficacy and the advantages of NaOCl but the problems arising from low penetration depth into the dentinal tubules have resulted in reduced desire to apply it.[9] The current literature suggests various techniques to improve the effectiveness of NaOCl as irrigating solution.[11],[12] For instance, the use of greater amount of irrigant and preheating of the irrigant. Cunningham has shown that a NaOCl solution at body temperature allows to carry out the sterilization in considerably less time compared to the same solution at room temperature.[13] Preheated NaOCl solution has greater ability to dissolve pulp tissue and cleanse the canal.[11] Woodmansey has shown that hypochlorite at boiling temperature is able to disintegrate the pulp tissue at speed 210 times higher compared to the solution at room temperature.[14]

However, according to Iandolo et al., NaOCl preheated to 50°C, 60°C, and 70°C stabilized in a few seconds to body temperature. Thus, the most critical area from a cleaning point of view, i.e., apical third was never able to reach 40°C. Whereas intracanal heating using system B gives a temperature rise of 40°C for 57 s.[15] Till date, there is a void in literature comparing the antimicrobial efficacy of intracanal heated NaOCl using system B against the most persistent E. faecalis.

Thus, the present in vitro study was performed to compare the disinfection ability of intracanal heated and preheated NaOCl in endodontic treatment of teeth infected with E. faecalis.

Sample preparation

This study was performed on 80 single-rooted, single-canal, intact mandibular premolars. Crowns of all teeth were cut off at cemento-enamel junction using a high-speed handpiece and the root lengths were standardized to a 15 mm length. All the root canals were prepared using the ProTaper Rotary System (Dentsply Maillefer, Ballaigues, Switzerland) up to F3.

After each instrument change, canals were passively irrigated with 2 ml of 5% NaOCl solution using 30 Guage side vented needle (NeoEndo, Orikam Health Care). The final irrigation was done with 5 ml of 5% NaOCl (Prime Dental Products Pvt. Ltd., India) followed by 5 ml of 17% ethylenediaminetetraacetic acid (EDTA) (Prevest Denpro Pvt Ltd, India) for 1 min. The apical foramen was sealed using a Glass Ionomer cement (GC America, Japan) and the external surfaces of the teeth were covered with two layers of colorless nail varnish to prevent liquid penetration.

For sterilization of the samples, each tooth was placed in a cryo-tube containing sterile brain-heart infusion (BHI) agar and was autoclaved under operating conditions of 121°C and 15 psi pressure for 30 min. One tooth was randomly selected and incubated to ensure the negative culture and sterilization of samples. Then, 50 μL of E. faecalis suspension (ATCC 29212) was inoculated in 5 mL of BHI and was kept in an incubator at 37°C for 24 h. The concentration of the test suspension was then adjusted to 0.5 McFarland standard turbidity. Under sterile condition, each tooth was brought out of the tube containing BHI and its root canal was dried using sterile paper points size F3 (Dentsply, Maillefer, Ballaigues, Switzerland). About 0.01 mL standard value of the bacterial suspension was then placed in to each root canal using sterile 1 ml insulin syringe. Thereafter, each sample was placed in a new tube containing BHI and was stored at 37°C for 2 weeks. During this period, the intracanal suspension was replaced with 0.01 mL of a new suspension every 48 h.

Root canal disinfection

After 2 weeks, specimens were randomly divided into four groups (n = 20):

  • Group A - The BHI broth inside the root canals was dried using sterile paper points (size F3) (Dentsply, Maillefer, Ballaigues, Switzerland). Followed by irrigation with 5 mL of 5% NaOCl (Prime Dental Products Pvt. Ltd., India) for 5 min using 30 Guage side vented needle (NeoEndo, Orikam Health Care Pvt., Ltd.)
  • Group B - Intracanal BHI broth was dried using sterile paper points (Size F3) (Dentsply, Maillefer, Ballaigues, Switzerland). Followed by irrigation with 5 ml of 60°C preheated (Temperature confirmed using Laboratory Thermometer) 5% NaOCl (Prime Dental Products Pvt. Ltd., India) in water bath for 5 min using 30 Guage side vented needle (NeoEndo, Orikam Health Care Pvt. Ltd.)
  • Group C - Intracanal BHI broth was dried using sterile paper points (Size F3) (Dentsply, Maillefer, Ballaigues, Switzerland). Followed by irrigation with 5 ml of 5% NaOCl (Prime Dental Products Pvt. Ltd., India), intracanal heated using diode lasers. For the laser treatment protocol, a diode laser was applied with the wave length of 980-nm, output power of 1.5 W, continuous wave mode, and 10-ms pulse duration and 10-ms pulse interval. The laser was irradiated into the canals to a depth of 1 mm shorter than the actual root canal length, using an optical fiber with a diameter of 320 μm.
  • Group D - Intracanal BHI broth was dried using sterile paper points (Size F3). Followed by irrigation with 5 ml of intracanal heated NaOCl (Prime Dental Products Pvt. Ltd., India) at 180°C using Elements System-B Heat Source.[16]


Root canal sampling

After disinfection of the root canal with NaOCl, the root canals were filled with sterile normal saline as a transfer fluid. Samples of dentinal shavings from each root canal were obtained using #30 H Files. In each specimen, a new file was used. The transfer fluid and dentinal shavings from each canal were collected using sterile paper points (Size F3).

#30 H-file and the paper point from each tooth were placed in a test tube containing 10 ml of normal saline. The test tube was then shaken in vortex mixer for 20 s. Finally, 50 μL of vortexed normal saline was inoculated in a plate containing nutrient agar.

Followed by incubation at 37°C for 24 hrs, the number of colony-forming units (CFU) was further determined by a colony counter.

Statistical analysis

Descriptive statistics were expressed as mean ± standard deviation for each group for bacterial count and mean reduction of bacterial count (CFU) - Four groups were compared for mean reduction of bacterial count by analysis of variance. Viable bacterial count among all four groups was done by Wilcoxon sign-rank test. Simple/ multiple bar charts; were used for data presentation in the above tests, P ≤ 0.05 was taken to be statistically significant. The data were entered into Microsoft Excel 2010. All analyses were performed using Statistical Package for Social Sciences software version 17 (IBM, Chicago).


  Results Top


In the present study, descriptive statistics for mean reduction of E. faecalis count was seen as Group A (1131.7500 ± 88.97361), Group B (1259.1500 ± 80.36121), Group C (1300.9500 ± 100.50005), and Group D (1320.0000 ± 69.58524) [Table 1] and [Graph 1]. ANOVA test for multiple comparison showed that there was statistically significant difference in between the groups Group A and Group B (−127.40000 ± 27.07240), Group A and Group C (−169.20000 ± 27.07240), and Group A and Group D (−188.25000 ± 27.07240) which was less than P < 0.001* [Table 2].
Table 1: Descriptive statistics for mean reduction bacterial count (colony forming unit) among four groups

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Table 2: Analysis of variance for multiple comparison

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Thus, from the results of the above test following the disinfection protocol significant reduction in E. faecalis count was seen in the groups irrigated using intracanal heated and extracanal heated NaOCl with that of NaOCl used at room temperature.


  Discussion Top


The present study evaluated and compared the antimicrobial efficacy of NaOCl at room temperature, NaOCl 60°C Preheated in water bath, and intracanal heated NaOCl using Diode Lasers and Elements System-B Heat Source.

The presence of bacteria within the complex network of the root canal system and the dentinal tubules is considered to be the most important reason for endodontic treatment failure. Thus, complete elimination of bacteria and their toxins is the fundamental of a successful endodontic treatment.[9]

During root canal preparation using chemomechanical approach, the superficial layers of dentin and a part of infected pulp tissue can be removed; moreover, the effect of chemical irrigants is limited to the superficial layers of the root dentin.[2],[3],[9] When it comes to the irrigating solutions, it is well known that NaOCl is a widely used solution due to its bactericidal activity, excellent lubrication, ability to dissolve organic components, and low surface tension.[15],[16]

It has been demonstrated that the antibacterial effectiveness of NaOCl is affected by its concentration, volume, contact time, and temperature in the root canal. Previous studies have shown that high concentrations of NaOCl are needed for the elimination of bacteria. Other than changing its concentration, NaOCl irrigants could be heated to increase its effectiveness. This appears to improve its immediate tissue-dissolution capacity as well as its effectiveness in removing organic debris and bactericidal action.[17],[18] In addition, the systemic toxicity of preheated NaOCl irrigants, once they have reached body temperature, is lower than nonheated counterparts with similar efficacy in the root canal.[16]

Many investigations have used E. faecalis to evaluate the disinfection potential of antibacterial agents or various kinds of irrigating solutions activation techniques [19],[20] as these cocci are highly resistant to many disinfecting agents and also is particularly important in persistent endodontic infections and failed root canal treatments.[7],[9] E. faecalis is very heat-resistant,[7],[9] hence, it is recommended to be used to evaluate the heat-dependent antimicrobial effect of NaOCl.

In order to compare the results with the previous investigations, a similar design with minimal differences in microbiologic methods and diode laser, Elements System-B Heat Source settings was used.

In the given study, colony counter has been used to assess CFUs as it is easily available, less time consuming, and cost-effective method. Other methods namely polymerase chain reaction and DNA-DNA hybridization can be used for similar studies but these methods are mainly used for large scale epidemiological studies to detect a target microorganisms.

In this study, the laser irradiation protocol was selected based on the manufacturer's setting applied with the wave length of 980-nm, output power of 1.5 W, continuous wave mode, 10-ms pulse duration, and 10-ms pulse interval. Similar to other studies by Sohrabhi et al.,[11] Ashofteh et al.[19] and Kanumuru & Subbaiah,[20] in the present study, the optical fiber of the laser (320 μm) was directly inserted into the root canal filled with NaOCl. Thus, the fiber tip was used to dissipate heat to the NaOCl.

Furthermore, Elements System-B Heat Source set at 180°C was used with X-fine tip (30/04) at 3 mm shorter than the working length; the tip was not in contact with dentinal walls and was activated for 8 s and then left nonactivated for 10 s; this activation procedure was repeated 10 times and NaOCl was refreshed with new solution at each cycle.[16]

In the present study, intracanal Heating using Elements System-B Heat Source at 180°C and Diode lasers with wavelength 980 nm showed effective antimicrobial efficacy against E. faecalis 100% and 99.9%, respectively. As compared to that of NaOCl heated at 60° in water bath and NaOCl at room temperature.

This increased efficacy can be attributed to the thermal effect of intracanal heating of NaOCl. As explained by Iandolo et al.[15] NaOCl preheated to 50°C, 60° C and 70°C stabilized in a few seconds to body temperature Furthermore, in the apical third, the most critical area from a cleaning point of view, was never able to reach 40°C. While with the intracanal heating technique of NaOCl using the heat carrier 30/04 of System-B managed to bring the irrigant to a higher temperatures for a longer time span 40°C for 57 s.[15]

Ashofteh et al.[19] observed a 97.56% reduction in the amount of bacteria using a 830-nm diode laser with output power of 1.5 W and claimed that diode laser can be considered as an alternative technique for root canal disinfection. In another study, Rahimi et al.[21] reported that laser is less effective in root canal disinfection compared to combined use of laser and NaOCl; hence, using laser in combination with root canal irrigants for intracanal heating was recommended.

The main difference between the present study and the other similar studies was that along with laser-assisted endodontics intracanal heating with Elements System-B Heat Source Element was also evaluated and compared.

In addition to the antimicrobial effects, intracanal heating of NaOCl using diode lasers and Elements System-B Heat Source could have some other beneficial effects. In the study by Parirokh et al.,[22] it was showed that application of diode laser after smear layer removal could successfully occlude the dentinal tubules particularly in apical third area which will decrease the risk of reinfection also study done by Iandolo et al.[16] concluded that the final irrigation protocol based on intracanal heated NaOCl at 180°C with System-B Heat Source shows to be better than preheated NaOCl at 50°C in obtaining clean canal walls without the use of EDTA as an irrigating solution.[16]

Thus, intracanal heating of NaOCl can give better clinical results than that of NaOCl at room temperature.


  Conclusion Top


Within the limitations of the present study, it can be concluded that intracanal heating using Elements System-B Heat Source and Diode Laser has better antimicrobial efficacy as compared to that of NaOCl extracanal heated (60°) and at room temperature.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Vahdaty A, Pitt Ford TR, Wilson RF. Efficacy of chlorhexidine in disinfecting dentinal tubules in vitro. Endod Dent Traumatol 1993;9:243-8.  Back to cited text no. 1
    
2.
Byström A, Sundqvist G. Bacteriologic evaluation of the efficacy of mechanical root canal instrumentation in endodontic therapy. Scand J Dent Res 1981;89:321-8.  Back to cited text no. 2
    
3.
Dalton BC, Orstavik D, Phillips C, Pettiette M, Trope M. Bacterial reduction with nickel-titanium rotary instrumentation. J Endod 1998;24:763-7.  Back to cited text no. 3
    
4.
Nair PR, Sjogren U, Krey G, Kahnberg KE, Sundqvist G. Intraradicular bacteria and fungi in root-filled, asymptomatic human teeth with therapy-resistant periapical lesions: A longterm light and electron microscopic follow-up study. J Endod 1990;16:580-8.  Back to cited text no. 4
    
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Haapasalo M, Orstavik D.In vitro infection and disinfection of dentinal tubules. J Dent Res 1987;66:1375-9.  Back to cited text no. 5
    
6.
Sundqvist G, Figdor D, Persson S, Sjögren U. Microbiologic analysis of teeth with failed endodontic treatment and the outcome of conservative re-treatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1998;85:86-93.  Back to cited text no. 6
    
7.
Evans M, Davies J, Sundqvist G, Figdor D. Mechanisms involved in the resistance of Enterococcus faecalis to Ca (OH). Int Endod J 2002;35:221-8.  Back to cited text no. 7
    
8.
Spratt DA, Pratten J, Wilson M, Gulabivala K. An in vitro evaluation of the antimicrobial efficacy of irrigants on biofilms of root canal isolates. Int Endod J 2001;34:300-7.  Back to cited text no. 8
    
9.
Heling I, Chandler NP. Antimicrobial effect of irrigant combinations within dentinal tubules. Int Endod J 1998;31:8-14.  Back to cited text no. 9
    
10.
Jeansonne MJ, White RR. A comparison of 2.0% chlorhexidine gluconate and 5.25% NaOCl as antimicrobial endodontic irrigants. J Endod 1994;20:276-8.  Back to cited text no. 10
    
11.
Soharabi K, Soortagar A, Zolfagharnasab K, Kharazifarad MJ, Afkhami F. Antibacterial activity of diode laser and NaOCl in Enterococcus faecalis-Contaminated Root Canals. Iran Endod J 2016;11:8-12.  Back to cited text no. 11
    
12.
Jeansonne MJ, White RR. A comparison of 2.0% chlorhexidine gluconate and 5.25% sodium hypochlorite as antimicrobial endodontic irrigants. J Endod 1994;20:276-8.  Back to cited text no. 12
    
13.
Cunningham WT, Joseph SW. Effect of temperature on the bactericidal action of NaOCl endodontic irrigant. Oral Surg Oral Med Oral Pathol 1980;50:569-71.  Back to cited text no. 13
    
14.
Woodmansey KF. Intracanal heating of NaOCl solution. An improve endodontic irrigation technique. Dent Today 2005;24:114-6.  Back to cited text no. 14
    
15.
Iandolo A, Simeone M, Orefice S, Rengo S. 3D cleaning, a perfected technique: Thermal profile assessment of heated NaOCl. Societa' Italiana di Endodonzia; 2017. p. 59-61.  Back to cited text no. 15
    
16.
Iandolo A, Amato M, Dagna A, Poggio C, Abdellatif D, Franco V, et al. Intracanal heating of NaOCl: Scanning electron microscope evaluation of root canal walls. J Conserv Dent 2018;21:569-73.  Back to cited text no. 16
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17.
Shahriari S, Kasraei S, Roshanaei G, Karkeabadi H, Davanloo H. Efficacy of sodium hypochlorite activated with laser in intracanal smear layer removal: An SEM study. J Lasers Med Sci 2017;8:36-41.  Back to cited text no. 17
    
18.
Siqueira JF Jr., Machado AG, Silveira RM, Lopes HP, de Uzeda M. Evaluation of the effectiveness of sodium hypochlorite used with three irrigation methods in the elimination of Enterococcus faecalis from the root canal, in vitro. Int Endod J 1997;30:279-82.  Back to cited text no. 18
    
19.
Ashofteh K, Sohrabi K, Iranparvar K, Chiniforush N.In vitro comparison of the antibacterial effect of three intracanal irrigants and diode laser on root canals infected with Enterococcus faecalis. Iran J Microbiol 2014;6:26-30.  Back to cited text no. 19
    
20.
Kanumuru NR, Subbaiah R. Bacterial efficacy of Ca (OH) 2 Against E. faecalis compared with three dental lasers on root canal dentin – An in vitro study. J Clin Diagn Res 2014;8:ZC135-7.  Back to cited text no. 20
    
21.
Rahimi S, Shahi S, Gholizadeh S, Shakouie S, Rikhtegaran S, Soroush Barhaghi MH, et al. Bactericidal effects of Nd: YAG laser irradiation and NaOCl solution on Enterococcus faecalis biofilm. Photomed Laser Surg 2012;30:637-41.  Back to cited text no. 21
    
22.
Parirokh M, Eghbal MJ, Asgary S, Ghoddusi J, Stowe S, Forghani F, et al. Effect of 808nm diode laser irradiation on root canal walls after smear layer removal: A scanning electron microscope study. Iran Endod J 2007;2:37-42.  Back to cited text no. 22
    



 
 
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