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Table of Contents   
ORIGINAL ARTICLE
Year : 2018  |  Volume : 8  |  Issue : 2  |  Page : 104-109
Antimicrobial efficacy of different endodontic sealers against Enterococcus faecalis: An In vitro study


Department of Conservative Dentistry and Endodontics, MGV's K.B.H. Dental College and Hospital, Nashik, Maharashtra, India

Date of Submission19-Jan-2018
Date of Acceptance15-Mar-2018
Date of Web Publication24-Apr-2018

Correspondence Address:
Dr. Swati Dalmia
12, Raviranjan, Gajpanth Society, Dindori Road, Mhasrul, Nashik - 422 004, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jispcd.JISPCD_29_18

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   Abstract 

Objective: The aim of this in vitro study is to compare the antimicrobial efficacy of four different endodontic sealers against Enterococcus faecalis.
Material and Method: Four different endodontic sealers, namely, resin based (AH Plus), zinc oxide-eugenol based (Tubliseal), calcium hydroxide based (Sealapex), and mineral trioxide aggregate (MTA Fillapex) based were tested for their antimicrobial efficacy against E. faecalis using agar diffusion method. Four wells were made by the removal of agar at equidistant points and filled with freshly mixed respective root canal sealers and were inoculated with E. faecalis. All the three plates were incubated for a period of 72 h at 37°C under aerobic conditions. The diameter of inhibition zones was measured at 24, 48, and 72 h time intervals. Data obtained were statistically analyzed using one-way analysis of variance and unpaired t-test.
Results: All the tested sealers showed some bacterial growth inhibition of
E. faecalis. Their efficacy in descending order of antibacterial activity was as follows: Sealapex > AH Plus > Tubliseal > MTA Fillapex. The efficacy of the root canal sealers decreased marginally with increase in their duration of action.
Conclusion: Antimicrobial efficacy of calcium hydroxide-based sealer was highest followed by resin-based sealer and was the least with MTA based sealer.


Keywords: AH plus, Enterococcus faecalis, Mineral trioxide aggregate Fillapex, Sealapex, Tubliseal


How to cite this article:
Dalmia S, Gaikwad A, Samuel R, Aher G, Gulve M, Kolhe S. Antimicrobial efficacy of different endodontic sealers against Enterococcus faecalis: An In vitro study. J Int Soc Prevent Communit Dent 2018;8:104-9

How to cite this URL:
Dalmia S, Gaikwad A, Samuel R, Aher G, Gulve M, Kolhe S. Antimicrobial efficacy of different endodontic sealers against Enterococcus faecalis: An In vitro study. J Int Soc Prevent Communit Dent [serial online] 2018 [cited 2018 Sep 25];8:104-9. Available from: http://www.jispcd.org/text.asp?2018/8/2/104/230911



   Introduction Top


For a successful endodontic treatment, complete chemicomechanical preparation, irrigation, obturation, and postendodontic restoration are essential to achieve optimal results, thus eliminating bacteria from the root canal.[1] Root canal disinfection is one of the main determinants which aids in the healing of the periapical tissues. Irrespective of thorough cleaning, shaping, and the use of intracanal medicaments, it is difficult to completely eradicate all microorganisms from the root canal system, which may lead to the failure of endodontic treatment. Microorganisms and their by-products are considered as primary etiological factors for pulp necrosis and apical periodontitis.[2]

Enterococcus faecalis is Gram-positive bacterium that can mostly resist endodontic therapy and has been frequently found in root canal-treated teeth with signs of chronic apical periodontitis. When lodged in the dentinal tubules of the canal, it is difficult to remove these species through root canal medicaments. Sundqvist et al.[3] related that 38% of failed root canal treatments were infected by E. faecalis. Although Enterococcus species comprise a small proportion of the initial flora in infected root canal, they are most commonly recovered from unsuccessful endodontic treatment and has also been associated with existing root canal infections. The success of obturation is directly related to the elimination of microorganisms through mechanical cleaning and shaping, supplemented by antibacterial irrigants, adequate filling of the empty space, and the use of antimicrobial dressings between appointments, if necessary.[3] Therefore, antimicrobial agents are incorporated to root canal sealers to enhance their antibacterial efficacy.

Grossman advocated that the ideal root canal filling material should be bacteriostatic.[4] The root canal sealers should be tissue compatible, provide an airtight seal, and possess antimicrobial effect. The antimicrobial activity of sealers may prevent persistent residual infection and microorganisms from re-entering through the oral cavity, thereby increasing the chances of a successful endodontic treatment outcome.

Root canal sealers can be classified according to the chemical composition as zinc oxide-eugenol-based, calcium hydroxide-containing, glass ionomer-based, epoxy resin-based, and mineral trioxide aggregate (MTA)-based sealers. Due to the variation in composition of the available sealers and considering the American National Standards Institute/American Dental Association standards, the objective of this study was to evaluate in vitro antimicrobial efficacy of sealers of different bases: zinc oxide eugenol (Tubliseal), calcium hydroxide (Sealapex), mineral trioxide aggregate (MTA Fillapex), and epoxy resin (AH Plus).

Aim

The aim of this in vitro study is to compare the antimicrobial efficacy of four endodontic sealers, i.e. resin based (AH Plus), zinc oxide-eugenol based (Tubliseal), calcium hydroxide based (Sealapex), and mineral trioxide aggregate based (MTA Fillapex) based against E. faecalis.


   Material and Method Top


In this study, antimicrobial efficacy of four root canal sealers was tested against E. faecalis. The sealers included in the study were as follows [Table 1] and [Figure 1]:
Table 1: Sealers used in the study

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Figure 1: Sealers used in the study

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  • Group I – Resin-based sealer (AH plus)
  • Group II - Zinc oxide-eugenol-based sealer (Tubliseal)
  • Group III - Calcium hydroxide-based sealer (Sealapex)
  • Group IV - MTA-based sealer (MTA Fillapex).


  • The microorganisms were grown in solid media, and culture containing broth suspensions was prepared and, thus, standard strains of E. faecalis were obtained (MTCC 2093). Microorganisms were subcultured in appropriate culture media to confirm their purity. Aliquots of the suspension containing E. faecalis were spread on three  Petri dish More Detailses containing Mueller-Hinton agar medium.

    Each agar plate was equally divided into four sections. In each section of each plate, a 4 mm diameter well was prepared with a sterile stainless steel cylinder by the removal of agar at equidistant points. The sealers were mixed according to the manufacturer's instructions. A freshly mixed sample of each sealer was placed into the wells in all the four sections of the three plates. All plates were incubated for 72 h at 37°C under aerobic conditions, and zones of inhibition were measured at 24, 48, and 72 h [Figure 2], [Figure 3], [Figure 4]. The diameter of the growth inhibition zones was analyzed statistically using one-way analysis of variance (ANOVA) and unpaired t-test.
    Figure 2: Zones of growth inhibition after 24 h

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    Figure 3: Zones of growth inhibition after 48 h

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    Figure 4: Zones of growth inhibition after 72 h

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       Results Top


    All four root canal sealers showed zones of growth inhibition. The mean diameters of inhibition zones caused by the four tested sealers are presented in [Table 2]. Sealapex exhibited the largest inhibition zone followed by AH plus while MTA Fillapex had the least effect on the tested microorganism. It also shows that the zone of inhibition decreased with time, the highest being at 24 h and the lowest at 72 h.
    Table 2: Mean diameter of inhibition zone

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    To evaluate the antimicrobial activity of the tested endodontic sealers, ANOVA and pairwise comparison were carried out using unpaired t-test at 95% confidence level and relevant degree of freedom. The obtained results are shown in [Table 3].
    Table 3: Mean inhibition, standard deviation, standard error, t value, and P value of the obtained readings

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    From the table, it can be observed that the average diameter of inhibition zone of Sealapex differs significantly when compared to Tubliseal, MTA Fillapex, and AH plus while, for all the remaining pairs, the difference in the average diameter of inhibition zones is not significant. Thus, it was seen that the highest value of the average diameter of the inhibition zones was for Sealapex.


       Discussion Top


    Chemomechanical preparation is undoubtedly one of the most important steps in successful endodontic treatment. However, this does not negate the importance of the quality of the obturation, in which the sealer has a role to play.

    Root canal sealers help by minimizing leakage, provide antimicrobial activity by reducing the possibility of residual bacteria, and resolve periapical lesion.[5] The persistence of bacteria in the root canal system often leads to failure of the root canal treatment. Enterococci have been shown to survive in root canals as single organisms.[6] It is difficult to completely remove microorganisms from the root canal system, even after debridement, shaping, and irrigation of the root canals with antimicrobial agents. Therefore, the use of root filling materials with antimicrobial activity might help to achieve this goal.[7]

    Although aerobic and facultative microorganisms usually constitute a minor proportion of primary endodontic infections, they are frequently found in cases with prolonged treatment, in flare-ups, and in endodontic failures. In the present study, E. faecalis was used as it is the most commonly used microorganism in various in vitro studies relevant to persistent periapical infections.[8] It is the most drug-resistant bacteria which has the ability to survive up to 12 months in the root canal even under nutrient-deficient conditions.[9]

    The agar diffusion test used in this study is one of the most commonly used methods to determine the antimicrobial efficacy of various endodontic sealers.[10] This method allows direct comparison of the root canal sealers against the microorganisms to be tested and the visual indication of which sealer has the potential to eradicate microorganisms in the local microenvironment of the root canal system.[11] The main disadvantage of agar diffusion test is that it cannot differentiate between bactericidal and bacteriostatic effect of the material, and results of this method are dependent on the antimicrobial activity of the test material for the particular microorganisms and is highly influenced by the diffusibility of the material across the medium.[12] Therefore, the absolute antimicrobial efficacy of the sealer is not determined by the size of the inhibition zones.

    A variety of endodontic sealers are available in the market which include zinc oxide-eugenol, calcium hydroxide, glass ionomer, silicon, resin, and bioceramic.[13],[14] These sealers have antimicrobial effect depending on their chemical composition.[15]

    Grossman, in 1936, introduced zinc oxide-eugenol-based sealers, to be used as root canal filling material along with gutta-percha or silver cones. Commonly used zinc oxide-eugenol-based sealers are Tubliseal, Endomethasone, and Endofill.

    The aim of introducing calcium hydroxide-containing sealers was to improve the biological properties and to ensure a good seal of the root canal system. The antimicrobial activity of hydroxide-based sealers may be attributed to its hydroxide ion-releasing property which creates an alkaline environment.[16] Among these, Apexit Plus (Ivoclar, Vivadent, Fürstentum, Schaan, Liechtenstein) and Sealapex (SybronEndo, Glendora, CA, USA) are available in a paste–paste presentation.

    Schröeder, in 1954, introduced the first resin-based sealer. Since then, studies were conducted which contributed to the improvement of the quality of sealers, which lead to the development of an epoxy resin-based sealer, AH Plus which has good physicochemical properties. Epoxy resin-based sealers have antimicrobial activity due to the presence of either bisphenol A diglycidyl ether or due to the release of formaldehyde during polymerization.[17]

    MTA Fillapex (Angelus, Londrina, PR, Brazil) is a recently introduced sealer. The philosophy behind manufacturing this sealer is the presence of MTA in its chemical structure. One of the properties of MTA that is present in the MTA Fillapex sealer is the alkaline pH and subsequent antibacterial activity.

    In this study, all sealers were tested using the agar diffusion test. After incubation, the diameter of zones of inhibition around the sealers was measured and the sealer which exhibited the maximum zone of inhibition was considered as having the most efficient antimicrobial activity.

    The results of this study showed that Sealapex showed highest antimicrobial activity whereas MTA Fillapex showed lowest antimicrobial activity measured at 24, 48, and 72 h. From the present study, it is also observed that the antibacterial activity of all the four sealers decreased with time, i.e. it was highest at 24 h and lowest at 72 h. Estrela et al.[18] hypothesized that the antimicrobial mechanism in calcium hydroxide-based sealers is influenced by its speed of dissociation into calcium ions and hydroxyl ions. This dissociated hydroxyl ion creates a high pH environment, which inhibits enzymatic activities which are essential for microbial metabolism, growth, and cellular division.

    In the present study, MTA Fillapex was found to be the least effective. Torabinejad et al.[19] detected the antimicrobial efficacy of MTA against some facultative bacteria; however, no antimicrobial activity was found against E. faecalis, Staphylococcus aureus, Bacillus subtilis, and  Escherichia More Details coli or against anaerobic bacteria. However, on assessing the antimicrobial properties of MTA, Stowe et al.[20] found that MTA inhibited the growth of both E. faecalis and Streptococcus sanguis. The antibacterial property of MTA is due to the presence of calcium oxide which forms calcium hydroxide on contact with water.[19],[21] Gilberto Debelian stated that MTA is hard to apply in narrow canals, thus making the material difficult to apply along with gutta-percha in canals.[22]

    MTA Fillapex had lower antimicrobial activity when compared to Sealapex due to lower pH which may be due to the differences between the percentage of extractable calcium hydroxide in accordance with the study conducted by Kuga et al.[23]

    The antibacterial efficacy of AH plus could be due to the presence of epoxy resin and amine ingredients. However, our results were similar to that of Zhang et al.[24] and Kayaoglu et al.,[25] who reported that freshly mixed AH plus killed E. faecalis effectively. Tandon et al.[26] suggested that freshly mixed AH plus had significant antibacterial effect whereas set samples did not show any antibacterial activity.

    Nirupama et al.[27] evaluated the antimicrobial efficacy of four endodontic biomaterials against E. faecalis, Candida albicans, and S. aureus and found that Tubliseal has shown maximum antimicrobial activity against C. albicans and S. aureus, which could be due to eugenol which is effective against mycotic and vegetative forms of bacteria. Antimicrobial effect of zinc oxide-eugenol sealer was mainly attributed to the action of eugenol, and a phenolic compound acts on microorganisms by protein denaturation, whereby the protein becomes nonfunctional.[10]

    The size of the inhibition zones depends mainly on two factors which include: (1) toxicity of the material to a particular strain of bacteria and (2) the ability of the material to diffuse through the particular medium. In turn, the diffusibility is mainly affected by three factors which include: (1) hydrophobicity or hydrophilicity of the material, (2) the rate of release from the matrix in which the material is placed, and (3) size. These factors are difficult to control in in vitro studies. The test results may vary according to the above-mentioned factors.[28] Wang et al.[29] evaluated the antibacterial efficacy of four endodontic root canal sealers on E. faecalis biofilm in dentinal tubules. It was found that zinc oxide-eugenol-based sealer has weaker antibacterial effect when compared to other sealers. The reason explained was on the basis of sensitivity of the technique used to test materials. Similarly, the study conducted by Tabrizizadeh and Mohammadi also demonstrated low antibacterial effect of zinc oxide-eugenol-based sealer.[30] It should be noted that the size of the inhibition zones does not determine the exact antimicrobial efficacy of a root canal sealer. Therefore, the root canal sealers tested in the present study may show differences in the zones of inhibition against E. faecalis when tested in vivo.

    The data presented here relate to in vitro conditions, and in vivo conditions such as the presence of dentin and serum might modify the antimicrobial activity of sealers. Hence, further in vivo studies are needed to evaluate the antimicrobial efficacy of sealers.


       Conclusion Top


    Under the limitations of this study, it can be concluded that Sealapex showed the highest antimicrobial activity against E. faecalis while MTA Fillapex showed the least. Moreover, the efficacy of the root canal sealers against E. faecalis decreased with time. Further research is required to compare their efficacy in vivo.

    Financial support and sponsorship

    Nil.

    Conflicts of interest

    There are no conflicts of interest.



     
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        Figures

      [Figure 1], [Figure 2], [Figure 3], [Figure 4]
     
     
        Tables

      [Table 1], [Table 2], [Table 3]



     

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        Abstract
       Introduction
       Material and Method
       Results
       Discussion
       Conclusion
        References
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