Antimicrobial effect of different calcium silicate – Based bioceramic endodontic sealers against Enterococcus faecalis: An in vitro study

Salah M Abduljabbar; Ammar Abumostafa

doi:10.4103/sjos.SJOralSci_95_20

ORIGINAL ARTICLE

Year : 2021 | Volume : 8 | Issue : 1 | Page : 48-52

Antimicrobial effect of different calcium silicate – Based bioceramic endodontic sealers against Enterococcus faecalis: An in vitro study

Salah M Abduljabbar, Ammar Abumostafa
Department of Restorative Dentistry, Division of Endodontics, Riyadh Elm University, Riyadh, Kingdom of Saudi Arabia

Date of Submission	12-Sep-2020
Date of Decision	10-Nov-2020
Date of Acceptance	27-Jan-2021
Date of Web Publication	17-Apr-2021

Correspondence Address:
Dr. Ammar Abumostafa
Department of Restorative Dentistry, Division of Endodontics, Riyadh Elm University, P.O Box 84891, Riyadh 11681
Kingdom of Saudi Arabia

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/sjos.SJOralSci_95_20

Abstract

Introduction: A successful root canal treatment ensures complete sealing of the root canal to abstain from re-infections. The efficiency of antimicrobial activity of the sealers is important for achieving long term success in root canal therapy.
Aim: This study compared the antibacterial properties of three calcium silicate-based root canal sealers against Enterococcus faecalis at varying incubation time periods.
Materials and Methods: This in vitro cross sectional experimental study was designed with three root canal sealers, namely, Endosequence/BC Sealer, CeraSeal, and BioRoot RCS. Freshly mixed selars were placed in agar plates infused with Enterococcus faecalis. Incubation at 37°C under anaerobic conditions was done for 1 week, during which measurements of the inhibition zones were recorded at 24 h, 48 h, 72 h, and 7 days. Data were compared using ANOVA, and Tukey's tests (P < 0.05).
Results and Discussion: Bacterial growth inhibition was demonstrated by all the sealers tested, with antimicrobial activity being highest for BioRoot RCS and least for CeraSeal. An increase in duration of incubation, positively affected the antimicrobial properties.
Conclusion: Significant difference (P < 0.05) of bacterial inhibition was observed among different time duration of 24 h compared to 48 h, 72 h, and 7 days, for all tested materials. Moreover, BioRoot RCS showed significant bacterial inhibition to tested microorganism.

Keywords: Bioactive materials, disinfection, Enterococcus faecalis

How to cite this article:
Abduljabbar SM, Abumostafa A. Antimicrobial effect of different calcium silicate – Based bioceramic endodontic sealers against Enterococcus faecalis: An in vitro study. Saudi J Oral Sci 2021;8:48-52

How to cite this URL:
Abduljabbar SM, Abumostafa A. Antimicrobial effect of different calcium silicate – Based bioceramic endodontic sealers against Enterococcus faecalis: An in vitro study. Saudi J Oral Sci [serial online] 2021 [cited 2023 Mar 21];8:48-52. Available from: https://www.saudijos.org/text.asp?2021/8/1/48/313931

Introduction

The essential goal of a successful root canal treatment, in addition to completely sealing it, is to render it devoid of infection. This is made possible by the combined functions of root canal sealers and solid core materials.^[1]

Microorganisms and their byproducts are the main etiologic factor for pulp necrosis and subsequent periapical diseases.^[2] Thorough debridement of the root canal system is important before completion of obturation and the antibacterial effect of both irrigants and sealers plays an essential role in this.^[3] These antimicrobial properties of sealers minimize the number of microbes for an extended period of time and prevent re-infection, especially since bacteriological sampling before root canal filling is not a routine practice nowadays.

The above facts makes it imperative to evaluate root canal sealers on the basis of their antimicrobial properties.^[4] Sealers comprise of certain components such as eugenol, thymol, and paraformaldehyde, which provide them their antimicrobial properties. However, the toxicity of such substances might damage the periapical tissues and outweigh their antimicrobial properties.^[5] Calcium silicate-based bioceramic sealers were recently introduced and have showed superior biocompatibility, along with less cytotoxicity compared with other sealers.^[6]

This study aimed at comparing the antibacterial properties of three calcium silicate-based bioceramic sealers: Endosequence/BC Sealer (Brassler, USA), CeraSeal (Meta Biomed Co., South Korea), and BioRoot RCS (Septodont, France) against Enterococcus faecalis. There has been no prior published literature comparing the effects of these sealers against Enterococcus faecalis. The null hypothesis was that there will be no significant difference among the tested groups.

Materials and Methods

An ethical approval from the research center in the institution was obtained (RC/IRB/2019/254). For the purpose of the study, 20 plates of Enterococcus faecalis agar base media were used. Inoculums of Enterococcus faecalis strains were prepared by harvesting 4 to 5 colonies with a circular loop. The bacteria were cultivated in Brain Heart Infusion (BHI) agar media, and a suspension of broth culture was prepared to obtain a turbidity equivalent to the 0.5 McFarland standards scale (approximately 1.5 × 10⁸ cfu/ml.). Aliquots of the suspension were evenly spread over Mueller-Hinton Agar contained in 100 mm diameter Petri dish ^{More Details}es (n = 20). The media-containing petri dishes were left to dry for 15 min at 37°C. Each petri dish was then evenly divided into two sections. A sterile stainless-steel cylinder was used to create wells of 4 mm depth and 5 mm diameter in each of the sections for all the plates.

The four tested sealers were divided into four groups (n = 10); Group 1: EndoSequence/BC Sealer, Group 2: CeraSeal sealer, Group 3: BioRoot RCS sealer, and Group 4 (control): AH26 sealer. The description of the sealers is shown in [Table 1].

Table 1: Type, trade name, manufacturer, and composition of tested sealers

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Compliant with the manufacturers' instructions, each sealer was mixed and, using a digital timer, its utilization was ensured to be within 20 min of mixing. One sample of each sealer was introduced into the wells in each section of the pre-labeled media-containing petri dishes. Five petri dishes per sealer type were used, amounting to 10 samples each. Incubation at 37°C under anaerobic conditions was done for 1 week, during which measurements of the bacterial inhibition zones were recorded at 24 h, 48 h, 72 h, and 7 days. The inhibition zones adjacent to the wells containing the sealer material depicted the lack of bacterial colonization. Measurements of only the most uniform diameter of each zone, after each incubation period, were recorded in millimeters.

Statistical analyses

The measurements collected on the bacterial growth inhibition zones were statistically analyzed using repeated measures analysis of variance (ANOVA). Inter-group analysis was done using one-way ANOVA while intragroup comparisons were made using Tukey's honestly significant difference test; wherein the statistical significance was set at P < 0.05. The statistical analyses of the raw data were performed using Statistical Package for the Social Sciences (SPSS) Version 21 (IBM^® Corp., Armonk, USA).

Results

Data were presented as mean and standard deviation values. The ANOVA test was applied to compare of means of microbial growth inhibition zones for sealers at varying time durations and revealed that the type of sealer tested significantly influenced the size of the inhibition zones (P < 0.001).

BioRoot sealer (BR) (19.51 ± 0.992 mm) displayed the best antibacterial effect against Enterococcus faecalis, followed by Endosequence/BC sealer (ES) (14.39 ± 0.366 mm), then CeraSeal (CS) (13.93 ± 0.621 mm), and the lowest inhibition numbers were shown by the control AH26 sealer (1.75 ± 0.225 mm). Significant statistical difference was present among the microbial inhibitions within the study material groups. Microbial inhibition by sealers increased gradually form 24 h to 7 days. For all groups, the results shown in [Table 2] indicate significant differences among tested materials during the same duration; highest to lowest scores were as follows, BioRoot RCS (BS), followed by Endosequence/BC sealer (BC), and lastly CeraSeal sealer (CS), when comparison with the control group Ah26 sealer (AH).

Table 2: Mean (standard deviation) of microbial inhibition in millimeters, statistically compared using Tukey multiple comparisons test within the same time duration for different materials

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Time duration of 24 h compared to 48 h, 72 h, and 7 days, [Table 3] shows statistically significant difference within the same material at different time duration. However, for Endosequence/BC sealer (ES) 72 h (14.13 ± 0.38 mm) and 7 days (14.39 ± 0.36 mm) were similar. In CeraSeal sealer (CS) group, 24 h (9.09 ± 0.85) was lowest and different from all groups, and 72 h (13.56 ± 0.63 mm) and 7 days (13.93 ± 0.63 mm) were similar. For BioRoot RCS (BR) 24 h (15.94 ± 1.07 mm) was the statistically higher than other durations, and 72 h (18.68 ± 1.00 mm) and 7 days (19.51 ± 0.99 mm) were also significant. In Ah26 sealer (AH) (control group), 24 h (1.14 ± 0.24 mm) and 48h (1.28 ± 0.26 mm) were statistically similar. In addition, 72 h (1.58 ± 0.29 mm) and 7 days (1.78 ± 0.22 mm) were also similar for AH26 sealer microbial inhibition.

Table 3: Mean (standard deviation) of microbial inhibition in millimeters, statistically compared using Tukey multiple comparisons test within the same material at different time durations

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Discussion

The findings of the study indicated that the bacterial inhibition of BioRoot RCS is significantly higher than the other tested materials. Furthermore, a significant difference was observed when comparing its bacterial inhibition after a time duration of 24 h with that after 48 h, 72 h, and 7 days.

A previous study has tested the chemical and physical properties of BioRoot RCS using environmental scanning electron microscopy, and reported that it demonstrated a higher and sustainably longer release of calcium ion (721 ppm) along with an increase in the pH (11-12) compared to MTA Fillapex (Angelus, Londrina, Brazil) and Pulp Canal Sealer (Kerr, Scafati, Italy).^[7] This ability of prolonged calcium ion release is further attributed to the presence of zirconium oxide and could possibly explain the result of our study regarding BioRoot RCS possessing the highest bacterial inhibition compared with the other groups.^[1] Alsubait et al. compared the antibacterial activity of BioRoot RCS with Totalfill BC and AH Plus sealers within dentinal tubules against Enterococcus faecalis

iofilms after incubation periods of 1, 7, and 30 days. A 50% morbidity rate of the bacteria was observed in the BioRoot RCS and Totalfill BC groups on the first and seventh days, respectively. By the end of 30 days, the BioRoot RCS group showed a significantly higher percentage of dead cells than the Totalfill BC group, and was also the highest percentage at 61.75%, among all tested materials.^[8] Our results are consistent with these findings pertaining to the efficacy of BioRoot RCS against Enterococcus faecalis.

The present study also reported that Endosequence/BC sealer exhibited a high inhibition zone size (mean = 14.39). This finding was consistent with the findings of Singh et al.^[9] in their study, who recorded a maximum mean inhibition zone of 14.4 mm. They attributed this efficient antimicrobial property to the high pH of the sealer material, as Enterococcus faecalis is incapable of surviving at pH levels above 11.5.

CeraSeal showed a significant increase in antibacterial effect between 24 h, 48 h and 72 h only. It was observed that, of the tested sealers (excluding the control), CeraSeal showed the least antibacterial effect. Kharouf et al.^[10] compared the physicochemical properties, filling ability, and antibacterial activity of CeraSeal and BioRoot against Enterococcus faecalis. BioRoot and CeraSeal showed no antibacterial effect against Enterococcus faecalis after 3 h, whilst both sealers showed antibacterial capacity after 24 and 72 h. BioRoot demonstrated higher antibacterial activity after 24 h, which is consistent with the findings of our study.

AH26 is a resin-based sealer that is reported to possesses antimicrobial properties owing to the release of formaldehyde which ensues the polymerization process.^[11] A similar report was documented which stated that the antibacterial effect of epoxy-resin-based sealers could be attributed to the release of either bisphenol-A diglyceryl ether or formaldehyde during polymerization. Furthermore, it was noted that freshly mixed AH26 was more potent in reducing bacterial growth than after 21 days.^[12] Collating these findings, AH26 root canal sealer was selected as a control material in this study.

Based on the results, the null hypothesis was rejected as BioRoot RCS showed significantly more bacterial growth inhibition compared to the other tested materials.

Enterococcus faecalis was used in this study as a model for testing the antibacterial effect of different root canal sealers because it is the most resistant intracanal bacteria and one of the main causes for endodontic failures and flare-up as stated in previous literature.^[2] Enterococcus faecalis is the most commonly implicated microorganism in asymptomatic persistent infections owing to its ability to thrive as a single organism, not being dependent on other bacteria for its existence within root canals.^[13]

The test method employed in this study, the agar diffusion test, is frequently used to assess the antimicrobial activity of root canal sealers. Nevertheless, the drawbacks of this test are that it does not account for anatomic variations in roots or the formation of biofilms by the microorganisms.^[14]

The calcium silicate-based bioceramic sealer is composed of calcium phosphate, calcium silicate cement, and calcium oxide. On contact with dentinal moisture, the calcium silicates undergo a hydration reaction which results in the formation of calcium silicate hydrogel and calcium hydroxide.^[15] This calcium hydroxide partially reacts with the calcium phosphate to form hydroxyapatite and water. The water formed, in turn, re-initiates the cycle to produce more calcium silicate hydrogel and calcium hydroxide, resulting in an increase in pH (> 12.5). By the time the sealer sets, its pH also reduces to about 9.14, consequently lessening its antibacterial efficacy. These sealers also exhibit hydrophilic properties, and it has been postulated that their antibacterial properties may be due to a combination of their high pH, active calcium hydroxide diffusion, and hydrophilicity. It has also been reported that their antimicrobial properties are greatly diminished at 7 days after mixing.^[16]

It has been reported that resin-based sealers displayed better antimicrobial properties than BioRoot RCS after 7 days of exposure, which may be explained by differences in experimental design. There are two factors that primarily govern the size of the inhibition zone, namely, (1) the level of diffusion of the sealer in a specific medium and (2) its toxicity to a specific bacterial strain. In turn, the amount of diffusion of the sealer is governed by three factors: its size, its rate of release from the matrix, and its hydrophilicity.^[17] As these factors are not easily controlled in in vitro studies, the test results may vary.^[18] In the present study, the plates were kept for 2 h at room temperature to allow the diffusion of the agents through the agar followed by incubation at 37°C under appropriate gaseous condition.

On reviewing the literature on various in vitro studies, it becomes apparent that there is a paradigm shift in the concept of merely using a root canal sealer to prevent reinfection by sealing the root canal, to a more biological one wherein antimicrobial properties of the material become important. Calcium silicate-based bioceramic sealers have the added advantage of providing a bioactive surface by the stimulation of hard tissue formation and good antibacterial properties,^[19] and can therefore be recommended for clinical use, particularly for the infected and retreatment cases.

Conclusion

Within the limitations of this study, it can be concluded that the tested calcium silicate-based bioceramic sealers showed antimicrobial properties. The BioRoot RCS showed significant bacterial growth inhibition to tested microorganism, followed by Endosequence/BC sealer, and CeraSeal sealer, respectively. Significant difference of bacterial inhibition was observed among different time duration of 24 h compared to 48 h, 72 h, and 7 days, for all tested materials. Moreover, the antimicrobial efficacy was seen to decrease with time.

Investigation of single microorganism in the study was considered a limitation. Future research recommendations should be directed on the diversified antimicrobial sensitivity of different microorganisms.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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