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J Conserv Dent Endod
J Conserv Dent Endod
JCDE
J Conserv Dent Endod
Journal of Conservative Dentistry and Endodontics
2950-4716
2950-4708
Wolters Kluwer - Medknow India

JCDE-27-737
10.4103/JCDE.JCDE_215_24
Original Article
Antimicrobial efficacy of calcium silicate-based bioceramic sealers against Enterococcus faecalis and Staphylococcus aureus – An in vitro study
Kirthiga M.
Thomas George
Jose Sunil
Adarsh V. J.
Nair Saron
Department of Conservative Dentistry and Endodontics, Mahe Institute of Dental Sciences and Hospital, Puducherry, India
Address for correspondence: Dr. M. Kirthiga, Department of Conservative Dentistry and Endodontics, Mahe Institute of Dental Sciences and Hospital, Chalakkara, Pallor Post, Mahe, Puducherry - 673 310, India. E-mail: kirthiga97@gmail.com
7 2024
08 7 2024
27 7 737742
17 4 2024
04 5 2024
23 5 2024
Copyright: © 2024 Journal of Conservative Dentistry and Endodontics
2024
https://creativecommons.org/licenses/by-nc-sa/4.0/ This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.
Context:

The main objective of endodontic therapy is to achieve a sterile environment and three-dimensional filling of the root canals. Endodontic sealers play an important role in the obturation of the root canal system as they may provide a seal, which prevents the penetration of bacteria. Sealers are meant to entomb residual bacteria, prevent leakage of nutrients, and ideally possess antibacterial properties.

Aim:

The aim of the study was to evaluate and compare the antimicrobial effectiveness of calcium silicate-based bioceramic root canal sealers against Enterococcus faecalis and Staphylococcus aureus using the agar diffusion method.

Materials and Methods:

The materials were divided into four groups of nine samples each for both E. faecalis and S. aureus. Group I (control group) – MTA-Fillapex (Angelus), Group II – BioRoot RCS (Septodont), Group III – Bio-C Sealer (Angelus), and Group IV – Dia-Root Bio Sealer (Diadent). To evaluate the antibacterial activity of the samples, the agar diffusion method was utilized. To begin, a bacterial suspension was evenly spread onto sterile Petri dishes using sterile swabs. Four wells of 4 mm diameter were then punched, and the sealers were mixed following the manufacturer’s instructions and placed onto the prepared wells. The Petri dishes were then incubated at 37°C for 72 h. After incubation, the zones of inhibition around each well were measured at 24 h, 48 h, and 72 h, and the resulting values were statistically analyzed.

Statistical Analysis Used:

The data were analyzed using the ANOVA and post hoc Tukey’s test.

Results:

Calcium silicate-based root canal sealers showed significantly better efficacy than the control group. BioRoot RCS (3.44 ± 1.13 mm) showed maximum antibacterial effect against E. faecalis at 24 h, 48 h, and 72 h time intervals. Bio-C Sealer (16.44 ± 1.42 mm) showed maximum antibacterial effect against S. aureus at 24 h, 48 h, and 72 h time intervals. The effectiveness of root canal sealants eventually decreased over a period. Based on analysis using the ANOVA test, a statistically significant difference (P ≤ 0.05) was observed between the mean values of antimicrobial efficacy of sealers against both E. faecalis and S. aureus.

Conclusions:

The study’s findings lead to the conclusion that BioRoot RCS showed a maximum antibacterial effect against E. faecalis and Bio-C Sealer showed maximum antibacterial effect against S. aureus at 24 h, 48 h, and 72 h.

Antimicrobial efficacy
bioceramic sealers
calcium silicate
Enterococcus faecalis
Staphylococcus aureus
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pmcINTRODUCTION

The primary goal of endodontic treatment is to meticulously eliminate all bacteria and their by-products from the root canal system to ensure complete prevention of any potential reinfection. This objective is achieved only through chemomechanical preparation using instrumentation and the use of chemical irrigants to eliminate bacteria. Although this process reduces the quantity of remaining microorganisms in the root canal, it does not entirely eradicate them.[1]

The endodontic microflora normally consists of both Gram-negative and Gram-positive bacteria and obligate anaerobes. Enterococcus faecalis is a facultative anaerobic bacteria that can endure extremely difficult conditions. It possesses several virulence factors that promote tissue invasion, influence immunomodulation, and result in toxin-mediated harm. These factors enable adhesion to host cells and extracellular matrix. Along with these factors, the presence of Staphylococcus aureus is one of the main reasons why root canal therapy fails. For this reason, a good root canal filler material with antibacterial properties would be helpful.[2]

An antibacterial sealer must ideally flow into the untreated spaces in the root canal and hermetically seal off all the voids in the root canal along with the primary core material. Therefore, the sealers play a vital role in achieving a three-dimensional obturation of the canal space. In addition, certain sealers exhibit antibacterial properties, which are thought to be advantageous for limiting and inhibiting the growth of leftover germs. Endodontic sealers play a critical role in endodontic infection control by engulfing the residual bacteria and preventing the leakage of nutrients that lead to reinfection. An ideal root canal filling material should meet the technical and biological standards as well as have antimicrobial and antibiofilm activity to get rid of any remaining bacteria and biofilm following instrumentation and irrigation.[3]

Additional long-term methods to limit bacterial growth are through the use of intracanal medicaments and bio-sealers with antimicrobial properties. The term “bio” refers to any material which exerts action similar to the biological properties of living tissue. These materials can be either bioactive or bio-inert depending on their function. Bioactive materials are a type of material that exhibits interaction with surrounding natural tissues to induce the growth of more durable tissues. Examples are glass and calcium phosphate. Bio-inert materials such as zirconia and alumina are known for their ability to elicit minimal or no biological or physiological responses from surrounding tissues.[4] These newer calcium silicate-based sealers offer numerous advantages over traditional materials such as calcium hydroxide, zinc oxide eugenol, and resin-based sealers. Some of the advantages include biocompatibility, chemical inertness, dimensional stability, bio-inductive properties, a strong antibacterial effect due to high pH, and excellent sealing abilities.

Bioceramic sealers are commonly composed of a blend of materials, including zirconium oxide, calcium silicates, calcium phosphate monobasic, calcium hydroxide, and various other filling and thickening agents. The hydrophilic nature of bioceramic sealers utilizes moisture within the canal to facilitate the setting reaction, while also minimizing any shrinkage that may occur during the setting process. The material is available in premixed calibrated syringes with intracanal tips, making it a convenient and reliable choice for endodontic procedures that require precision and accuracy.[5] This study aimed to evaluate the antimicrobial effectiveness of contemporary calcium silicate-based bioceramic sealers against E. faecalis and S. aureus.

MATERIALS AND METHODS

Four different types of sealers were used in this study, MTA-Fillapex (Angelus), BioRoot RCS (Septodont), Bio-C Sealer (Angelus), and Dia-Root Bio Sealer (Diadent). The sample size was calculated using the software G*Power version 3.1.

The sealers were divided into four groups:

Group I (control group) – MTA-Fillapex (Angelus)

Group II – BioRoot RCS (Septodont)

Group III – Bio-C Sealer (Angelus)

Group IV – Dia-Root Bio Sealer (Diadent).

This study utilized E. faecalis (ATCC 29212) and S. aureus (ATCC 25923) as the primary microorganisms, which were procured from “HiMedia.” The sealers were divided into four groups of nine samples each for both E. faecalis and S. aureus. The microorganisms were cultured in brain heart infusion broth for 24 h at 37°C. To confirm the purity of the cultures, a single colony of each E. faecalis and S. aureus was selected from the cultured plate and subjected to Gram’s staining. The morphology was confirmed by observing the stained cells under a microscope. The bacterial suspension was standardized by comparing it to a broth with a density equivalent to the 0.5 McFarland units of barium sulfate standard, which corresponds to 1.5 × 108 colony-forming units per milliliter. In total, 18 Muller Hinton Agar (MHA) plates were utilized in this study. Nine MHA plates were inoculated with the E. faecalis suspension using sterile swabs, while another nine plates received the S. aureus suspension.

Agar diffusion test

Microbiological assessments were performed in aseptic conditions within a laminar flow chamber. Each MHA plate was divided into four sections, and 4-mm diameter wells were made in each section by removing agar at equidistant points using a needle hub. The sealers were mixed according to the manufacturer’s instructions and poured into the wells. Petri dishes containing the sealers along with E. faecalis and S. aureus microorganisms were incubated for 72 h at 37°C in an incubator. Zones of inhibition were measured at 24h,48h and 72 h [Figure 1]. The size of the growth inhibition zone was measured in millimeters using a digital caliper, and the recorded values were noted. The point at which growth abruptly diminished, indicating complete growth inhibition, was considered the zone edge.

Figure 1 (a) Zones of inhibition for Enterococcus faecalis at 24 h, (b) Zones of inhibition for E. faecalis at 48 h, (c) Zones of inhibition for E. faecalis at 72 h, (d) Zones of inhibition for Staphylococcus aureus at 24 h, (e) Zones of inhibition for S. aureus at 48 h, (f) Zones of inhibition for S. aureus at 72 h

RESULTS

BioRoot RCS showed a maximum antibacterial effect against E. faecalis, followed by Bio-C Sealer and MTA-Fillapex, and the minimum zone of inhibition was shown by Dia-Root Bio Sealer. The effectiveness of root canal sealants eventually decreased over a period. The zone of inhibition was maximum during the first 24 h and minimum during the 72-h time interval [Table 1].

Table 1 Mean zone of inhibition for E. faecalis and S.aureus at three time points

Materials	24 h	48 h	72 h	
	
Mean±SD of microbial inhibition in millimeters for Enterococcus faecalis	
MTA-Fillapex	13.89±0.78	13.56±0.88	13.00±1.00	
BioRoot RCS	23.44±1.13	23.44±1.13	22.22±1.39	
Bio-C Sealer	16.44±1.42	15.89±1.54	15.78±1.56	
Dia-Root Bio Sealer	13.78±0.97	13.67±1.00	12.56±0.53	
Mean±SD of microbial inhibition in millimeters for Staphylococcus aureus	
MTA-Fillapex	13.78±1.20	13.33±1.00	12.56±1.42	
BioRoot RCS	13.56±1.51	12.89±1.36	12.22±1.39	
Bio-C Sealer	19.22±1.39	19.00±1.50	18.33±1.22	
Dia-Root Bio Sealer	15.67±1.12	15.56±1.01	14.78±0.97	
SD: Standard deviation

Against S. aureus, Bio-C Sealer showed the maximum zone of inhibition, followed by Dia-Root Bio Sealer and MTA-Fillapex, and the least amount of zone of inhibition was shown by BioRoot RCS [Table 1].

Statistical analysis of data

The results were statistically analyzed using IBM, SPSS (Statistical Package for the Social Sciences, IBM Co., Armonk, NY, USA) version 26 statistical software. One-way ANOVA test and Tukey’s post hoc analysis were used to compare the mean zone of inhibition (in mm) between four groups at different time intervals. An ANOVA test was conducted on four distinct sealers to evaluate their efficacy against E. faecalis and S. aureus at different time intervals. The findings revealed a highly significant difference (p ≤ 0.05) among the sealer groups for their antimicrobial activity against E. faecalis and S. aureus at three different time intervals [Table 2].

Table 2 Comparison of groups based on zone of inhibition for E. faecalis and S. aureus at three time points

ANOVA	
Time points	Comparisons	Sum of squares	df	Mean square	F	p*	
	
Comparison of groups based on Enterococcus faecalis inhibition zones at three time points	
24 h	Between groups	556.67	3	185.56	152.69	0.000	
	Within groups	38.89	32	1.22			
	Total	595.56	35				
48 h	Between groups	586.97	3	195.66	144.49	0.000	
	Within groups	43.33	32	1.35			
	Total	630.31	35				
72 h	Between groups	536.22	3	178.74	126.17	0.000	
	Within groups	45.33	32	1.42			
	Total	581.56	35				
Comparison of groups based on Staphylococcus aureus inhibition zones at three time points	
24 h	Between groups	185.56	3	61.85	35.77	0.000	
	Within groups	55.33	32	1.73			
	Total	240.89	35				
48 h	Between groups	210.53	3	70.18	45.73	0.000	
	Within groups	49.11	32	1.53			
	Total	259.64	35				
72 h	Between groups	213.64	3	71.21	44.39	0.000	
	Within groups	51.33	32	1.60			
	Total	264.97	35				
*Considered significant at p≤0.05 based on One-way ANOVA

A graphical representation of the mean zones of inhibition at 24 h, 48 h, and 72 h against E. faecalis and S. aureus was represented in Figure 2.

Figure 2 (a) Comparison of the mean zones of inhibition at 24 h, 48 h, and 72 h among the study groups for Enterococcus faecalis, (b) Comparison of the mean zones of inhibition at 24 h, 48 h, and 72 h among the study groups for Staphylococcus aureus

DISCUSSION

In this study, four different sealers were used, namely, MTA-Fillapex (control group), BioRoot RCS, Bio-C Sealer, and Dia-Root Bio Sealer. The main focus of the study was to evaluate the antimicrobial efficacy of these sealers against E. faecalis and S. aureus. E. faecalis is a type of Gram-positive facultative anaerobic bacteria that can thrive in environments with or without oxygen and is commonly detected in endodontic failures. It can easily survive in root canals and can resist a high alkaline pH of 11.5.[6] A study conducted by Byström et al. has shown that E. faecalis was the species examined that was most resistant to calcium hydroxide.[7] Its prevalence ranges from 40% to 77%, and its highly invasive nature enables it to infiltrate dentinal tubules and adhere to collagen, making it difficult to eliminate. This presents a significant challenge in the field of endodontic treatment.[8,9] S. aureus is a microorganism that has demonstrated significant resistance to treatment after repeated root canal procedures; the act of leaving the root canal open during root canal therapy is a significant etiological factor that contributes to the development of primary endodontic infections and chronic infections that occur between treatment sessions.[10] Hence, we chose these two organisms for our study. It is not possible to completely eliminate bacteria from the root canal system, and therefore, root canal filling comprising a core and a sealer with antimicrobial activity is employed to reduce microorganisms and prevent infections. Bioceramic-based endodontic sealers have gained popularity in recent years due to their biocompatibility, stability, and minimal postoperative pain, and they also possess the ability to promote biomineralization.[11,12]

Antibacterial action was assessed using the agar diffusion test (ADT). The ADT refers to a method in which an inhibition zone, also known as a halo, forms around the item being tested on the agar plate. The size of the zone corresponds to the antibacterial effect of the sealer. It has several advantages, including simplicity, affordability, and ease of use, allowing for the testing of a vast number of microorganisms and antimicrobial agents, and leading to straightforward interpretation of results.[13] In this study, BioRoot RCS showed maximum antibacterial effect against E. faecalis, which is in accordance with a study conducted by Abduljabbar and Abumostafa[14] and Arias-Moliz and Camilleri.,[15] where they found that the antimicrobial activity being the highest for BioRoot RCS. There are no studies in the literature that have been done with BioRoot RCS against S. aureus. Siboni et al., in 2017, stated that the antimicrobial efficacy of BioRoot RCS is due to the release of mineralizing ions, such as Ca++, which bind to physiologically active ions and promote the nucleation of apatite precursors on the material surface, demonstrating bioactivity.[16] In our present study, the least zone of inhibition has been shown by BioRoot RCS against S. aureus compared to other sealers, and this can be explained by the size of the zone of inhibition. This size is determined by two crucial factors. First, the level of sealer diffusion in a specific medium is a significant factor in determining the size of the inhibition zone. Second, the toxicity of the sealer to particular bacterial strains significantly impacts the size of the inhibition zone. Furthermore, the amount of diffusion of the sealer is influenced by three key factors: the size of the sealer, its rate of release from the matrix, and its hydrophilicity. Because of the above-mentioned factors, the test results may differ.[14]

It is noteworthy to mention that MTA-Fillapex has been extensively researched and analyzed across numerous studies, emerging as the most commonly investigated bioceramic material within the current literature. In our present study, MTA-Fillapex showed a minimum zone of inhibition against E. faecalis and S. aureus. This might be due to the presence of salicylate resin in MTA which should not be considered a tricalcium silicate sealer, with the mineral trioxide aggregate constituting just a small part.[17] The result we got from this study is similar to a study conducted by Dalmia et al., in 2018.[18] However, it is contradictory to a study conducted by Torabinejad et al. who observed that MTA was effective against a few facultative bacteria but had no effect against anaerobic bacteria, Escherichia coli, S. aureus, Bacillus subtilis, and E. faecalis.[19]

Bio-C Sealer showed the second-most antibacterial effect against E. faecalis and the maximum zone of inhibition against S. aureus. The results we obtained from our study is similar to a study conducted by Barbosa et al. where they found that Bio-C Sealer exhibited antibacterial effects against E. faecalis, E. coli, Pseudomonas aeruginosa, and E. faecalis strains, but not against Streptococcus mutans. Despite the sealer’s short setting time and high solubility, the considerable release of calcium hydroxide may further contribute to the antibacterial properties of fresh Bio-C Sealer.[20] Whereas the results are contradictory to a study conducted by Raouf et al., where they found that Bio-C Sealer demonstrated the best antibacterial activity against E. faecalis but was weaker against S. aureus, whereas MTA-Fillapex showed maximum antibacterial effect against S. aureus.[21]

In this in vitro study, Dia-Root Bio Sealer shows minimal antibacterial effect against E. faecalis. El-sayed et al. stated that the antibacterial activity of DiaRoot BioAggregate might be attributed to the presence of more calcium hydroxide and hydroxyapatites, which are powerful antibacterial inhibitors when mixed with fluids.[22] There are no studies in the literature that have been done with Dia-Root Bio Sealer against S. aureus. In this study, the antimicrobial effect of Bio-C Sealer was the maximum against S. aureus, followed by Dia-Root Bio Sealer, MTA-Fillapex, and BioRoot RCS.

It has been observed from the present study that the antimicrobial activity of all four sealers eventually decreases with time. It was the maximum at 24 h and the minimum at 72 h. The present study has shown that BioRoot RCS has a maximum antibacterial effect against E. faecalis at 24 h, 48 h, and 72 h, followed by Bio-C Sealer, MTA-Fillapex, and Dia-Root Bio Sealer. Therefore, based on the findings of this study, Bio-C Sealer can be the sealer of choice in cases of persistent root canal infections. It is important to note that this study is not conclusive as it was conducted in vitro and not in vivo. The presence of dentin can significantly modify the antimicrobial efficacy of sealers. Therefore, further clinical studies are required to be conducted in vivo to validate the effectiveness of these sealers.

CONCLUSIONS

The study’s findings lead to the conclusion that all the sealers exhibited antibacterial effects on the test microorganisms. BioRoot RCS showed maximum antibacterial effect against E. faecalis and Bio-C Sealer against S. aureus at 24 h, 48 h, and 72 h, respectively.

Bio-C Sealer showed promising results against both the test organisms, thereby proving its worth as a recommended sealer in the retreatment of infected root canals.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.
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