Short-Term Antibacterial Efficacy of Three Bioceramic Root Canal Sealers Against Enterococcus Faecalis Biofilms.

OBJECTIVES: The aim of the study was to evaluate the antimicrobial efficacy of three bioceramic root canal sealers against Enterococcus faecalis (E. faecalis) biofilm.
MATERIAL AND METHODS: E. faecalis bacterial suspension was grown on filter paper discs on agar plates. After the incubation period, the discs were covered with four different root canal sealers: 1) Premixing bioceramic root canal sealer (TotalFill BC Sealer); 2) Dual component bioceramic sealer (BioRoot RCS); 3) Mineral trioxide agreggate based sealer (MTA Fillapex); 4) Epoxy resin-based selar (AH Plus). After contact time of 60 minutes, the sealers were removed, and the discs were transferred into sterile tubes containing phosphate buffered saline. After serial dilutions, the aliquots of the suspension were cultivated for 24 hours. After the incubation period, the colony forming units (CFUs) were counted.
RESULTS: There were no significant differences in antibacterial efficacy between the Total Fill BC Sealer and the AH Plus sealer (p=0.386). Both sealers showed better antibacterial efficacy compared to the BioRoot RCS and the MTA Fillapex (p<0.001).
CONCLUSION: The Total Fill BC Sealer and AH Plus had better antibacterial efficacy than the BioRoot RCS and the MTA Fillapex sealers.

Introduction

The main goal of root canal treatment is the elimination of intracanal infection and obturation of root canal system in order to prevent reinfection (). Although chemo-mechanical preparation reduces the number of microorganisms significantly, 40-60% of the root canals still remain positive for bacterial presence (, ). The residual microorganisms are usually located in dentinal tubules, lateral canals and apical ramifications (), thus preventing the healing of periradicular lesion, or decreasing the long-term success of root canal treatment (, ). Therefore, the root canal obturation needs to entomb these residual bacteria and to prevent their access to periradicular tissues, and to block any other communications between the oral cavity and periradicular tissues. Besides, the antimicrobial activity of root canal sealers could contribute to further reduction of viable microorganisms (, ).

The newest generation of root canal sealers are based on tricalcium silicate or calcium phosphate and they are called bioceramic sealers. They have attracted attention due to their alkaline pH, biocompatibility, bioactivity, non-toxicity, dimensional stability, sealing ability and potential to increace root strenght after obturation (). Their antimicrobial efficacy has been evaluated in few past in vitro studies using different microbiological methods and obtaining different results (-). A recent study by Wang et al. () revealed same efficacy of Endosequence BC Sealer as AH Plus against Enterococcus faecalis (E. faecalis) in dentinal tubules after one day, seven days and 30 days of contact time. Another in vitro study reported superiority of the epoxy resin based sealer over a bioceramic sealer ().

The aim of this study was to evaluate the short-term antimicrobial effect of three bioceramic root canal sealers and epoxy resin based sealer against 72 h old E. faecalis biofilm after 60 minutes of contact time.

Materials and methods

Enterococcus faecalis cultivation

In this study, we used a wild type strain of E. faecalis, which had been isolated from a root canal of the lower first human molar with symptomatic chronic apical periodontitis. The bacteria were grown in brain heart infusion (BHI) broth at 4˚ C. Subsequently, few colonies were isolated and grown up on trypticase soy broth (TSB) at 37˚C overnight. The density of the bacterial suspension was adjusted to 1.0 McFarland measured by a densitometer (Densimat, Bio Mérieux, Marcy l'Etoile, France).

For the evaluation of antimicrobial efficacy of bioceramic sealers and epoxy resin based sealer, we used the protocol with filter paper discs described by Barros et al. (). The 10 µL of the E. faecalis suspension was grown on 81 filter paper discs (Whatman GmbH, Dassel, Germany), which were placed on Mitis Salivarius agar plates (Sigma-Aldrich, Taufkirchen, Germany). The paper discs with bacteria were incubated for 48 hours at 37˚C and 100% of humidity. The discs were randomly choosen for scanning electron microscopy (SEM) in order to confirme biofilms growth. The selected discs were fixed in 2.5% glutaraldehyde (Sigma-Aldrich, Taufkirchen, Germany) for 24h and then dehydrated in an ascending ethanol solutions (60%, 70%, 80% and 96%), for 30 minutes in each. After 24 hours of drying on air, the samples were sputter-coated with gold and observed by the SEM (Tescan Vega TS5136LS, Tescan, Brno, Czech Republic).

Placement of sealers on filter discs

The filter discs with biofilms were divided into four experimental groups (n=17/each) according to the tested sealer used and the positive control group (n=10):

Group 1: Total Fill Bioceramic Sealer (TotalFill BC Sealer, FKG, Switzerland)

Group 2: BioRoot Root Canal Sealer (BioRoot RCS, Septodont, Saint Maur Des Fosses, France)

Group 3: MTA Fillapex (Angelus, Londrina, PR, Brasil)

Group 4: Epoxy resin based sealer, AH Plus (Dentsply, Konstanz, Germany)

The tested sealers were prepared according to the manufacturers` instructions. Approximately, 40 µL of each freshly prepared sealer, which was measured by a spoon excavator (Premium instruments, New York, USA) of the same volume, was placed on the whole contaminated surface of filter disks. The sealer was spread over each disc using a single use sterile microbiological inoculating loop (Thermo Fisher Scientific, Waltham, MA, USA).

In the positive control group (n=10), the paper discs with biofilms remained without any sealers. In the negative control group, the sterile discs were not contaminated, but they were covered with the tested sealers (n= two samples for each sealer).

Microbiological analysis of antibacterial efficacy

After 60 minutes of contact time, the sealers were carefully removed from each disc using a sterile Heidelmann instrument (Hu-Friedy Mfg. Co., Frankfurt am Main, Germany). Some minor remnants remained on the surface of few discs. Each disc was placed in a sterile tube containing 1 ml od phosphate-buffered saline (PBS) and slightly agitated for 5 seconds (Vortex mixer X-HD, Sinosource, Guandgong, China) to remove unattached bacterial cells. Then, the discs were transferred to another sterile test tube, which contained 1 ml of PBS, and agitated for 30 seconds. After 8-fold serial dilutions, 10 µL of each dilution was grown on Mitis-Salivarius agar plate (Sigma-Aldrich, Taufkirchen, Germany) and incubated for 24 h on 37˚C and 100% humidity. After the incubation period, the colony forming units (CFUs) were counted and transformed into actual count based on the dilution factor.

Statistical analysis

The obtained data were analysed using the Mann-Whitney U test. All p- values lower than 0.05 were considered statistically significant. The program IBM SPSS Statistics version 23.0 () was used.

Results

The SEM analysis confirmed the presence of biofilms of E. faecalis on filter discs (Figure 1, Figure 2).

Figure 1

Scanning electron microscopy of Enterococcus faecalis colonization (1002x) of filter paper disk

Figure 2

Scanning electron microscopy of Enterococcus faecalis colonization (6000x) of filter paper disk

All tested materials reduced the number of CFUs significantly compared with the positive controls (p<0.001) (Table 1). There was no statistically significant difference between the Total Fill BC Sealer and the AH Plus (p=0.386), nor between the BioRoot RCS and the MTA Fillapex (p=0.931) regarding the reduction of the CFUs. The Total Fill BC Sealer and the AH Plus showed greater antibacterial efficacy than the BioRoot RCS and the MTA Fillapex (p<0.001) (Figure 3).

Table 1

Number of CFUs after 60 minutes of exposure to the tested root canal sealers and comparison to the positive control group

	Mean	SD	Minimum	Maximum	Percentiles			Reduction compared to positive control
Groups					25th	50th (Median)	75th
TotalFill BCS	3.72E+07	1.20E+08	1.00E+04	5.00E+08	1.00E+06	8.00E+06	1.00E+07	99.94%
BioRoot RCS	3.58E+09	2.56E+09	1.00E+09	9.00E+09	1.40E+09	2.90E+09	6.00E+09	79.29%
AH Plus	1.78E+08	6.51E+08	1.00E+05	2.70E+09	1.00E+06	1.00E+07	4.75E+07	99.93%
MTA Filapex	3.51E+09	2.50E+09	4.00E+08	7.90E+09	1.45E+09	2.80E+09	6.05E+09	80.00%
Positive control	2.44E+10	2.75E+10	2.10E+09	9.00E+10	4.75E+09	1.40E+10	3.53E+10

Figure 3

Number of Enterococcus faecalis colony forming units after the effect of each sealer and in the positive control group

Discussion

Although all tested sealers showed significantly lower nuber of CFUs, there was no complete eradication of bacteria in any group. Firstly, these results can be explained due to the source of bacteria. In this study, wild-type strain of E. faecalis was used. Wild-type strains are less susceptible to sealers compared to most commonly used ATCC strain of E. Faecalis, and they have been recommended for the evaluation of antimicrobial efficacy of sealers (). Also, E. faecalis has a significant role in etiology of persistent periradicular lesions () due to its virulence factors and ability to survive the conventional chemo-mechanical root canal procedures ().

Secondly, the type of bacteria can affect antibacterial efficacy of sealers. Most previous studies evaluated the antibacterial efficacy of root canal sealers against planktonic bacteria (, , ). However, since planktonic bacteria do not represent a real clinical situation in infected root canals, the use of older bacterial biofilms which express greater resistance has been recommended (, , ). In this study, we used 48 hours old bacterial biofilm in a microbiological protocol described by Barros et al. (). Therefore, the recommendation for further studies is to investigate the antibacterial efficacy of sealers on older biofilms.

Our finding of no complete eradication in all groups is cosistent with few previous studies (, ). On the contrary, Zhang et al. () reported complete eradication of E. faecalis after application of both, the bioceramic root canal sealer (iRoot SP Sealer) (for 2 minutes) and the AH Plus (for 5 to 20 minutes) sealer. This result could be related to the planktonic species of bacteria used in suspension. These bacteria are more susceptible than biofilms ().

The results of this study also showed similar antibacterial activity of the TotalFill BC Sealer and the epoxy resin based AH Plus against E. faecalis biofilms (eradication of more then 99.9%) after contact time of 60 minutes. The same conclusions have been reached in a recent study by Wang et al (), in which no difference between the bioceramic sealer (Endosequence BC Sealer) and the AH Plus was found even after longer contact time of one day, seven days and 30 days using confocal microscopy evaluation. The antibacterial efficacy of bioceramic sealers is considered to be related to the release of Ca2+ions and high pH (, , ). In another study by Nirupama et al (), the AH Plus and the bioceramic iRoot SP sealers showed statistically significant antibacterial efficacy against E. faecalis. However, the AH Plus presented longer antibacterial effect (18 hours), probably due to its releasing of bisphenol-A-diglycidyl ether during polymerization (). In this study, we evaluated the antimicrobial effect of the sealers only within 60 minutes of application since the direct contact test (DCT) on filter paper discs is solely intended for testing the short-term antimicrobial effect of the sealers (). Namely, the DCT studies, where dentin is not preset, have shown that the sealers lose their antibacterial effect within a few hours or days from mixing (, ). Also, durations shorter than 60 minutes proved to be too short to affect resisting bacteria such as E. faecalis ().

An interesting finding of this study is superior results of the Total Fill BC Sealer over the BioRoot RCS since both materials are bioceramics and of similar composition. The BioRoot RCS is the newest two-component root canal sealer. The powder component is composed of tricalcium silicate, zirconium dioxide and povidone, and the liquid is composed of water, calcium chloride and polycarboxylate. There have been only few studies published so far on its antibacterial activity (, ). Poggio et al. () reported lower antibacterial activity of the BioRoot RCS compared with epoxy resin sealers against 18h old E. faecalis, which was explained due to its shorter working (minimum 10 min) and setting time (maximum 4 h) (, ).

Another reason could be related to smaller homogeneity of the BioRoot RCS after hand mixing when compared with the “premixing” bioceramic sealers. In the most recent study of Arias-Moliz et al. (), the BioRoot RCS showed better intratubular antibacterial efficacy compared with the MTA Fillapex and the AH Plus.

Different results of the same materials in the above mentioned studies could be explained due to different microbial tests used (, ). The most commonly used microbial tests in this kind of studies are DCT and agar diffusion test (ADT) (, , ). ADT has been suitable for soluble materials but due to its limitations (dependence on diffusion and physical properties of tested materials, does not distinguish bacteriostatic or bactericidal effect of the material) () this test is no longer recommended (). DCT is a quantitative and reproducible method to simulate the contact of microorganisms with endodontic sealers, providing information about bactericidal effect (), thus being more reliable and relevant (, ).

In conclusion, Total Fill BCS and AH Plus presented similar antibacterial efficacy against E. faeaclis, which was superior compared to BioRoot RCS and MTA Fillapex.