BASIC RESEARCH – TECHNOLOGY

Sarah Bukhari, BDS, MS,* and

The Antimicrobial Effect of Bekir Karabucak, DMD, MS†

Bioceramic Sealer on an
8-week Matured Enterococcus
faecalis Biofilm Attached to
Root Canal Dentinal Surface

ABSTRACT
SIGNIFICANCE
Introduction: The aim of this study was to test the antibacterial activity of bioceramic sealer in
comparison with AH Plus (Dentsply International Inc, York, PA) on 8-week-old Enterococcus The recently introduced
faecalis biofilms attached to root canal surfaces using a dentin infection model. bioceramic sealer exhibited a
Methods: The canal surfaces of single-rooted intact extracted teeth were infected by significantly potent
growing E. faecalis biofilms for 8 weeks. AH Plus sealer and EndoSequence BC Sealer antibacterial effect against
(Brasseler USA, Savannah, GA) were placed on the root canal wall of the dentin specimens for 8-week-old Enterococcus
24 hours and 2 weeks in humid conditions at 37 C. Infected samples incubated with no faecalis biofilms over AH Plus
sealers for similar periods were used as the negative controls. Specimens were labeled with sealer in the presence of
fluorescent viability staining, and confocal laser scanning microscopy was used as an dentin.
assessment tool of the proportions of dead and live bacteria on canal walls after exposure to
root canal sealers for the determined times. Results: EndoSequence BC Sealer killed
significantly more E. faecalis in biofilm attached to the canal surfaces when compared with AH
plus sealer and control at both time points (P , .05–.0005). Conclusions: EndoSequence
BC Sealer exhibited significant antimicrobial capacity in the presence of dentin for up to 2
weeks on an 8-week-old E. faecalis biofilm in comparison with AH Plus sealer. (J Endod
2019;-:1–6.)

KEY WORDS
Bioceramic sealer; confocal microscopy; Enterococcus faecalis biofilms

The role of microorganisms in the development of apical periodontitis has been well recognized in the
endodontic literature1. Furthermore, recent evidence indicates that endodontic biofilm within the canal
system plays a significant role in the apical disease process2. Thereby, the goal of root canal treatment is
to reduce the microbial load as much as possible through chemomechanical preparation and intracanal
medicaments3. However, complete bacterial eradication from root canal spaces using current From the *Department of Dental Services,
King Abdulaziz Medical City, Ministry of
therapeutic strategies may not be attainable because of the limitation of mechanical instrumentation4,5. It
National Guard Health Affairs, Jeddah,
would be advantageous if the root canal filling materials had an antimicrobial substantivity to eliminate Saudi Arabia; and †Department of
residual microbes that survive the initial root canal disinfection process within the dentinal tubules or as a Endodontics, School of Dental Medicine,
biofilm on untouched dentinal surfaces6. University of Pennsylvania, Philadelphia,
A bioceramic-based root canal sealer (EndoSequence BC Sealer; Brasseler USA, Savannah, GA) Pennsylvania
was recently introduced in endodontics as an alternative root canal sealer promising superior biological Address requests for reprints to Dr Bekir
and physiochemical characteristics7. EndoSequence BC Sealer has been shown to have better Karabucak, Department of Endodontics,
School of Dental Medicine, University of
biocompatibility with significantly less cytotoxicity and genotoxicity compared with other endodontic
Pennsylvania, 240 South 40th Street,
sealers8,9. Because of its bioactive nature, EndoSequence BC Sealer may exhibit a positive effect on hard Philadelphia, PA 19104.
tissue deposition by forming hydroxyapatite10. Moreover, it is radiopaque11, easy to handle, E-mail address: [email protected]
hydrophilic12, expands after setting13, insoluble14, and possesses antimicrobial properties8,9,15,16. 0099-2399/$ - see front matter
The antimicrobial activity of different sealers has been previously evaluated8,9,15,16. However, there Copyright © 2019 American Association
is little to no information available on the antimicrobial efficacy of EndoSequence BC Sealer using an of Endodontists.
experimental environment with biofilm attached to dentinal root canal surfaces. https://doi.org/10.1016/
j.joen.2019.04.004

JOE  Volume -, Number -, - 2019 Antimicrobial Effect Go Bioceramic Sealer 1

 Because of the inherent limitations of the 0.5 absorbance at 600 nm. Modified bacterial Microsystems GmbH, Mannheim, Germany)
agar diffusion test (ADT), it has been suspension was then added to each well with a 20XLPlan water immersion objective
substituted with the direct contact test (DCT) (Olympus 24-well plate, 3.5 mL; Genesee lens (AmScope, Irvine, CA) without zooming.
to assess the antimicrobial activity of Scientific, San Diego, CA) filled with 2 mL BHI Each sample was examined and scanned at 4
endodontic sealers17. Despite being widely and the dentinal specimen. All samples were different random areas chosen at the
accepted in the endodontic literature, it does incubated aerobically at 37 C in BHI broth for 8 microscope stage. Optical sectioning at these
not mirror clinical reality in terms of evaluating weeks. The medium was changed every 48 selected areas produced a confocal image
sealer efficacy against biofilm adhered to hours. Two cylinders were used for the series. Scans with a depth of 80mm (1-mm step
dentinal surfaces. confirmation of biofilm formation using SEM. size) at a resolution of 512 ! 512 pixels were
Recent modifications on the dentin acquired.
block model of Haapasalo and Ørstavik have Two-dimensional images showing
Sealer Placement
been introduced using viability staining and biofilms with live/dead bacteria were created
After 8 weeks of incubation, the infected dentin
confocal laser scanning microscopy as using Amira 5.4.1 software (Visage Imaging,
samples were washed with sterile water for 1
assessment tools for evaluation of the San Diego, CA) for visualization. Pixels from
minute and placed in cell culture wells. Each
antimicrobial efficacy of different agents18. In confocal image stacks were converted to
semicylindrical half was then reassembled with
this study, the aforementioned model has been numeric values using ImageJ software (https://
its corresponding half using a hemostat.
used to evaluate the antimicrobial potency of imagej.nih.gov/ij/; National Institutes of Health,
Twenty-four canals were then randomly filled
EndoSequence BC Sealer in comparison with Bethesda, MD) to analyze the images
with either EndoSequence BC Sealer or AH
AH Plus sealer (Dentsply International Inc, quantitatively. Manual setting of the red and
Plus sealer (Dentsply International Inc, York,
York, PA) on 8-week-old Enterococcus green fluorescence thresholds were done
PA) using a plastic syringe tip according to the
faecalis biofilms attached to a dentinal root thereafter and maintained constant for all
manufacturers’ instructions. Eight samples
canal wall after 24 hours and 2 weeks of samples. Automatic calculation of the live/
were left without sealers to serve as the
contact. dead bacterial cell ratio in the biofilm attached
negative controls. Samples were reincubated
to the dentinal root canal surface was
again at 37 C and divided into the following 6
performed. The amount of the killed bacteria
MATERIALS AND METHODS groups:
was determined by calculating the volume ratio
Sample Preparation 1. Group 24hNC (n 5 4): the negative control of red fluorescence to green and red
Thirty-five caries-free single-rooted extracted for 24-h groups fluorescence. Statistical analysis of the data
teeth were included in this experiment. Teeth 2. Group 24hAHPlus (n 5 6): filled with AH was conducted using 1-way analysis of
were prepared to make a uniform 4-mm dentin Plus sealer and evaluated after an variance (ANOVA) followed by the 2-tailed
cylinder by decoronation at 1 mm down from additional 24 hours of incubation unpaired t test to compare 2 sets of data at a
the cementoenamel junction. A detailed 3. Group 24hBC (n 5 6): filled with time when the ANOVA test was significant. All
protocol of this process has been previously EndoSequence BC Sealer and evaluated tests were performed using GraphPad Prism
described by Bukhari et al19. All dentinal after an additional 24 hours of incubation software (Version 5.0; GraphPad Software Inc,
cylinders were then split into 2 halves. The 4. Group 2wNC (n 5 4): the negative control San Diego, CA) at a significance level of P ,
smear layer was subsequently cleared by for the 2-week groups .05 with the 95% confidence interval.
submerging specimens in a 5.25% sodium 5. Group 2wAHPlus (n 5 6): filled with AH Plus
hypochlorite and 17% EDTA ultrasonic bath sealer and evaluated after an additional 2
respectively for 4 minutes each. Scanning weeks of incubation
RESULTS
electronic microscopy (SEM) (FEI [Hillsboro, 6. Group 2wBC (n 5 6): filled with Verification of smear layer removal was
OR] Quanta FEG 250) was used thereafter for EndoSequence BC Sealer and evaluated performed using SEM. Furthermore, scanning
confirmation of smear layer removal. All after an additional 2 weeks of incubation electron microscopic analysis of the infected
samples were flushed with sterile water for 10 surfaces was conducted to confirm biofilm
minutes and autoclaved for 20 minutes at Specimens were then disassembled
formation on the canal surface. The data
121 C. Successful sterilization was into 2 halves to expose the canal surfaces and
showed bacterial aggregates (typically found in
substantiated by 24-hour monitoring of the sealer for confocal laser scanning microscopic
biofilms20) bound to the canal surface after 8
incubated specimens in brain-heart infusion examination.
weeks of infection with E. faecalis
broth (BHI) (Dot Scientific, Inc, Burton, MI) at One sample from group 2 was lost
37 C for the development of turbidity. Confocal Laser Scanning because of contamination. The contamination
Sterilized and confirmed samples were Microscopic Analysis was confirmed when turbidity was observed in
considered as the positive control. Following manufacturer’s instructions, each the medium of the sample in question. The
semicylindrical half was stained using the reason for contamination was not known.
Dentin Infection with E. faecalis fluorescent LIVE/DEAD BacLight Bacterial Table 1 shows the mean values of dead E.
E. faecalis OG1RF (a gift from Dr Brenda Viability Kit (Molecular Probes, Eugene, OR) faecalis for each sealer. The 1-way ANOVA test
Gomes, State University of Campinas, S~ao consisting of green and red fluorescent nucleic showed a significant effect of sealer type on
Paulo, Brazil), which was extracted from acid stain (SYTO 9 and propidium iodide, killing E. faecalis (P , .0001). Statistical
retreatment cases, was selected as the test respectively). Samples underwent 1 minute of analysis of the data obtained from image
organism. Overnight produced colonies that rinsing with PBS and were fixed on the sequence analysis indicated a significant
were cultured on BHI agar plates in an aerobic microscope stage before being examined with antibacterial effect of EndoSequence BC
condition at 37 C in an atmosphere of 5% CO2 confocal laser scanning microscopy. Sealer over the control without sealer and AH
were suspended in BHI and adjusted Samples were then viewed using the Plus sealer groups. EndoSequence BC Sealer
spectrophotometrically to an optical density of multiphoton Leica SP5 microscope (Leica eradicated 76.33 6 5.348 (P , .0005) and

2 Bukhari and Karabucak JOE  Volume -, Number -, - 2019

TABLE 1 - Percentage of Dead Enterococcus faecalis Cells in the Canal Exposed to Different Sealers after 24 Hours and a significant additive effect when mixed with
2 Weeks of Contact for Each Sample and the Overall Percentage of Dead E. faecalis Cells in the Canal Exposed to bioactive glass in amplifying the killing of
Different Sealers after 24 Hours and 2 Weeks of Obturation E. faecalis25.
The antibacterial effect of
24hBC 24hAHPlus 2wBC 2wAHPlus EndoSequence BC Sealer could be caused by
Sample 1 (%) 67 25 80 43 the high pH, which was evident in several
Sample 2 (%) 79 17 89 1.4 experiments9,15. However, Zhang et al15 have
Sample 3 (%) 54 49 74 16 shown that even though EndoSequence BC
Sample 4 (%) 89 20 94 46 Sealer maintained an alkaline pH value up to 7
Sample 5 (%) 83 19 56 21 days, its antimicrobial activity dropped 3 days
Sample 6 (%) 86 56 28 after setting regardless of its high pH. Hence,
Overall 76.33 6 5.348 26.07 6 5.956 74.79 6 6.612 25.84 6 6.906
the antibacterial effect of EndoSequence BC
percentage
Sealer cannot be entirely explained by the
of dead cells
24 hours 2 weeks alkaline pH. Another potential mechanism of
Negative control 4.750 6 1.022 6.382 6 0.9015 action is the increased bioactive glass
dissolution catalyzed by dentin causing an
24hAHPlus, filled with AH Plus sealer and evaluated after an additional 24 hours of incubation; 24hBC, filled with increased silica level. This may interfere with
EndoSequence BC Sealer and evaluated after an additional 24 hours of incubation; 2wAHPlus, filled with AH Plus sealer bacterial cellular integrity via Ca/P
and evaluated after an additional 2 weeks of incubation; 2wBC, filled with EndoSequence BC Sealer and evaluated after an
additional 2 weeks of incubation.
precipitations25,26. This might explain why the
significant antimicrobial effect of
EndoSequence BC Sealer started within 24
74.79 6 6.612 (P , .0005) of E. faecalis in 24 Our study has shown the significant hours of application and lasted for 2 weeks in
hours and 2 weeks, respectively. On the other antibacterial effect of EndoSequence BC this study and up to 30 days in another
hand, AH Plus killed only 26.07 6 5.956 (P , Sealer over AH Plus sealer at all tested time experiment, whereas it significantly dropped 3
.05) and 25.84 6 6.906 (P . .05) of the same points (24 hours and 2 weeks). days after setting in the study by Zhang
bacteria within similar periods. The EndoSequence BC sealer was over 2 times et al15,16. On the other hand, the antimicrobial
antibacterial effect of 24 hours of AH Plus was more potent antimicrobially than AH Plus. capacity of AH Plus sealer was insignificant for
more significant than the control (P , .05). This difference in superiority was statistically the entire experiment, except for the 24-hour
There was no significant difference between significant. These findings are consistent with time point in comparison with the negative
either time points (24 hours vs 2 weeks) within other studies. Using DCT, Zhang et al15 and control.
the EndoSequence BC and AH Plus groups Colombo et al9 showed a significant To our knowledge, this is the first study
(P ..05). Representative confocal laser antibacterial effect of EndoSequence BC that evaluated the effect of EndoSequence BC
scanning microscopic images are presented in Sealer in comparison with AH Plus for up to Sealer on 8-week-old biofilm grown on the
Figure 1A–F, and group analysis is shown in 60 minutes as both freshly mixed and set dentin wall of the main root canal. Realistically,
Figure 2A and B. In general, EndoSequence samples. the endodontic biofilm is more likely to be
BC Sealer was superior in killing E. faecalis Contrary to our study, Candeiro et al8 minimally a few weeks or months old by the
compared with AH Plus regardless of the and Wang et al16 using DCT and a dentinal time treatment is initiated. Previous studies
period (2 weeks vs 24 hours) with a statistically tubule infection model, respectively, found no have shown that mature biofilms are more
significant difference (P , .0005) (Table 1). significant difference in the antimicrobial resistant to the effect of disinfectants than
efficiency between EndoSequence BC Sealer young biofilms27. In this study, we tried to
and AH Plus sealer. However, these create a relevant model by testing the effect of
DISCUSSION contrasting results might be caused by 2 different sealers on mature biofilm,
The antimicrobial efficacy of EndoSequence different methodologies. In DCT, planktonic considering the chemical environment of the
BC Sealer has been previously assessed using bacteria in the form of suspension are placed root canal (dentin). The E. faecalis strain was
ADT or DCT6,7,13. The use of ADT for directly over the sealer spread in wells of a the organism of choice because of its
antimicrobial analysis of endodontic sealers microtiter plate. Although consistency is a established ability to tolerate high pH levels28.
has been discouraged17 because it reflects the significant advantage of this experimental In addition, it has been frequently isolated from
diffusion capacity of the tested material rather setting, it is clinically unrealistic. Intracanal persistent cases of apical periodontitis29.
than its antimicrobial potentials, conceivably biofilm has been found to be the predominant Because the desired advantage of having an
generating false results. Therefore, it has been form of endodontic bacterial invasion20. antimicrobial sealer is to eradicate surviving
replaced with DCT, which gives more reliable Bacteria in the biofilm have been found to be microbes after biomechanical preparation6,
results21. However, DCT does not take into 100- to 1000-fold more resistant to antibiotics this makes the E. faecalis strain an ideal
consideration the presence of dentin and its when compared with their planktonic candidate to evaluate the antimicrobial efficacy
potential effect as part of the complex nature of correspondents23. Moreover, the effect of of obturation materials. On the other hand,
root canal anatomy or for biofilm formation. For dentin and the subsequent influence of its having a single-species biofilm, although well
this reason, a modified Haapasalo and chemistry are not incorporated in the DCT accepted as a model, does not represent the
Ørstavik dentin infection model has been used experiment design. Dentin has been multimicrobial nature of the true endodontic
in this study for evaluation of the antimicrobial well-known to have an inhibitory impact on infection30. The antimicrobial efficacy of a
effect of EndoSequence BC Sealer in some endodontic antimicrobial agents24. disinfectant may differ when tested on a
comparison with AH Plus sealer22. Interestingly, dentin powder was found to have polymicrobial model because of the synergistic

JOE  Volume -, Number -, - 2019 Antimicrobial Effect Go Bioceramic Sealer 3

FIGURE 1 – Representative confocal laser scanning microscopic images of a dentinal canal wall infected by an 8-week- old E. faecalis biofilm. Samples were stained with the LIVE/
DEAD BacLight Bacterial Viability Kit containing SYTO 9 green fluorescent nucleic acid stain and the red fluorescent nucleic acid stain propidium iodide. (A) Twenty-four–hour negative
control, (B ) 2-week negative control, (C ) 24-hour AH Plus, (D ) 2-week AH Plus, (E ) 24-hour EndoSequence BC Sealer, and (F ) 2-week EndoSequence BC Sealer.

FIGURE 2 – Quantitative analysis of the confocal laser scanning microscopic imaging data. (A) The overall percentage of dead E. faecalis cells in the canal exposed to different sealers
after 24 hours and 2 weeks of contact with sealers. EndoSequence BC Sealer significantly killed more bacteria than AH Plus sealer and the negative control (***P , .0005–*P , .05)
at both time points. (B ) The overall ratio of dead to live cells.

4 Bukhari and Karabucak JOE  Volume -, Number -, - 2019

interactions in multispecies biofilms31. In conclusion, within the limitations of ACKNOWLEDGMENTS
Thereby, future research efforts should focus this study, EndoSequence BC Sealer exhibited
on creating a polymicrobial biofilm model that significant antimicrobial capacity in the The authors deny any conflicts of interest
better reflects the complex makeup of presence of dentin for up to 2 weeks on an related to this study.
endodontic infections rather than using an 8-week-old E. faecalis biofilm in comparison
oversimplified single-species model. with AH Plus sealer.

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