Journal of Dental Sciences (2018) 13, 54e59

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Original Article

Comparison of antimicrobial activity of

traditional and new developed root sealers
against pathogens related root canal
Joo-Hee Shin a, Dong-Yul Lee b, Sung-Hoon Lee c*

a
Department of Conservative Dentistry, Korea University Medical Center Guro Hospital, Republic of
Korea
b
Department of Orthodontics, Korea University Medical Center Guro Hospital, Seoul, Republic of
Korea
c
Department of Oral Microbiology and Immunology, College of Dentistry, Dankook University,
Cheonan, Republic of Korea

Received 23 January 2017; Final revision received 24 October 2017

Available online 1 February 2018

KEYWORDS Abstract Background/purpose: Bacterial infection is closely associated with the failure of
Root canal sealer; endodontic treatment, and use of endodontic sealer with antimicrobial activity and biological
Antimicrobial compatibility is necessary for the success of root canal treatment. The purpose of this study
activity; was to investigate and to compare the antibacterial effect of two calcium silicate-based root
Oxide compound; canal sealers (Endoseal and EndoSequence BC sealer) as recent development sealers and with
E. faecalis three conventional root canal sealers (AH Plus, Sealapex, and Tubli-Seal), before or after
setting, on Porphyromonas endodontalis, Porphyromonas gingivalis, and Enterococcus faeca-
lis.
Materials and methods: The sealers were soaked in phosphate buffered saline to elute its com-
positions after and before setting, and the elutes were performed the antimicrobial assay.
Also, X-ray fluorescence analysis was carried out to compare compositions of two calcium
silicate-based sealers.
Results: The conventional root canal sealers have strong antibacterial activity against the
Gram-negative bacteria, P. endodontalis and P. gingivalis. Endoseal sealer showed antibacte-
rial activity against not only the Gram-negative bacteria, but also against the Gram-positive
bacteria, E. faecalis. However, Endosequence BC sealer exhibited a weak antibacterial effect
on all bacteria in this study. X-ray fluorescence analysis exhibited that Endoseal contained
more types and more amount of the oxide compound known to have strong antimicrobial
activity such as Al2O3, Fe2O3, MgO, Na2O, NiO, and SO2 than Endoseqeunce BC.
Conclusion: Endoseal, which contains various types of oxide compounds, seems to be a suit-
able sealer for preventing bacterial infection in both treated and untreated root canals.

* Corresponding author. Department of Oral Microbiology and Immunology, College of Dentistry, Dankook University, 119 Dandae-ro,
Dongnam-gu, Cheonan 31116, Republic of Korea. Fax: þ82 41 550 1859.
E-mail address: [email protected] (S.-H. Lee).

https://doi.org/10.1016/j.jds.2017.10.007
1991-7902/ª 2018 Taiwan Association of Obstetrics & Gynecology. Publishing services by Elsevier B.V. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Comparison of antimicrobial activity 55

ª 2018 Taiwan Association of Obstetrics & Gynecology. Publishing services by Elsevier B.V. This
is an open access article under the CC BY-NC-ND license (http://creativecommons.org/
licenses/by-nc-nd/4.0/).

Introduction calcium silicate-based root canal sealers (Endoseal and

EndoSequence BC sealer) as recent development sealers
Bacterial infection into the root canal plays an important and with three conventional root canal sealers (AH Plus,
role in the induction of pulpal and periapical inflammation Sealapex, and Tubli-Seal), before or after setting, against
and is closely associated with the failure of endodontic P. endodontalis, P. gingivalis, and E. faecalis.
treatment.1 Although individual cases differ, averages of
five to seven different species per canal have been detec- Materials and methods
ted, and the bacterial species most frequently isolated
from necrotic pulps are Porphyromonas gingivalis and The bacteria in this study were purchased from American
Porphyromonas endodontalis.2e4 P. gingivalis and P. endo- Type Culture Collection. E. faecalis ATCC 29221 was aero-
dontalis are associated with initial infection of the root bically cultivated in brain heart infusion (BHI) broth (BD
canal, and Enterococcus faecalis has been detected in Bioscience, Sparks, MD, USA) at 37  C, and P. endodontalis
apical periodontitis lesions in root canal-treated teeth.5 ATCC 35406, and P. gingivalis ATCC 33277 were cultured in
Because the root canal system varies in the anatomical BHI broth supplemented with hemin (1 mg/mL) and vitamin
features including fins, isthmi, and accessory canals, com- K (0.2 mg/mL) at 37  C in an anaerobic condition (5% H2, 10%
plete elimination of the bacteria in the root canals is CO2, 85% N2).
difficult. In treating the root canal, along with mechanical Table 1 shows the composition of the root canal sealers.
cleaning, various intracanal irrigants and medicaments, Sealers tested for antibacterial activity were prepared ac-
such as calcium hydroxide, sodium hydroxide, and chlor- cording to the manufacturers’ directions. Each sealer was
ohexidine, are used in attempts to eradicate bacteria in the dispensed into each well of 12-well polystyrene microplates
infected root canal, However, some bacteria may remain in (SPL Life Science, Gyeonggi, South Korea), and phosphate
the root canal systems.6 Therefore, a hermetic seal of the buffer solution (PBS) was then added, for a sealer con-
root canal space is required to entomb any residual bac- centration of 200 mg/mL. The microplates were agitated on
teria and ultimately kill them in the filled root canal. a shaker (50 rpm) for 4 h at room temperature. To compare
Root canal sealers are used to overcome the limitations the antibacterial activity between set and unset materials,
of gutta-percha (GP) cones and obturation techniques by eluates from each sealer were also collected after setting.
filling the space between the GP and the dentinal wall. The sealers were placed into the inside wells of the 12-well
Hence, root canals sealers that possess superior sealing microplates, and PBS was added in the outside wells of the
ability and antibacterial activity would be clinically bene- microplates to ensure stable humidity levels. The sealers
ficial by preventing bacteria from re-entering the canal were solidified for 24 h at 37  C, and PBS was then added
and by inactivating bacteria remaining in the canal after into the wells. Based on the initial mass, the concentration
root canal obturation. Traditional root canal sealers are of the sealer was adjusted to 200 mg/mL by adding PBS into
categorized as zinc oxide eugenol (ZOE), epoxy resin each well. The microplates were agitated on a shaker for
(ER), or calcium hydroxide (CH) on the basis of their 4 h at room temperature. Each eluate was transferred to a
composition.7e9 Recently, calcium silicate-based cement fresh 15-mL conical tube, which was then centrifuged at
with the addition of various oxide compounds have been 5000 g for 10 min to remove any remaining insoluble
developed for root sealer and are called mineral trioxide particles.
aggregation (MTA).10 This cement is known to bioactive
properties that have stimulation of tissue repair and in-
duction of mineralization.11,12 For these reasons, the
Table 1 The used root canal sealers in this study and its
cement has been considered suitable for application to root
characterization.
canal sealer and have led to the development of root canal
sealers. Antimicrobial activity is also an important factor in Materials Corporation/ Product information
investigating dental materials for application to root sealer Country
because bacterial infection is closely associated with the Sealapex Kerr/USA Calcium hydroxide based
failure of endodontic treatment. Although the antimicro- sealer
bial activity of these products against Lactobacillus aci- Tubli-Seal Kerr/USA Zinc oxide eugenol based
dophilus, Staphylococcus aureus, and E. faecalis has been sealer
studied,13 the evaluation has been limited to the antibac- AH plus Dentsply/USA Epoxy resin based sealer
terial effect on Gram-positive bacteria notwithstanding the EndoSequence Brasseler/USA Calcium silicate based
isolation of P. endodontalis, P. gingivalis, and E. faecalis BC sealer
from necrotic pulps, and the antibacterial activity has been Endoseal MARUCHI/Korea Calcium silicate based
examined only before setting of the sealer. Therefore, we sealer
investigated and compared the antibacterial activity of two
56 J.-H. Shin et al

Antimicrobial assays were performed according to Results

the protocol of Clinical and Laboratory Standards
Institute (CLSI). The incubated bacteria level was assessed Figure 1 shows the growth of E. faecalis as a function of the
using a bacterial counting chamber (Marienfeld, Lauda- sealers’ concentration. The antibacterial activity against
Konigshofen, Germany). The concentration of E. faecalis E. faecalis was the greatest in Endoseal, followed by
was adjusted to a density of 1  106 cell/mL by adding fresh Sealapex, Tubli-Seal, AH Plus and EndoSequence BC sealer.
BHI broth. The BHI broth supplemented with hemin and Endoseal exerted an inhibitory effect at 25 mg/mL,
vitamin K was added to adjust the level of P. endodontalis whereas Sealapex, Tubli-Seal, and AH Plus inhibited the
and P. gingivalis to 1.5  106 cell/mL. Subsequently, 180 mL bacterial growth at 50 mg/mL. All the sealers had less
of the specific media for each test microorganism was inhibitory effect against E. faecalis after the materials
dispensed into each well of 96-well polystyrene plate, and were set, and EndoSequence BC sealer was found to have
160 mL of the specific media plus 20 mL of the prepared no antibacterial activity.
sealer eluate were added to the first row of the plate, and As shown Fig. 2, the growth of P. endodontalis was
serial two-fold dilution was performed using a multi- significantly inhibited when the concentration of AH Plus
channel micropipette. Next, 20 mL of each bacterial sus- and Sealapex was greater than 6.4 mg/mL (P < 0.05). Tubli-
pension was inoculated to the wells containing the eluates Seal and Endoseal showed the bacterial growth at 25 mg/
from the sealers. The plates were incubated for 24 h at mL. When the materials were set, the antibacterial activity
37  C, aerobically for E. faecalis, and anaerobically for of Tubli-Seal was greater, whereas AH Plus, Sealapex, and
P. endodontalis and P. gingivalis. Bacterial growth was Endoseal showed less antibacterial activity. EndoSequence
monitored by measuring the absorbance at 600 nm in a BC sealer exhibited the least antibacterial activity regard-
microplate reader (BioTek, Winooski, VT, USA). less of whether or not the material was set.
To investigate the difference in the antimicrobial ac- The inhibitory effects of unset sealers against P. gingi-
tivity between the two calcium silicate-based sealers, valis decreased in the order of AH Plus, Sealapex, Tubli-
Endoseal and EndoSequence BC sealer, the chemical com- Seal, Endoseal, and EndoSequence BC sealer. When the
positions of the sealers were analyzed using an X-ray fluo- materials were set, the antibacterial effect of AH Plus was
rescence (XRF) spectrometer (ZSX primus II, Rigaku Co., significantly reduced (Fig. 3).
Tokyo, Japan). The sealers were loaded on micro-carry Endoseal sealer contained more types and larger amount
paper and dried at 55  C. The XRF spectrometer was of the oxide compound known to have strong antimicrobial
outfitted with X-ray tubes with Rh anodes and was operated activity such as Al2O3, Fe2O3, MgO, Na2O, NiO, and SO3 than
at 60 kV and 150 mA. The XRF patterns for the sealers were Endoseqeunce BC sealer in XRF analysis (Table 2). The main
obtained using SC and F-PC diode detectors and analyzed compounds were zirconium dioxide, calcium oxide, and
using EZ Scan (Rigaku Co., Tokyo, Japan). silicon dioxide according for approximately 97% of the total
The data were analyzed non-parametrically by using the mass of EndoSequence BC sealer and 86% of Endoseal. Both
KruskaleWallis and ManneWhitney tests. IBM SPSS Statistics EndoSeqeunce BC sealer and Endoseal are the sealer on the
Ver. 23 (IBM, Armonk, NY, USA) was used for statistical basis of calcium oxide and zirconium dioxide and have large
analysis. Statistical significance was defined by a P value of amount of the two molecules. However, higher levels of
less than 0.05. metal oxide such as soidum oxide, aluminum oxide, ferric

Figure 1 The antibacterial activity of traditional and calcium silicate-based root canal sealers against E. faecalis. The eluate
from traditional and calcium silicate-based root canal sealers before (A) or after setting (B) was prepared using a PBS, and
E. faecalis was cultivated with and without the prepared eluate of various sealers at various concentrations in a 96-well polystyrene
plate. The growth of E. faecalis was measured using a microplate reader at 600 nm. The experiments were conducted three times
in duplicate, and data are represented as the mean  S.D. * Statistically significant differences compared with cultures not treated
with the spent culture medium (p < 0.05).
Comparison of antimicrobial activity 57

Figure 2 The antibacterial activity of traditional and calcium silicate-based root canal sealers against P. endodontalis. The
eluate from traditional and calcium silicate-based root canal sealers before (A) or after setting (B) was prepared using a PBS, and
P. endodontalis was cultivated with and without the prepared elute of various sealers at various concentrations in a 96-well
polystyrene plate under anaerobic conditions. The growth of P. endodontalis was measured using a microplate reader at
600 nm. The experiments were conducted three times in duplicate, and data are represented as the mean  S.D. * Statistically
significant difference compared with cultures not treated with the spent culture medium (p < 0.05).

oxide, and silcon dioxide in Endoseal were detected than and their antibacterial activity against some Gram-positive
EndoSequence BC sealer. bacteria, including E. faecalis, has been examined.13,14
However, other bacteria, such as P. gingivalis and P. endo-
dontalis, are related to pulpal inflammations, and the
Discussion antibacterial activity of the sealers after setting has not
been evaluated. Therefore, this study investigated and
Control of the bacteria determines the success or failure of compared the antibacterial activity of traditional sealers
root canal treatment. Although, chemomechanical proced- and calcium silicate-based sealers against P. gingivalis,
ures, cleaning, and disinfectant treatment are performed to P. endodontalis, and E. faecalis before and after setting.
reduce the number of bacteria when treating the root canal, In the susceptibility test, before setting, the traditional
some bacteria often remain in the root canal systems.6 sealers showed stronger antimicrobial activity on P. gingivalis
Therefore, the root-filling materials with antibacterial and P. endodontalis than the calcium silicate-based. Endo-
activity are required and are advantageous. Recently, cal- seal showed the strongest antibacterial activity against
cium silicate-based root canal sealers have been developed, E. faecalis, whereas, Endosequence BC sealer showed weak

Figure 3 The susceptibility of P. gingivalis for various root canal sealers. The eluate from traditional and calcium silicate-based
root canal sealers before (A) or after setting (B) was prepared using a PBS, and P. gingivalis was cultivated with and without the
prepared eluate of various sealers at various concentration in a 96-well polystyrene plate under anaerobic conditions. The growth
of P. gingivalis was measured using a microplate reader at 600 nm. The experiments were conducted three times in duplicate, and
data are represented as the mean  S.D. * Statistically significant differences compared with cultures not treated with the spent
culture medium (p < 0.05).
58 J.-H. Shin et al

molecules into the cytoplasm through electrostatic inter-

Table 2 Comparison of compositions of calcium silicate-
action.19e22 Finally, various oxide compounds with antimi-
based root canal sealers by X-ray fluorescence analysis.
crobial activity in Endoseal may damage the cell wall of
EndoSequence BC Endoseal bacteria and help the penetration of Ca(OH)2 into the
Components Mass % Components Mass % cytosol, and then Ca(OH)2 may denature DNA and protein.
Because Endosequence BC contained relatively low amount
Al2O3 0.0035 Na2O 0.0706
of oxide compounds with antimicrobial activity, Endo-
SiO2 5.77 MgO 1.20
sequence BC may weakly damage the cell walls of bacteria,
P2O5 1.80 Al2O3 2.84
and Ca(OH)2 may penetrate less. This indicates that cal-
K2O 0.0358 SiO2 7.56
cium silicate-base sealers containing oxide compounds
CaO 37.8 SO3 1.27
may show the strong antimicrobial activity against Gram-
MnO 0.0157 K2O 0.574
negative and Gramepositive bacteria.
SrO 0.0046 CaO 25.1
P. gingivalis and P. endodontalis are related to un-
ZrO2 53.3 TiO2 0.141
treated root canal infection,23 and E. faecalis is associated
HfO2 1.04 Cr2O3 0.105
with re-infection of treated root canals.4,23 According to
MnO 0.0365
the results of this study, traditional sealers may be effec-
Fe2O3 1.29
tive in treating primary root canal infections, whereas
NiO 0.0029
Endoseal, a calcium silicate-base sealer that contains oxide
SrO 0.0503
compounds, is more effective in preventing re-infection
Y2O3 0.0592
with E. faecalis. However, considering that traditional
ZrO2 53.0
sealers have a cytotoxic effect on human pulp cell
HfO2 1.05
in vitro.17,24,25 Endoseal may be useful for preventing
Bi2O3 5.66
bacterial infection in untreated and treated root canals.
Within the limitations of the present study, all of the
freshly mixed sealers exhibited higher antibacterial activity
antibacterial activity against all bacteria. The comparison of than the set sealers. The antibacterial activity of the tested
antimicrobial activity among the sealers against the bacteria sealers was found to be material- and bacteria-dependent.
or the comparison of susceptibility among the bacteria Endoseal continues to exhibit antibacterial activity after
for the sealer is possible by performing broth method using setting and may be the most effective in eliminating
the elute. The used elutes were extracted after measuring E. faecalis in the root canal. Finally, Endoseal may be the
same weight of the sealer at one time and then carried most useful sealer for preventing bacterial infection when
out the test of the antimicrobial activity. These data may treating the root canal.
be not obtained by the experiment of agar diffusion assay.
In the present study, AH plus, an epoxy resin-based
sealer, showed the strongest antibacterial activity against
Conflicts of interest
P. gingivalis and P. endodontalis but was weaker against
E. faecalis. Epoxy resin-based sealers exhibit antibacterial The authors have no conflicts of interest relevant to this
activity through bisphenol A diglycidyl ether and formal- article.
dehyde during polymerization.15 Therefore, Gram-negative
bacteria, which have thin cell walls, are sensitive to Acknowledgments
chemicals because of easy penetration into the bacterial
cytosol. Formaldehyde penetrates into the interior of
This research was financially supported by the “the product
bacteria and inhibits metabolism of bacteria by reacting
diagnosis & improvement for rising medical device manu-
with cytosolic proteins, RNA, and DNA.16 Endoseal is a
facturer support program” through the Gangwon Province,
calcium silicate-based sealer and showed an antibacterial
Wonju City and Wonju Medical Industry Techno Valley(WMIT).
effect against E. faecalis before or after setting. Endoseal
showed the strongest antimicrobial effect against bacteria
under alkaline conditions because of calcium silicate.17 References
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