Basic ResearchTechnology
Setting Time Affects In Vitro Biological Properties
of Root Canal Sealers
Carlos Henrique R. Camargo, DDS, PhD, Tatiana R. Oliveira, DDS, MSc, Gleyce O. Silva, DDS, MSc,
Sylvia B. Rabelo, DDS, PhD, Marcia C. Valera, DDS, PhD, and Bruno N. Cavalcanti, DDS, PhD
Abstract
Introduction: Biocompatibility of root canal sealers is
important because of the long-term contact of their
eluates and/or degradation products with periapical
tissues. The literature still lacks studies about the geno-
toxic effects of these materials and the influence of
setting time on biological properties. The cytotoxicity
and genotoxicity of an epoxy resinbased sealer (AH
Plus), a single methacrylate-based sealer (EndoRez),
and a silicone-based sealer (RoekoSeal) were assessed.
Methods: Chinese hamster fibroblasts (V79) were
cultured and exposed to different dilutions of extracts
from the sealers that were left to set for 0, 12, and 24 hours
before contact with culture medium. Cell viability was
measured by the methyl-thiazol-diphenyltetrazolium
assay. Genotoxicity was assessed by the comet assay.
Data were statistically analyzed by Kruskal-Wallis and
Dunn tests (P < .05). Results: Root canal sealers were
statistically more cytotoxic than the untreated control
group, except for the silicon-based sealer. Cell viability
ranking was the following (from the most to the least cyto-
toxic): methacrylate-based > epoxy resinbased >
silicone-based. The setting time influenced the epoxy
resinbased sealer cytotoxicity (decreased at 12 hours)
and the general genotoxicity (increased at 24 hours).
DNA damage ranking was the following (from the most
to the least genotoxic): methacrylate-based > silicone-
based = epoxy resinbased. Conclusions: The setting
time had influence on the cytotoxicity of the epoxy resin
based sealer and genotoxicity of all tested sealers. The
methacrylate-based sealer was the most cytotoxic, and
the silicone-based sealer was not cytotoxic. Genotoxicity
was observed for all sealers. (J Endod 2014;40:530533)
Key Words
Biocompatibility, cytotoxicity, endodontic sealer, geno-
toxicity
From the Department of Restorative Dentistry, Institute of
Science and Technology, Universidad Estadual Paulista (UN-
ESP), Sao Jose dos Campos, Brazil.
Address requests for reprints to Dr Carlos Henrique R.
Camargo, Department of Restorative Dentistry, Institute of
Science and Technology, Universidad Estadual Paulista, Av.
Eng. Francisco Jose Longo, 777, Sao Jose dos Campos, SP,
12245-000 Brazil. E-mail address: chrcamargo@icloud.com
0099-2399/$ - see front matter
Copyright 2014 American Association of Endodontists.
http://dx.doi.org/10.1016/j.joen.2013.08.009
530 Camargo et al.
T he development of endodontic sealers aims to achieve good mechanical properties
and biocompatibility. This property is necessary because of the long-term contact of
their eluates and/or degradation products with periapical tissues (1, 2). Toxic materials
can damage periapical cells and affect the DNA, leading to carcinogenic transformations
and/or genome instability (3).
It is known that epoxy resinbased sealers could induce an initial mild inflamma-
tory reaction on surrounding tissues, as well as cytotoxicity (46), with slight mutagenic
capacity (7). Single methacrylate resinbased sealers were also studied. Whereas some
authors reported them as well-tolerated by connective tissues (8), others observed an
intense and long-lasting inflammatory reaction (9). A possible cause for this effect
could be the presence of urethane dimethacrylate in the structure of the sealer (10).
Previous studies also reported that high concentrations of methacrylate monomers
might induce DNA damage (11). Recently, silicone-based sealers were introduced,
with none or minimal cytotoxicity (12); however, there are no current studies regarding
its genotoxic effects. There are a number of studies assessing the cytotoxicity of
endodontic sealers, although only a few authors observed the effects of setting time
on the genotoxicity of root canal sealers (5, 1315). With this gap in the knowledge,
we hypothesized that different setting times would change both cytotoxicity and
genotoxicity of root canal sealers.
The objective of this study was to evaluate the cytotoxic and genotoxic effects of 3
different endodontic sealers by using the methyl-thiazol-diphenyltetrazolium (MTT)
assay and the comet assay in a setting timedependent manner.
Materials and Methods
Cell Culture
Chinese hamster fibroblasts (V79) were cultured in Dulbecco modified
Eagle medium supplemented with 10% fetal bovine serum, penicillin (10,000 IU/
mL), and 1% streptomycin (10 mg/mL). Cultures were incubated under an atmosphere
of 5% CO2 at 37
C until confluency. Cells were then detached from the flasks,
seeded according to the assay (cytotoxicity or genotoxicity), and incubated again for
24 hours.
Preparation of Extracts
Three sealers were tested, each one with a specific chemical base (Table 1).
Sealers were manipulated following the manufacturers instructions, layered into
24-well plates, and covered by 2.5 mL cell culture medium after waiting 0, 12, and
24 hours for setting. Samples were then reincubated for 24 hours at 37
C. Original
extracts (1:1) were serially diluted in the cell culture medium (1:2, 1:4, 1:8, 1:16,
and 1:32).
Cytotoxicity Analysis
Cells seeded in 96-well plates (5  103 cells/well) were exposed to 200 mL/
well of the serial dilutions of the extracts. Untreated cells were used as controls. Cells
under the experimental conditions were incubated at 5% CO2 37
C for 24 hours.
The culture medium in each well was replaced by 100 mL MTT solution (Sigma
JOE Volume 40, Number 4, April 2014

TABLE 1. Main Components, Setting Time, and Manufacturer of Tested Sealers
Sealer Composition
AH Plus Paste A: bisphenol-A epoxy resin, bisphenol-F epoxy resin,
calcium tungstate, zirconium oxide, silica, iron oxide
pigments; Paste B: dibenzyldiamine, aminoadamantane,
tricyclodecane-diamine, calcium tungstate, zirconium
oxide, silica, silicone oil
RoekoSeal Polydimethysiloxane, silicone oil, paraffin-base oil,
hexachloroplatinic acid (catalytic agent), zirconium
dioxide
EndoRez 30% urethane dimethacrylate, zinc oxide, barium sulfate,
resins, pigments
Aldrich Co, Munich, Germany) and incubated for 1 hour. This solu-
tion was removed, and resulting formazan crystals were dissolved by
the addition of 100 mL dimethyl sulfoxide (Sigma Aldrich). Plates
were shaken for 10 minutes at room temperature and read at 570
nm in a spectrophotometer (ASYS Hitech GmbH, Eugendorf, Austria).
Four wells were exposed to each dilution of the extracts in 3 inde-
pendent experiments. Absorbance readings were normalized to the
untreated control cultures and represent the inhibition of succinyl
dehydrogenase activity (cellular metabolism). Statistical differences
were analyzed by Kruskal-Wallis complemented by Dunn multiple
comparisons test, both with significance of P < .05.
Genotoxicity Analysis
The comet assay was performed following a standard protocol
(16). Chemicals were obtained from Sigma (St Louis, MO). Dilutions
that allowed cell viability of approximately 50% in the MTT assay
were chosen for this test. The basic principle of the single-cell gel
(comet) assay is the migration of DNA fragments in an agarose matrix
under electrophoresis. Under a microscope, cells resemble a comet,
with a nucleus and a tail containing DNA fragments or strands migrating
toward the anode.
The negative control group was treated with cell culture medium
(Dulbecco modified Eagle medium), and positive control group was
Figure 1. Graphical representation of the sealers cytotoxicity in V79 cells after exposure to extracts, according to dilution and setting time. Columns represent the
median cell viability expressed as percentage. Bars represent 25% and 75% percentiles. Original extracts (1:1) were serially diluted in fresh medium. Cell cultures
were exposed for 24 hours, and cellular survival in treated and untreated cell cultures was determined in quadruplicate in 3 independent experiments (n = 12).
Symbols (*, #) indicate the statistical differences among groups.
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Basic ResearchTechnology
Setting time Manufacturer
8h Dentsply De Trey GmbH, Konstanz, Germany
4550 min Coltene-Whaledent, Langenau, Germany
1520 min Ultradent, South Jordan, UT
established by using ethyl methane sulfonate at 5 mmol/L. Three slides
were prepared per treatment. After incubation with the extracts, cells
were suspended in culture medium (approximately 1  104 cells/
mL), and 10 mL of this suspension was added to 120 mL 0.5% low
melting point agarose at 37
C. This mixture was layered onto a pre-
coated slide with 1.5% regular agarose and covered with a coverslip.
The agarose was gelled in a refrigerator, the coverslip was removed,
and slides were immersed in lysis solution (2.5 mol/L NaCl, 100
mmol/L EDTA, 10 mmol/L Tris-HCl buffer pH 10, with 1% Triton
X-100, and 10% dimethyl sulfoxide) for 2 hours at 4
C. After immersion
in alkaline buffer (0.3 mmol/L NaOH, 1 mmol/L EDTA pH >13) for 20
minutes, slides were electrophoresed for another 20 minutes at 25 V
(0.86 V/cm, 300 mA), neutralized in 0.4 mol/L Tris-HCl (pH 7.5) for
15 minutes, and fixed in absolute ethanol. Staining was performed
with 300 mL DAPI solution (40 ,6-diamidino-2-phenylindole dihydro-
chloride) for 5 minutes. At least 50 randomly captured comets per treat-
ment (25 cells from each slide) were examined at 400 magnification
by using a fluorescence microscope (Leica Leitz, Wetzlar, Germany).
Images were analyzed by software (Comet Assay IV v4.3; Perceptive
Instruments Ltd, Bury St Edmunds, UK). Tail moment was calculated
by the image analysis system as the product of the tail length (DNA
migration) and the fraction of DNA in the comet tail (% DNA in the
tail). At least 2 slides derived from independent experiments were
analyzed, and the statistical differences were analyzed by Kruskal-
Biological Properties of Root Canal Sealers 531
Basic ResearchTechnology
Figure 2. DNA damage in V79 cells after exposure to sealers. (A) Representative photomicrographs for the comet assay, according to setting time and sealer tested.
(B) Median percentage of tail moment (DNA damage), according to the tested groups. Bars represent 25% and 75% percentiles. Median DNA damage was also
calculated for 5 mmol ethyl methane sulfonate (EMS). NC, negative control.
Wallis complemented by Dunn multiple comparisons test, both with
significance of P < .05.
Results
Cytotoxicity Assay
The silicone-based sealer demonstrated cell viability close to
100% for all setting times. On the other hand, the methacrylate-based
sealer was highly cytotoxic in more concentrated dilutions (1:1 and
1:2), independently of the setting time. The epoxy-based sealer was
highly cytotoxic in lowest dilutions, but 12-hour samples presented
low cytotoxicity (1:2 and 1:4 dilutions). At 1:8 dilution, it was highly
cytotoxic only at 0 hours. None of the sealers were cytotoxic at highest
dilutions (1:16 and 1:32). Cytotoxicity data are summarized in Figure 1.
The ranking of cell viability from the most to the least cytotoxic material
was the following: methacrylate-based > epoxy resinbased > silicone-
based.
Genotoxicity Assay
Figure 2A shows representative DNA damage caused after expo-
sure to the extracts. Percentage of tail moment has increased through
532 Camargo et al.
setting times for all sealers, suggesting an influence of this factor on
sealer genotoxicity. Statistical differences were observed for the
methacrylate-based sealer at 24 hours (Fig. 2B). The ranking of DNA
damage verified at the comet assay from the most to the least genotoxic
was the following: methacrylate-based > silicone-based = epoxy resin
based.
Discussion
In the present study, the setting time affected the cytotoxicity of an
epoxy resinbased sealer, reducing its effects at 12 hours. In addition,
as the most remarkable data, genotoxicity was increased through time
for all sealers, with statistical differences for the methacrylate resin
based sealer at 24 hours.
Cytotoxicity was evaluated by the MTT assay, which has been used
to test the biocompatibility of endodontic sealers (7, 12, 1719).
Testing freshly mixed sealers is relevant, because sealers are
clinically used right after mixing and incompletely set. However,
evaluation in different periods of time is important, because the
diffusion of eluates and subproducts into the tissues may lead to
changes in cytotoxicity and genotoxic levels (5, 17, 19, 20). Our
JOE Volume 40, Number 4, April 2014

study confirmed this hypothesis, because all tested sealers showed
different levels of toxicity in different times. In this context, the use of
Chinese hamster fibroblasts (V79) is also corroborated by others,
because these cells have been extensively used for cytotoxicity and
genotoxicity evaluation of biomaterials (7, 17), with similar results to
those obtained with 2 primary human oral fibroblasts (21, 22).
We observed that the epoxy resinbased sealer (AH Plus; Dentsply
DeTrey GmbH, Konstanz, Germany) presented immediate high level of
cytotoxicity, which is in disagreement with another study (13). Accord-
ing to the manufacturer, AH Plus does not release formaldehyde;
however, other studies (5, 20, 23) reported that this sealer preserves
amines to accelerate polymerization, which may be the reason for
strong initial cytotoxicity. In addition, it is suggested that a cytotoxic
by-product appears later during setting (4). This may be the reason
for the decreased cytotoxicity at 12 hours for this sealer, with a recovery
of the cytotoxic effect at 24 hours. The methacrylate-based sealer
showed high cytotoxicity, probably because of the presence of urethane
dimethacrylate in the structure (24). Other important data from our
study are that the methacrylate-based sealer (EndoRez; Ultradent Prod-
ucts, South Jordan, UT) was not completely set, even following all the
manufacturers instructions. This may explain its harmful biological
effects on the cultures. On the other hand, the silicone-based sealer
(RoekoSeal; Colt
ene-Whaledent, Langenau, Germany) was not cytotoxic
at any of the tested times. This is in agreement with another study (19).
The comet assay test has been used to determine the genotoxicity
of the biomaterials. It is a rapid, simple, and reliable biochemical tech-
nique for evaluating DNA damage in mammalian cells (16). It is impor-
tant to emphasize that this experiment demands viable cells. Thus, the
use of diluted extracts, besides presenting more comprehensive data on
sealer cytotoxicity (for example, simulation of the diffusion of the sealer
on different layers of tissue), provides an adequate concentration to be
used on the cells for additional experiments. Our data demonstrate that
the methacrylate-based sealer was able to induce DNA damage with
significant genotoxicity at 24 hours. This suggests the possibility that
the components of this sealer can induce a late toxic reaction, probably
because of increased generation of reactive oxygen species, which
may be responsible for genotoxic and mutagenic effects (25, 26).
The epoxy resinbased sealer was shown to be genotoxic as the
cytotoxicity increases or decreases. This was not found in another
study, where no genotoxic evidence with EndoRez or AH Plus was
found in contact with the cells (20). Despite not showing cytotoxic
effects, the silicone-based sealer presented genotoxicity. It is important
to emphasize that this is the first study to report data on the potential
genotoxic effects of the silicone-based RoekoSeal.
With all data exposed, it is clear that the setting time plays an
important role in the biological effects of endodontic sealers. Further
studies may evaluate longer time-dependent effects to present a wider
picture on the cytotoxicity and/or genotoxicity of endodontic sealers.
In addition, cytotoxicity and genotoxicity are not necessarily interdepen-
dent, and the cellular mechanisms involved in both processes may be
different.
Conclusions
The setting time has influenced the cytotoxicity of the epoxy resin
based sealer and genotoxicity of all sealers. The methacrylate-based
sealer was the most cytotoxic, mainly in more concentrated dilutions, fol-
lowed by the epoxy resinbased sealer, with the silicone-based sealer not
cytotoxic at any concentration. Genotoxicity was observed for all sealers,
again with harmful effects observed for the methacrylate-based sealer.
JOE Volume 40, Number 4, April 2014
Basic ResearchTechnology
Acknowledgments
Supported by the Fundac~ao de Amparo
a Pesquisa do Estado de
S~ao Paulo (FAPESP 2009/13285-8).
The authors deny any conflicts of interest related to this study.
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Biological Properties of Root Canal Sealers 533