In Vitro Cytotoxicity of Calcium Silicate-containing Endodontic Sealers

Basic Research—Biology
In Vitro Cytotoxicity of Calcium Silicate–containing
Endodontic Sealers
Hui-min Zhou, PhD,*† Tian-feng Du, DDS, PhD,†‡§ Ya Shen, DDS, PhD,†jj
Zhe-jun Wang, DDS, PhD,† Yu-feng Zheng, PhD,*¶ and Markus Haapasalo, DDS, PhD†
Abstract
Introduction: The cytotoxicity of 2 novel calcium silicate–containing endodontic sealers to human gingival
fibroblasts was studied. Methods: EndoSequence BC
(Brasseler, Savannah, GA), MTA Fillapex (Angelus
Industria de Produtos Odontologicos S/A, Londrina, PR,
Brazil) and a control sealer (AH Plus; Dentsply DeTrey
GmbH, Konstanz, Germany) were evaluated. Human
gingival fibroblasts were incubated for 3 days both with
the extracts from fresh and set materials in culture medium and cultured on the surface of the set materials in
Dulbecco-modified Eagle medium. Fibroblasts cultured
in Dulbecco-modified Eagle medium were used as a control group. Cytotoxicity was evaluated by flow cytometry,
and the adhesion of the fibroblasts to the surface of the set
materials was assessed using scanning electron microscopy. The data of cell cytotoxicity were analyzed statistically using a 1-way analysis of variance test at a
significance level of P < .05. Results: Cells incubated
with extracts from BC Sealer showed higher viabilities
at all extract concentrations than cells incubated with extracts from freshly mixed AH Plus and fresh and set MTA
Fillapex, esspecially for the high extract concentrations
(1:2 and 1:8 dilutions). Extracts from set MTA Fillapex
of 2 weeks and older were more cytotoxic than extracts
from freshly mixed and 1-week-old cement. With extract
concentrations of 1:32 and lower, MTA Fillapex was no
longer cytotoxic. After setting, AH Plus was no longer
cytotoxic, and the fibroblast cells grew on set AH Plus
equally as well as on BC Sealer. Conclusions: BC Sealer
and MTA Fillapex, the 2 calcium silicate–containing endodontic sealers, exhibited different cytotoxicity to human
gingival fibroblasts. (J Endod 2015;41:56–61)
Key Words
Calcium silicate–containing sealer, cell attachment,
cytotoxicity, flow cytometry, mineral trioxide aggregate
M
ineral trioxide aggregate (MTA) is a calcium silicate–based hydraulic cement
(1–3). MTA has gained wide acceptance in dentistry since its introduction in
the early 1990s (4) because of its good biological and physical properties. It was
initially used as a root-end filling material but is now used in a variety of challenging
clinical situations such as pulp capping, pulpotomy, apexogenesis, apical barrier formation in teeth with open apexes, repair of root perforations, and as a root canal filling
material (1, 5). The excellent properties of MTA such as good biocompatibility,
bioactivity, and osteoconductivity have encouraged scientists worldwide to develop
other MTA-based endodontic materials. Recently, new calcium silicate–based endodontic sealers have been introduced, such as MTA Fillapex (Angelus Industria de Produtos Odontologicos S/A, Londrina, PR, Brazil). According to the manufacturer, it
consists of 2 main components (MTA-like bioceramic mixture and resinous components), and after mixing it, contains MTA, salicylate resin, natural resin, bismuth oxide,
and silica. It is also claimed by the manufacturer that MTA Fillapex has excellent radiopacity, easy handling, and good working time.
Another relatively new bioceramic root canal sealer, Endosequence BC Sealer
(Brasseler, Savannah, GA), was launched a few years ago. It is composed of calcium
silicates, zirconium oxide, calcium phosphate monobasic, calcium hydroxide, filler,
and thickening agents. It requires the presence of water for setting (6). BC Sealer
has been reported to have antimicrobial activity possibly because of a high pH, hydrophilicity, and active calcium hydroxide diffusion (7). Both of these recently
introduced endodontic sealers (MTA Fillapex and BC Sealer) contain calcium silicate, which is biocompatible (8) and reacts with water to generate a calcium silicate
hydrate phase during setting (5). The development of new types of endodontic
sealers containing calcium silicate is based on the pursuit of sealers with good
biocompatibility that induce the formation of mineralized tissue and suitable physical properties including flow rate, sealing ability, manipulation, and faster setting
reaction (9, 10).
Endodontic sealers are supposed to fill the irregularities between the dentinal
walls and the gutta-percha core as well as the lateral or accessory canals and bond
both to gutta-percha and dentin (11, 12). In addition to defined requirements of
mechanical and physical properties (13), the biological compatibility of root canal
sealers is important because they come into contact with periapical tissues (14) and
the tissue response to the sealers may influence the final outcome of the root canal treatment (15). The sealers with good biocompatibility are beneficial to aid or stimulate the
repair of injured tissues (14). In MTA Fillapex, calcium silicate is mainly combined with
From the *Center for Biomedical Materials and Engineering, Key Laboratory of Superlight Material and Surface Technology, Ministry of Education, Harbin Engineering University, Harbin, China; †Division of Endodontics, Department of Oral Biological and Medical Sciences, Faculty of Dentistry, The University of British Columbia,
Vancouver, Canada; ‡Department of Stomatology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, PR China; §Department of Stomatology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; jjDepartment of Materials Engineering, The University of British Columbia,
Vancouver, Canada; and ¶State Key Laboratory for Turbulence and Complex Systems and Department of Materials Science and Engineering, College of Engineering,
Peking University, Beijing, China.
Address requests for reprints to Prof Markus Haapasalo, Department of Oral Biological and Medical Sciences, UBC Faculty of Dentistry, 2199 Wesbrook Mall, Vancouver, BC, Canada V6T 1Z3. E-mail address: markush@dentistry.ubc.ca
0099-2399/$ - see front matter
Copyright ª 2015 American Association of Endodontists.
http://dx.doi.org/10.1016/j.joen.2014.09.012
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Basic Research—Biology
a resinous component, whereas in EndoSequence BC Sealer, calcium
silicate is combined with an inorganic component, making it a true bioceramic cement. Several studies have been done to evaluate the cytotoxicity and physicochemical properties of MTA Fillapex (9, 10, 16–18)
and BC Sealers (6, 19). However, to our knowledge, no studies that
have made a direct comparison of the cytotoxicity between these
2 new types of calcium silicate–containing root canal sealers have
been published. The purpose of this study was to compare the
in vitro cytotoxicity of 2 calcium silicate–containing endodontic
sealers (MTA Fillapex and BC Sealer) immediately after mixing and
after complete setting. The results are compared with an epoxy
resin–based sealer (AH Plus; Dentsply DeTrey GmbH, Konstanz,
Germany), which has a long clinical track record and has been
extensively studied (20–23).
Materials and Methods
EndoSequence BC (batch #12003SP) and MTA Fillapex (batch #
25612) were examined in the present study together with an epoxy
resin–based sealer (AH Plus, batch #1210000912) used as a control
material.
Cell Culture
In vitro cytotoxicity of the endodontic sealers was evaluated using
human gingival fibroblasts. The fibroblasts were obtained from previously established stocks cultured from healthy patients who underwent
oral surgery (7, 24). Standard protocols were followed in establishing
and maintaining the cultures. Dulbecco-modified Eagle medium
(DMEM) (Gibco, Grand Island, NY) supplemented with 100 mg/mL
penicillin G, 50 mg/mL streptomycin, 0.25 mg/mL Fungizone (Gibco),
and 10% fetal bovine serum (Gibco) was used as the cell culture medium. Fibroblasts of the seventh to eighth passage were used for both
cytotoxicity and cell adhesion assays.
Preparation of Specimens and Extracts
Three sealers (AH Plus, EndoSequence BC Sealer, and MTA
Fillapex) were mixed in strict compliance with manufacturers’ instructions and shaped with 3-mm-thick nonreactive plastic molds
with a diameter of 10 mm under aseptic conditions. Sealers that
were used immediately after mixing were designated as fresh specimens, and those that were incubated (37 C, 100% relative humidity) for 3 times the setting time as given by the manufacturers were
designated as set specimens. The set specimens were further
exposed to ultraviolet light for 20 minutes on each surface to
ensure sterility.
For the fresh specimens, a disc was placed at the bottom of 24-well
tissue culture plates containing culture medium and incubated (37 C
and 95% relative humidity) for 24 hours. The surface area to volume
ratio used for extract preparation was approximately 250 mm2/mL according to ISO standard 10993-5 (25), and the extract was collected for
the evaluation of cytotoxicity. For the set specimens, a disc was placed in
the well of 24-well plates containing the DMEM culture medium and
incubated for 24 hours at 37 C and 100% humidity (week 0). After
the collection of extract, fresh culture medium with the same volume
was added to the same well where the same sealer disc used in week
0 was placed. The specimen was incubated for another 6 days at
37 C and 100% humidity, and a new extract was collected (week 1).
Every weekly cycle was performed as described earlier until 4 weeks.
The extract collected at each time point was diluted to 1:2, 1:8, 1:32,
and 1:128 with DMEM to achieve a total of 4 concentrations of each
extract. DMEM without the materials incubated for 24 hours was
used as the control.
JOE — Volume 41, Number 1, January 2015
Cytotoxicity Assay
The cytotoxicity of sealers was evaluated by cell viability as
measured by a quantitative flow cytometry test. Human gingival fibroblasts were plated at a density of 1 105 cells/mL in a 12-well plate
containing 2 mL culture medium per well and incubated with or without
different concentrations of extracts diluted in DMEM for 3 days. After
culture for the indicated time, cells from different test groups and controls were washed twice with phosphate buffered saline (PBS; SigmaAldrich, St Louis, MO) and detached from the culture wells with
0.25% trypsin. The collected cells were centrifuged to discard the supernatant and resuspended at 1 105 cells/mL in PBS. The cells
were stained with fluorescein Calcein AM (Molecular Probes Inc, Eugene, OR) (labs/lem = 494/517 nm, green fluorescence) and ethidium
homodimer-1 (EthD-1) (Molecular Probes Inc) (labs/lem = 528/
617 nm, red fluorescence) according to the flow cytometry protocol
for a viability assay (LIVE/DEAD Viability/Cytotoxicity Kit for mammalian
cells; Molecular Probes Inc, Eugene, OR) and incubated for 20 minutes
at room temperature and protected from light. The stained cells were
analyzed using a flow cytometer (BD FACSCalibur; BD Biosciences,
San Jose, CA) using 488-nm excitation and measuring green fluorescence emission for calcein and red fluorescence emission (617 nm)
for EthD-1. Single-color stained cells and nonstained cells were used
to standardize the settings and to determine background autofluorescence, respectively. The percentage distributions of viable and dead
cells were determined by FlowJo software (Tree Star, Inc, Ashland,
OR). The cell viability was expressed as a percentage of the mean of
DMEM controls, which was taken to represent 100% cell viability. Experiments were performed in triplicate.
Cell Adhesion Assay
The morphology of human gingival fibroblasts attached to the
surface of endodontic sealers after culturing for 1, 3,and 7 days
was observed by scanning electron microscopy (Stereoscan 260;
Cambridge Instruments, Cambridge, UK). The specimens of EndoSequence BC, MTA Fillapex, and AH Plus were shaped into 1.6-mmthick disks of 5 mm in diameter using rubber molds under the
same conditions as the cytotoxicity assay. Fifteen disks of each material were prepared and subdivided into 3 groups. Each group contained 5 parallel samples (n = 5). To remove the toxic byproducts,
all disks were first incubated at 37 C in the wells of 24-well tissue
culture plates (Sarstedt, Inc, Montreal, Canada) containing 1 mL
distilled water that was changed daily for 5 days. The disks were
then incubated in DMEM culture medium without cells for 7 days
and then seeded with gingival fibroblasts (5 104 cells/well with
1 mL DMEM) for 1, 3, and 7 days, respectively.
Specimens for scanning electron microscopic (SEM) examination were prefixed with phosphate buffered 2.5% glutaraldehyde
(Sigma-Aldrich) for 30 minutes before further fixation in 1%
osmium tetroxide (OsO4) for 1 hour. The specimens were subsequently rinsed in PBS and dehydrated in sequential-graded concentrations of ethanol (50%, 70%, 80%, and 90%) for 5 minutes each
and in pure ethanol (100%) for 10 minutes. The dehydrated specimens were dried using a critical point drier (Samdri-795; Tousimis
Research Corporation, Rockville, MD) and then sputter coated with
gold-palladium. Finally, specimens were examined with a scanning
electron microscope at a magnification of 480–500 and an accelerating voltage of 8–10 kV.
Cell viability data were expressed as the mean standard deviation. Differences between groups were analyzed statistically using 1-way
analysis of variance (SPSS for Windows 11.0; SPSS, Chicago, IL) at a significance level of P < .05.
Calcium Silicate–containing Endodontic Sealers
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Basic Research—Biology
Results
Cytotoxicity of the Sealers
Representative flow cytometry histograms obtained from the
stained human gingival fibroblasts after culture with various concentrations of extracts derived from AH Plus, BC Sealer, MTA Fillapex, and
DMEM cultural medium for 3 days are shown in Figure 1. The percentage of viable cells was determined by flow cytometry analysis, and cell
viabilities of various concentrations of extracts derived from both fresh
and set calcium silicate–containing (MTA Fillapex and BC) and epoxy
resin–based (AH Plus) endodontic sealers after culture for 3 days
are summarized in Figure 2A–D. Fresh AH Plus was strongly cytotoxic
at a high extract concentration (1:2), but the cytotoxic effect was
reduced compared with the control at an extract dilution of 1:128.
MTA Fillapex showed similar concentration-dependent cytotoxicity to
AH Plus, but the cytotoxicity of MTA Fillapex was reduced to control
levels when the extract was diluted to 1:8 (Fig. 2). BC Sealer exhibited
mild toxicity at a high extract concentration, and there was no significant
difference in the cell viabilities at various extract concentrations
(P > .05). Cells incubated with extracts from BC Sealer showed the
highest viabilities at all extract concentrations.
With sealers set for 2 weeks or more, extracts from AH Plus
had no cytotoxic effect (P > .05). BC Sealer remained noncytotoxic
throughout the 4-week testing period (fresh and set material), and
there were no significant differences in viabilities of cells exposed
to extracts from BC Sealer at all extraction times and extract concentrations (P > .05). Contrary to AH Plus and BC Sealer, extracts
from MTA Fillapex set for more than 1 week at concentrations of
1:2 and 1:8 were significantly more cytotoxic than extract from
fresh cement and 1-week-old cement (Fig. 2). With extract concentrations of 1:32 and lower, MTA Fillapex no longer was cytotoxic.
Cell Adhesion Assay
Representative morphologies of human gingival fibroblast adhesion on the surfaces of BC Sealer, MTA Fillapex, and AH Plus after culture in DMEM with the 3 sealers for 1, 3, and 7 days are shown in
Figure 3. The morphology of the cells seeded on AH Plus and BC Sealer
showed similar characteristics; the fibroblasts attached to and spread
over the material surface displaying the typical spindle-shaped fibroblast morphology after an overnight culture (Fig. 3A and D). As the
Figure 1. Representative 2-dimensional dot plots of the flow cytometry data derived from CAM- and EthD-1 stained human gingival fibroblasts after exposure to
extracts from set AH Plus, EndoSequence BC Sealer, MTA Fillapex (week 4), and DMEM control after culture for 3 days. The dot plots in each of the 13 images below
represent the distribution of viable (right), early apoptotic (lower left), and dead cells (upper left), respectively.
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Figure 2. Cell viability of extracts with various concentrations derived from the set (A) AH Plus, (B) EndoSequence BC Sealer, and (C) MTA Fillapex after culture
for 3 days and (D) the fresh sealers. The extracts of set specimens were collected at different experimental periods (0, 1, 2, 3, and 4 weeks). The results show
mean standard deviation of 3 parallel experiments performed in triplicate.
culture time increased, the number of attached cells increased, and the
cells began to connect with each other on the surface of sealers (Fig. 3B,
C, E, and F). After culture for 7 days, the cells covered the entire surface
of AH Plus, and the cell population density of fibroblasts grown on AH
Plus was higher than those grown on BC Sealer (Fig. 3C and F). For MTA
Fillapex, the cells were round and seemed weakly attached to the material surface at all determined time points (Fig. 3G–I).
The SEM images also revealed that the surface of both MTA Fillapex
and BC Sealer showed uneven crystalline surface structures where
fibroblasts grew, whereas AH Plus had a relatively smooth, noncrystallized surface.
Discussion
The cytotoxicity of endodontic sealers may cause cellular degeneration and delayed wound healing because of the direct contact of sealers
with periapical tissues (26, 27). Most conventional endodontic sealers
have shown inadequate biological activity and have been cytotoxic in cell
Figure 3. Scanning electron micrographs of the morphology of human gingival fibroblasts attached on the surface of (A–C) AH Plus, (D–F) EndoSequence BC
Sealer, and (G–I) MTA Fillapex after culture in DMEM culture medium for (A, D, and G) 1, (B, E, and H) 3, and (C, F, and I) 7 days.
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Basic Research—Biology
cultures, especially when freshly mixed (27).Calcium silicate cements
have exhibited good biocompatibility, bioactivity, and osteoconductivity
(5); therefore, it has appeared interesting to develop endodontic
sealers based on calcium silicate hydraulic cements. Consequently,
new types of calcium silicate–containing root canal sealers have been
introduced, such as MTA Fillapex and BC Sealer. However, there are
some limitations resulting from the physical characteristics of calcium
silicate cements such as their long setting times (28). Such drawbacks
can be overcome by adding other components to the sealers to adjust
their physical properties. Thus, it is important to investigate the influence of the added components on the biocompatibility of the newly
developed endodontic sealers. Therefore, in the present study, the cytotoxicity of MTA Fillapex, which is a combination of calcium silicate with
a resin component and a pure bioceramic sealer, and the cytotoxicity of
BC Sealer, which is a combination of calcium silicate with an inorganic
component, were evaluated with an epoxy resin–based sealer (AH Plus)
used as a control material.
In the present study, the in vitro cytotoxicity assessment of both
freshly mixed and set sealers over a period of 4 weeks was performed
using cell culture and sealer extracts in various concentrations The extracts obtained by incubating the freshly mixed materials with culture
medium were used to evaluate the short-term cytotoxic effect because
clinically the sealers are applied in this form (29). The extracts
collected by incubating the set materials with culture medium over
the 4-week period were used to assess the cytotoxicity of set sealers.
In addition, under the clinical condition, it is assumed that the apical
and periapical tissues come into contact with progressively lower concentrations of the leachable cytotoxic compounds because of the setting
reaction and because the leached components are continually cleared
by extracellular fluids (29). Therefore, the cytotoxicity of the extracts
with various concentrations was investigated in the present study.
Among the methods and strategies available for cytotoxicity assay
of materials, flow cytometry provides a fast, cost-effective, safe, and sensitive assessment to the cytotoxic events (13). The determination of cell
viability depends on the physical and biochemical properties of cells.
Calcein AM is a detection probe for live cells, which readily enters cells
and is converted to calcein by intracellular esterase activity of live cells
producing an intense green fluorescence. EthD-1 is a detection probe
for dead cells, which enters cells with damaged membranes and binds
to nucleic acids to produce bright red fluorescence. Therefore, in the
present study, the cell viability was determined by flow cytometry analysis using calcein AM and EthD-1 fluorescent stains.
Cell viability depends on the type of material, culture medium, and
incubation time to which the cells are exposed. Calcium silicate is 1 of
the main components in both MTA Fillapex and BC Sealer; it reacts with
water to form C-S-H gel and contributes to good cytocompatibility (5).
However, fresh MTA Fillapex was clearly more cytotoxic than BC Sealer,
and the set Fillapex was more cytotoxic than both BC Sealer and AH Plus.
This indicates the different influence of constituent components other
than calcium silicate on the cytotoxicity of the sealers. In agreement
with earlier studies, BC Sealer showed excellent biocompatibility at
all extract concentrations as both fresh and set material (Figs. 1–3).
There was no significant difference of cell viabilities exposed to
extracts of various concentrations and extraction times. The
significantly higher cytotoxicity of MTA Fillapex may be caused by the
resin component or by some other component of the sealer.
A previous study found that both MTA Fillapex and BC Sealer
produced an alkaline pH after being freshly mixed and set (30), suggesting that the pH does not explain the difference in cytotoxicity. AH
Plus is an epoxy resin–based sealer; however, after initial cytotoxicity
of the freshly mixed AH Plus sealer in high extract concentrations, set
AH Plus was no longer cytotoxic, unlike MTA Fillapex (Figs. 2 and 3).
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Zhou et al.
The 3 sealers each had their own, characteristic timeline of cytotoxicity. BC Sealer was not cytotoxic at any stage of the setting (from
fresh to 4 weeks after mixing). The freshly mixed AH Plus was cytotoxic
in a concentration-dependent manner. This is in agreement with previous studies that have documented the moderate to severe cytotoxic effect of AH Plus immediately after mixing (20–22). This initial
cytotoxicity has been attributed to a minimum formaldehyde release
from amines added to accelerate the epoxy polymerization (21–23),
which decreases after setting (21, 23), and the epoxy resin
component (21, 23).Two weeks after mixing, no cytotoxic effect was
measured from AH Plus extracts. In fact, the growth of gingival
fibroblasts on set AH Plus sealer was comparable with that on BC
Sealer (Fig. 3), which was perhaps somewhat unexpected and further
proof of the biocompatibility of set AH Plus. MTA Fillapex was cytotoxic
throughout the 4-week test period. It can be speculated that the initial
cytotoxicity is caused by the resin component and the long-lasting cytotoxic effect is caused by other substances released from the sealer, such
as lead (31). SEM images of cell culture experiments revealed round,
damaged fibroblasts on the surface of the MTA Fillapex sealer (Fig. 3).
Conclusions
Within the limitations of the present study, freshly mixed and set BC
Sealer showed better cytocompatibility to human gingival fibroblasts
than MTA Fillapex. The long-lasting cytotoxic effect by MTA Fillapex
may be caused by lead released only from the set sealer. AH Plus was
cytotoxic only as freshly mixed sealer and allowed growth of gingival fibroblasts on the surface of the set material.
Acknowledgments
Hui-min Zhou and Tian-feng Du contributed equally to this
study.
The authors thank Angelus Soluç~oes Odontologicas for
donating the materials used in this study.
Supported in part by the National Natural Science Foundation
of China (grant no. 81300864), the China Postdoctoral Science
Foundation (grant no. 2013M541350), startup funds provided by
the Faculty of Dentistry, University of British Columbia, Canada,
and the Canada Foundation for Innovation (CFI fund; Project
number 32623).
The authors deny any conflicts of interest related to this study.
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