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RESEARCH Open Access
In vitro analysis of the cytotoxicity and the
antimicrobial effect of four endodontic sealers
Ines Willershausen, Angelika Callaway, Benjamin Briseño and Brita Willershausen*
Abstract
Introduction: The aim of this study was to investigate in vitro the cytotoxicity and antibacterial properties of four
different endodontic sealers using human periodontal ligament fibroblast cell proliferation and visual analysis of
growth inhibition.
Methods: A silicone (GuttaFlow), silicate (EndoSequence BC), zinc oxide eugenol (Pulp Canal Sealer EWT) and
epoxy resin (AH Plus Jet) based sealer were incubated with PDL fibroblasts (104 cells/ml, n = 6) up to 96 h. Cell
proliferation (RFU) was determined by means of the Alamar Blue assay. Cell growth and morphology was visualized
by means of fluorescent dyes. Possible antibacterial properties of the different sealers were visualized by means of
SEM (Enterococcus faecalis; Parvimonas micra).
Results: Fibroblast proliferation depended on sealer and cultivation time. After 72 and 96 h GuttaFlow and
EndoSequence BC showed relatively non-cytotoxic reactions, while Pulp Canal Sealer EWT and AH Plus Jet caused
a significant decrease of cell proliferation (p < 0.001). Visualization of cell growth and morphology with various
fluorescent dyes supplemented the results. No antibacterial effect of EndoSequence BC to P. micra was found,
whereas GuttaFlow showed a weak, Pulp Canal Sealer EWT and AH Plus Jet extensive growth inhibition. Also, no
antibacterial effect of GuttaFlow, EndoSequence BC or AH Plus Jet to E. faecalis could be detected.
Conclusions: These in vitro findings reveal that GuttaFlow and EndoSequence BC can be considered as
biocompatible sealing materials. However, prior to their clinical employment, studies regarding their sealing
properties also need to be considered.
Keywords: in vitro study, root canal sealer, E. faecalis P. micra, cytotoxicity
Introduction
In recent decades, a considerable Improvement in endo-
dontic methods, devices, and also in root canal filling
materials, has occurred. Thus, patients as well as dental
professionals are more inclined to favour tooth preserva-
tion over extraction of disputable teeth [1,2]. In conse-
quence, since increased technical knowledge and
scientific improvements have lead to higher treatment
success rates, endodontic treatment and the subsequent
restoration of the tooth should be considered as a ther-
apy superior to implantation [3,4].
The choice of a biocompatible sealing material is cru-
cial to the clinical success of endodontic therapy [5].
Although sealers were developed to be confined within
the root canal system, their extrusion over the apical
constriction is frequently observed [6,7]. Therefore,
these materials should have good biocompatibility and
be well tolerated by the peri-apical tissues [8]. The
induction of a mild tissue reaction, together with cellu-
lar resorption of the sealing material in the case of
extrusion over the apical foramen, needs to be evalu-
ated. Several in vitro, in vivo and clinical studies [9-13]
indicate that AH Plus, an epoxy resin-based root canal
sealer, is suitable for successful endodontic therapy. This
sealer remains popular despite its well-documented
mutagenicity [14], cytotoxicity and the induction of a
severe inflammatory response [15-17]. Besides cell dys-
functionality as a reaction to the epoxy resin-based seal-
ing material [16], an intense inflammation characterized
by the presence of lymphocytes, macrophages, giant for-
eign body cells as well as necrotic bone fragments in
* Correspondence: brita.willershausen@unimedizin-mainz.de
Department of Operative Dentistry, University Medical Centre of the
Johannes Gutenberg University Mainz, Germany
Willershausen et al. Head & Face Medicine 2011, 7:15
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HEAD & FACE MEDICINE
© 2011 Willershausen et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
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maxilla of guinea pigs after AH Plus implantation was
observed. Due to its severe initial inflammatory reaction
that diminished over time but persisted throughout the
entire observation period, the authors [17] claim that
this material does not possess enough biocompatible
properties to be considered as an acceptable sealer for
clinical use. Based on these contradictory results con-
cerning an endodontic sealing material with a “gold
standard” status [13], the tissue reaction induced by
alternative sealers needs to be investigated in similar
study designs to decide upon their potential clinical
usage. GuttaFlow is a relatively new sealing material,
which combines gutta-percha and sealer into an inject-
able system. According to the manufacturer, this system
is based on polydimethylsiloxane with added gutta-
percha and nano-silver particles (< 30 μm). Due to its
viscosity, it is more likely to be extruded into the peri-
apical tissue when placed under pressure [18]. However,
it remains unclear which tissue reaction is caused by
this material. In the study of AlAnezi et al. [19], the
possible cytotoxicity of Endosequence BC Root Repair
Material and grey and white MTA was evaluated. When
exposed to these materials, the cells showed no signifi-
cant difference in viability, while the cells in contact
with AH 26 were significantly reduce in their viability.
Cleaning and shaping procedures are used to eliminate
microorganisms from the root canal system during
endodontic treatment. However, quite often a complete
removal of bacteria is not possible [20]. In such cases it
would be desirable that sealing materials have antimi-
crobial properties. Using either the agar diffusion test or
the direct contact test or both, different endodontic sea-
lers have already been assessed for a possible antibacter-
ial effect, most often measured against strains of
E. faecalis [21-28].
Baer and Maki [29] demonstrated that AH Plus and
Pulp Canal Sealer EWT were not able to inhibit the
growth of E. faecalis.
Therefore, the present in vitro study aimed at compar-
ing the biocompatibility and the possible antibacterial
effect on E. faecalis and P. micra of the four different
root filling materials GuttaFlow, Endosequence BC, Pulp
Canal Sealer EWT and AH Plus Jet.
Materials and methods
Sealing materials
For this in vitro study four different root canal sealers
were chosen: GuttaFlow (Roeko, Coltène Langenau Ger-
many, Batch No. 240412) consists of a polydimethylsilox-
ane matrix, is a cold flowable and self-curing sealer,
which combines sealer and gutta-percha in one product;
Endosequence BC Sealer (Brasseler, Savannah, GA, USA,
Batch No. 0900458) is a premixed ready-to-use injectable
material, based on a calcium silicate composition; Pulp
Canal Sealer EWT (Pulp Canal Sealer EWT; SybronEndo,
Orange, CA, USA, Batch No. 9-1222) is a zinc oxide
eugenol based sealer; AH Plus Jet (Dentsply/Detrey, Kon-
stanz, Germany, Batch No. 1004002041) is an epoxy resin
based root canal sealer and consists of a paste-paste sys-
tem, with paste A containing epoxy resin and iron oxide,
and paste B containing amines and silicone oil.
The sealers were prepared according to the manufac-
turers’ recommendations. For the cell culture experi-
ments, the materials (1.3 mg ± 0.1 mg) were placed at
the junction between the base and wall of each multi-
well cylinder (16 mm diameter; Greiner Bio-One, Frick-
enhausen, Germany), thus covering only a small area of
the well. The amount of sealer was determined accord-
ing to preliminary experiments and calculated by weigh-
ing the sealers with an analytical balance (Pioneer PA64,
Ohaus, Pine Brook, USA, Figure 1, left). The sealing
materials were allowed to set for 24 h.
To determine the bacterial colonization of root canal
sealers, discs of equal size (Ø 12.5 ± 0.5 mm; thickness
2 ± 0.5 mm) were prepared under sterile conditions
from the materials and allowed to set for 24 h.
Cell culture
Human Periodontal Ligament Fibroblasts (Clonetics®
HPdLF Lonza, Switzerland) were cultured in Dulbecco’s
Figure 1 Analytical balance (Pioneer PA64, Ohaus, Pine Brook, USA), left; inverted fluorescence microscope (Axiovert 40C/Carl Zeiss,
Göttingen, Germany), middle; fluorescence/luminescence reader (Synergy HT-Reader, Biotek, Winooski, VT, USA), right.
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Modified Eagle Medium, supplemented with 10% foetal
bovine serum, 2 mM L-Glutamine and 100U/100 μg/ml
Penicillin/Streptomycin (Invitrogen, Paisley, UK), incu-
bated at 37°C, in a humidified atmosphere containing
5% CO2, and a bidaily medium change. To assess the
interaction of the sealing agents with the fibroblasts,
different in vitro assays were carried out.
Cell fluorescence
To demonstrate the interactions between cells and seal-
ing materials, cells (20,000 cells/well) were stained with
various fluorescent dyes and viewed with an inverted
fluorescence microscope (Axiovert 40C/Carl Zeiss,
Göttingen, Germany) at magnifications of × 25-400
(Figure 1, middle).
Phallacidin (BODIPY® FL phallacidin; Invitrogen, Pais-
ley, UK) selectively labels F-actin and was used to visua-
lize the cytoskeleton. The blue-fluorescent DAPI nucleic
acid stain (4’,6-Diamidine-2’-phenylindole dihydrochlor-
ide; Roche Diagnostics, Mannheim, Germany) preferen-
tially stains double stranded DNA. It yields highly
fluorescent nuclei and no detectable cytoplasmic fluores-
cence. Blue fluorescence contrasts vividly with the green
phallacidin staining.
Calcein-AM/Ethidium homodimer II staining (LIVE/
DEAD® Viability/Cytotoxicity Kit; Invitrogen, Paisley,
UK), a two-colour fluorescence-based method, was used
to measure the viability of the cultured cells, and to
detect a possible cytotoxic effect of the sealers. Calcein
AM is a fluorogenic esterase substrate that is hydrolysed
intracellularly to a green fluorescent product, which is
an indicator of live cells. Ethidium homodimer II enters
cells through damaged membranes and intercalates with
the DNA in the nucleus, emitting a red fluorescent
signal.
Cell viability assays
The four sealers were tested for possible effects on cell
proliferation and metabolic activity of the PDL fibro-
blasts. Cell proliferation was quantitatively measured
by means of the Alamar Blue assay (Alamar Blue Cell
Viability Reagent; Biozol, Eching, Germany), which is
based on detection of metabolic cell activity. The Ala-
mar Blue reagent contains an indicator dye, which
fluoresces in response to cell growth. The cells were
incubated in a 96-well plate (10,000 cells/well) under
standard conditions, and with 10% Alamar Blue for 96
h. At 0, 1, 6, 24, 48, 72, 96 h the fluorescence was mea-
sured at a wavelength of 560/20 and 620/40 nm with a
fluorescence reader (Synergy HT-Reader, Biotek,
Winooski, VT, USA). Cells without sealing material
served as control. Logarithmic signals were converted
to a linear scale and expressed as relative fluorescence
units (RFU).
The cytotoxic potential of the four sealing materials
was also investigated by means of the ToxiLight® BioAs-
say Kit (Lonza Rockland, Rockland, ME, USA). This
assay is a non-destructive, bioluminescent cytotoxicity
assay, which quantitatively measures the release of Ade-
nylate Kinase (AK) from damaged cells. The PDL fibro-
blasts were incubated under standard conditions in a 96-
well plate (30,000 cells/well). After incubating the cells
with the sealing agent for 24 h, the supernatants were
mixed with AK detection agent. After 5 min incubation,
the emitted light intensity is measured in a luminometer
(Synergy HT-Reader, Biotek, Winooski, VT, USA, Figure
1, right). Logarithmic signals were converted to a linear
scale and expressed as relative luminescence units (RLU).
Bacterial colonization of root canal sealers
Enterococcus faecalis DSM 20478 was grown anaerobi-
cally for 24 h at 37°C in Schaedler broth (Becton Dick-
inson, Sparks, MD, USA). Parvimonas micra ATCC
33270 was grown anaerobically for 48 h at 37°C in
Anaerobe Basal Broth (Oxoid, Basingstoke, Hampshire,
England). Discs of equal size, prepared from the
cements and set, were placed into Petri dishes, contain-
ing 25 ml of nutrient broth, inoculated with E. faecalis
or P. micra, and incubated anaerobically at 37°C. After
24 h (E. faecalis) or 48 h (P. micra) of incubation, the
discs were removed. To make the bacteria visible in a
scanning electron microscope (SEM), the samples were
fixed for 30 min in 3% formaldehyde at room tempera-
ture, and dehydrated by sequential washes through a
series of 50 to 96% graded ethanol baths. After sputter-
ing in a cold sputter unit, the samples were viewed in a
DSM 962 SEM (Zeiss, Oberkochen, Germany) at an
accelerating voltage of 10 kV.
Statistical analysis
Six replicates per sealing material were used in the cell
proliferation and cytotoxicity assays, and the results are
presented as means ± standard deviation. The statistical
analysis was performed using SPSS 15.0 (SPSS Inc., Chi-
cago, IL) and SAS 9.2 (SAS Institute Inc., Cary, NC).
The data were analysed by the Mann-Whitney-Test; p <
0.05 was chosen to define statistical significance, p <
0.01 was termed as highly significant.
Results
The Alamar Blue assay yields information about the
proliferation rate of the PDL fibroblasts incubated with
the different sealers over a period of 96 h. In this assay,
high cellular proliferation rates were expressed as high
relative fluorescence units (RFU). The here-investigated
sealers influenced the proliferation and viability of the
human periodontal ligament fibroblasts in different
degrees (Figure 2). After an incubation time of 24 h,
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Pulp Canal Sealer EWT and AH Plus Jet significantly
inhibited cell growth (p < 0.001). In contrast, incubation
with GuttaFlow produced proliferation rates of the same
order of magnitude as were found for the control group,
and even promoted cell growth at 96 h. The prolifera-
tion rate of the cells in contact with Endosequence BC
was significantly lower (p < 0.001) than of the controls,
but significantly higher than cells in contact with Pulp
Canal Sealer EWT and AH Plus Jet (p < 0.001).
With the use of the ToxiLight® BioAssay, it is possible
to measure the quantitative release of Adenylate Kinase
(AK) from damaged cells. High relative luminescence
units (RLU) indicate a high release of Adenylate Kinase,
which again is an indicator for damaged cells. The RLU
are measured after the cells have been incubated with the
respective sealing agents for 24 h. PDL fibroblasts with-
out sealing material served as controls. Figure 3 shows
the amounts of Adenylate Kinase released from the PDL
fibroblasts incubated with the different sealing materials.
Cells in contact with AH Plus Jet showed a significantly
higher cytotoxicity than the controls and those incubated
with the other sealing materials (p < 0.001).
The application of Phallacidin/DAPI was utilized to
visualize nucleus and cytoplasm (Figure 4A-D). This
revealed that the PDL fibroblasts in contact with the sealing
materials were partially altered in shape, appearing round
with no visible cytoplasmic structures. Hardly any cells are
visible in close proximity to Pulp Canal Sealer EWT and
AH Plus Jet (Figure 3C-D). Similar results were obtained
when the cells were stained with Calcein-AM/Ethidium
homodimer II (Figure 4E-H). Ethidium homodimer II
enters into cells through damaged membranes, binding to
nucleic acids, thereby producing a bright red fluorescence
in dead cells. The intact cell membrane of live cells is not
permeable for Ethidium homodimer II. In close proximity
to Pulp Canal Sealer EWT and AH Plus Jet, most of the
cells are damaged, as can be observed by the red colour in
nearly all cells close to the sealers (Figure 4G-H).
Bacterial growth
No antibacterial effect of GuttaFlow, EndoSequence BC
or AH Plus Jet to E. faecalis DSM 20478 could be
detected by scanning electron microscopy. After 24 h of
incubation, on GuttaFlow, EndoSequence BC and AH
Plus Jet short chains, micro-colonies or layers of the bac-
teria, covering the complete surface, can be seen (Figure
5A-B, D). In contrast, Pulp Canal Sealer EWT is more
sparsely colonized and only short chains of the cells can
be detected (Figure 5D). The visual analysis of the scan-
ning electron micrographs of the root canal sealers incu-
bated for 48 h with P. micra ATCC 33270 shows on
GuttaFlow only few bacteria organized in micro-colonies,
whereas EndoSequence BC is uniformly colonized by the
bacteria (Figure 6A-B). On Pulp Canal Sealer EWT and
AH Plus Jet only at a magnification of 2000 or higher a
few bacteria can be detected (Figure 6C-D).
Discussion
The need for endodontic treatment is often associated
with an inflammation caused by bacterial infection. For
0
2000
4000
6000
8000
10000
12000
14000
0h 1h 6h 24h 48h 72h 96h
R
FU
Control GuttaFlow EndoSequence BC Pulp Canal Sealer EWT AH Plus Jet
Figure 2 Results of the Alamar Blue proliferation assay with PDL cells in contact with GuttaFlow, Endosequence BC, Pulp Canal Sealer
EWT and AH Plus Jet, and with cells without root canal sealers (controls). After an incubation time of 96 h, the root canal sealers Pulp
Canal Sealer EWT and AH Plus Jet significantly inhibited cell growth compared to GuttaFlow, Endosequence BC, and the control cells.
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the successful root canal treatment, minimizing the pos-
sible inflammatory reaction caused by sealing materials,
and suppressing bacterial growth are fundamental con-
ditions. The goal of the endodontic treatment is to treat
the teeth before a bacterial infection develops, and to
use a biocompatible sealing agent. In case of an infec-
tion leading to pulp necrosis or of a bacterial contami-
nation of the apical tissue, it is crucial for the outcome
of the endodontic treatment to have a successful micro-
bial elimination from the infected root canal system or
0
500
1000
1500
2000
2500
3000
3500
4000
Control GuttaFlow EndoSequence BC Pulp Canal Sealer EWT AH Plus Jet
RL
U
Figure 3 Results of the ToxiLight® BioAssay with PDL cells after 24 h. The root canal sealer AH Plus Jet lead to a significantly higher release
of Adenylate Kinase in comparison to the control cells and the other materials.
Figure 4 The reaction of the PDL fibroblasts to GuttaFlow, Endosequence BC, Pulp Canal Sealer EWT and AH Plus Jet, stained with
Phallacidin/DAPI (magnification A, B and D ×200, C ×100, bar = 100 μm) (A-D, upper panels) and with Calcein-AM/Ethidium
homodimer II (magnification E, G and H ×200, F ×100, bar = 100 μm) (E-H, lower panels) is shown. DAPI- stains the nucleus blue, and
Phallacidin counterstains the cytoplasm green. The intact membrane of live cells is not permeable for Ethidium homodimer II.
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