Basic Research—Technology
Cytotoxicity of GuttaFlow Bioseal, GuttaFlow2,
MTA Fillapex, and AH Plus on Human Periodontal
Ligament Stem Cells
Mar Collado-Gonz
alez, BSc, MBB, MSc,* Christopher J. Tom

as-Catal

Ricardo E. O~ anchez, MD, DDS, PhD,† Jos
e M. Moraleda, MD, PhD,*
nate-S

and Francisco J. Rodr
ıguez-Lozano, DDS, PhD*†
Abstract
Introduction: The aim of the present study was to eval-
uate the in vitro cytotoxicity of endodontic sealers
(GuttaFlow Bioseal, GuttaFlow2, and MTA Fillapex) on
human periodontal ligament stem cells (hPDLSCs). As
a reference, AH Plus was compared with the more recent
endodontic sealers regarding cell viability and cell
attachment. Methods: Biological testing was carried
out in vitro on hPDLSCs. Cell viability assay was per-
formed by using eluates from each endodontic sealer.
To assess cell morphology and attachment to the
different sealers, the hPDLSCs were directly seeded
onto the material surfaces and analyzed by scanning
electron microscopy. Chemical composition of the
sealers was determined by energy-dispersive x-ray,
and eluates were analyzed by inductively coupled
plasma mass spectrometry. Statistical differences were
assessed by analysis of variance and Tukey test
(P < .05). Results: Cell viability was evident after
24 hours in the presence of GuttaFlow Bioseal and Gut-
taFlow 2 but not in the case of AH Plus or MTA Fillapex.
At 168 hours, GuttaFlow Bioseal and GuttaFlow 2 ex-
hibited high and moderate cell viability, respectively,
whereas AH Plus and MTA Fillapex revealed low rates
of cell cell viability (P < .001). Finally, scanning electron
microscopy studies revealed a high degree of prolifera-
tion, cell spreading, and attachment, especially when
using GuttaFlow Bioseal disks. Conclusions: GuttaFlow
Bioseal and GuttaFlow2 showed lower cytotoxicity than
MTA Fillapex and AH plus. Further in vitro and in vivo
investigations are required to confirm the suitability of
GuttaFlow Bioseal for clinical application. (J Endod
2017;43:816–822)
From the *Cellular Therapy and Hematopoietic Transplant Unit, Hematology Department, Virgen de la Arrixaca Clinical University Hospital, IMIB-Arrixaca, and
†
School of Dentistry, Faculty of Medicine, University of Murcia, Murcia, Spain.
Address requests for reprints to Dr Francisco J. Rodr
ıguez-Lozano, School of Dentistry, University of Murcia, Hospital Morales Meseguer 2pl., Av. Marqu
es de los
V
elez s/n, 30008 Murcia, Spain. E-mail address: fcojavier@um.es
0099-2399/$ - see front matter
Copyright ª 2017 American Association of Endodontists.
http://dx.doi.org/10.1016/j.joen.2017.01.001
816 Collado-Gonz
alez et al.
a, DDS, MSc,*†
Key Words
Adhesion, calcium silicate-based endodontic sealers, cytotoxicity, human periodontal
ligament stem cells, mineral trioxide aggregate
T he aim of endodontic
treatment, after clean-
ing and shaping the root
Significance
These results may suggest that GuttaFlow Bioseal
canal system, is three- may achieve better biological response and heal-
ing when compared with GuttaFlow2, MTA Filla-
dimensional filling and
pex, and AH Plus.
sealing of all root canals
and their accessory spaces
to avoid the presence of microorganisms and promote periradicular tissue repair (1).
Combined with gutta-percha, endodontic sealers have an important role within
obturation techniques (2). It is known that such techniques may involve the noninten-
tional extrusion of the obturation materials beyond the apical foramen (3, 4).
Endodontic sealers are expected to have at least a minimum contact with
periradicular tissues when sealing the apex. However, when extruded into the
periradicular spaces, sealers may not always be removed by periradicular tissue
reaction (5) and may alter tissue repair time (6) and cause different tissue reactions
depending on the sealer’s composition (7). As a result, in vitro cytotoxic studies of
these endodontic sealers are the first step toward an evaluation of their safety (8).
The biological properties of endodontic sealers have been tested in vitro for a
long time by using animal cell lines such as L-929 mouse fibroblast, RPC-C2A rat
pulp cells, osteoblastic cells, and more recently, human periodontal ligament stem cells
(hPDLSCs), which are a multipotent stem cell population able to differentiate into ce-
mentoblasts and to develop a new periodontal ligament, including cementum, alveolar
bone, and periodontal fibers (9–12). It has been reported that PDLSCs express
mesenchymal stem cell (MSC) markers, CD105, CD73 and CD90, and have
immunosuppressive properties (13).
Calcium silicate–based materials are recognized as bioactive materials through
their capacity to induce hard tissue formation in both the dental pulp and bone, defining
a new treatment approach for dentin remineralization, vital pulp therapy, and bone
regeneration (14). The excellent properties of mineral trioxide aggregate (MTA)
(including their biocompatibility, bioactivity, osteoconductivity) have fostered a race
to develop new calcium silicate–based endodontic sealers incorporating MTA.
JOE — Volume 43, Number 5, May 2017

MTA Fillapex (Angelus Ind
ustria de Produtos Odotontol
ogicos S/
A, Londr
ına, PR, Brazil) is a resin MTA-based root canal sealer with MTA
natural resin, salicylate resin, diluting resin, bismuth trioxide, nanopar-
ticulated silica, and pigments (4). According to the manufacturer, the
properties of MTA Fillapex include good radiopacity, good working
time, and ease of handling. Biocompatibility studies showed that MTA
Fillapex has long-lasting cytotoxic effects on human gingival fibroblasts
(15). Although it shows good physicochemical properties, MTA Fillapex
appears to be even more cytotoxic than epoxy resin–based sealer AH
Plus (16).
Silicone-based endodontic sealers have been seen to have good
biological properties (17). The silicone-based sealer GuttaFlow
(Coltene Whaledent, GmBH + Co KG, Langenau, Switzerland) contains
a mixture of gutta-percha powder and polydimethylsiloxane with
nanometer-sized silver particles added as a preservative. GuttaFlow2
(Coltene Whaledent, GmBH + Co KG), an evolution of its predecessor
GuttaFlow, is also a cold flowable system combining gutta-percha pow-
der form with a particle size of less than 30 mm and sealer. Both Gutta-
Flow and GuttaFlow2 are silicone-based endodontic sealers that differ in
the form of the silver particles used (17, 18). Several biocompatibility
assays have been carried out to demonstrate that both silicone-based
sealers have good biological properties on human ligament periodontal
fibroblasts (18, 19).
A novel formulation of polydimethylsiloxane with gutta-percha
powder combined with calcium silicate particles was launched in late
2015 and named GuttaFlow Bioseal (Coltene/Whaledent AG, Altstatten,
Switzerland). Although the physicochemical properties of this material
have already been tested, no biocompatibility test has been carried out
(20).
This study was designed to assess and compare the cytotoxicity of
different endodontic sealers in contact with hPDLSCs: MTA-based end-
odontic sealer (MTA Fillapex) and 2 novel silicone-based sealers (Gut-
taFlow2, GuttaFlow Bioseal). A commonly used root canal sealer, AH
Plus (Maillefer Dentsply, Ballaigues, Switzerland), was used as refer-
ence material for comparison. The null hypothesis was that the compo-
sition of these sealers is not a determining factor in their
biocompatibility, whereas the reason for performing the experiment
was to evaluate the differences in cell viability resulting from increasing
concentrations of different tested materials over time.
Materials and Methods
Sealer Extracts
The materials tested were GuttaFlow Bioseal (Coltene/Whaledent
AG), GuttaFlow2 (Coltene/Whaledent AG), MTA Fillapex (Angelus
Ind
ustria de Produtos Odontol
ogicos S/A), and AH Plus (Dentsply
TABLE 1. Tested Materials
Materials Manufacturer
GuttaFlow Bioseal ne/Whaledent AG,
Colte
Altstatten, Switerland
GuttaFlow 2 ne/Whaledent AG,
Colte
Altstatten, Switerland
MTA Fillapex Angelus, Londrina, Parana, Brazil
AH Plus Dentsply DeTrey, Konstanz, Germany
JOE — Volume 43, Number 5, May 2017
Basic Research—Technology
DeTrey GmbH, Konstanz, Germany). Their complete compositions
are described in Table 1.
The sealers were mixed according to the manufacturers’ instruc-
tions. Disks of each sealer were formed under aseptic conditions in ster-
ile cylindrical rubber molds 5 mm in diameter and 2 mm high, sterilized
by using ultraviolet irradiation for 15 minutes, and stored in an incu-
bator at 37
C for 48 hours to achieve complete setting. To simulate
the clinical situation whereby endodontic sealer is present in the apical
region of the root and extruded over the apex, a region where different
fluids are found, the materials were evaluated by incubating cultured
cells with extracts or eluates of the sealers according to the International
Organization for Standardization 10993-5. The eluates of the different
materials were extracted in sterile conditions by using Dulbecco modi-
fied Eagle medium (DMEM) culture medium as extraction vehicle. The
extraction procedure is as follows. The materials were stored in the cul-
ture medium for 24 hours at 37
C in a humid atmosphere containing
5% CO2. In accordance with the International Organization for Stan-
dardization standards, the ratio between the surface of the sample
and the volume of the medium was 6 cm2/mL. The extraction media
were collected at the end of this period and passed through a 0.22-
mm filter (Merck Millipore, Billerica, MA). Subsequently, various dilu-
tions (1/1, 1/2, 1/4 vol/vol) of these extraction media were prepared
with fresh DMEM medium.
Assessment of pH, Osmotic Pressure, and Inductively
Coupled Plasma Mass Spectrometry of Sealer Extracts
Specimens measuring 5 mm in diameter and 2 mm high were pre-
pared from each sealer type. They were then immersed in Hank’s
balanced salt solution (HBSS) without calcium or magnesium for
24 hours. The pH of the HBSS and HBSS after immersion of the test
sealers was assessed in triplicate (GLP21 + from Crison, Barcelona,
Spain). The results correspond to the average and standard deviation.
The osmolality of HBSS and HBSS after immersion of the test
sealers was determined directly on 10 mL HBSS (Osmometer Vapro
5520, Wescor, UT). The analysis of the presence of silicon, phosphorus,
calcium, and strontium was assessed by using inductively coupled
plasma mass spectrometry (ICP-MS) (Agilent 7900, Stockport, UK).
Isolation and Culture of hPDLSCs
Human periodontal ligaments (hPDLs) were obtained from
impacted third molars (n = 12) from 10 healthy subjects. Donors
gave written informed consent according to the guidelines of the Ethics
Committee of our institution. The hPDL was scraped from the middle
third region of the root surface. After extraction, hPDL was washed
with Ca2+/Mg2+-free HBSS (Gibco, Gaithersburg, MD) and subjected
Composition
Gutta-percha powder, polydimethylsiloxane, platinum
catalyst, zirconium dioxide, silver (preservative),
coloring, bioactive glass ceramic
Gutta-percha powder, polydimethylsiloxane, silicone oil,
paraffin oil, platinum catalyst, zirconium dioxide, micro-
silver (preservative), coloring
Paste-paste: salycilate resin, diluting resin, natural resin,
bismuth trioxide, nanoparticulated silica, MTA, pigments
Epoxy paste: diepoxy, calcium tungstate, zirconium oxide,
aerosol, and dye
Amine paste: 1-adamantane amine, N.N’dibenzyl-5
oxanonandiamine-1,9, TCD-diamine, calcium tungstate,
zirconium oxide, aerosol, and silicone oil
Calcium Silicate Sealers and Human Periodontal Stem Cells 817
Basic Research—Technology
to collagenase-A digestion (3 mg/mL) (Sigma-Aldrich, St Louis, MO) for
1 hour at 37
C. The hPDL cells were cultured as described previously
(21). Red blood cells and other non-adherent cells were removed and
washed 2–3 times with phosphate-buffered saline (PBS), and fresh me-
dium was added to allow further growth. The adherent cells were grown
to 80% confluence and were defined as passage zero. For subsequent
passaging, cells were washed with Ca2+/Mg2+-free PBS (Gibco Invitro-
gen, Waltham, MA) and detached by incubating with 0.25% trypsin-
EDTA solution (Gibco Invitrogen) for 2–5 minutes at 37
C. Culture me-
dium was added to inactivate the trypsin activity. Finally, hPDLSCs were
centrifuged at 1200 rpm for 5 minutes and plated in 75-cm2 flasks at a
density of 5  103 cells/cm2. Cells were subcultured once a week. This
study was carried out with cells from passage 4 and on.
Characterization of hPDLSCs
Before the experiments, hPDLSCs were characterized by immuno-
fluorescence using specific antibodies for CD90 (1:250) (Becton-Dick-
inson, San Jose, CA), CD73 (1:200) (Santa Cruz Biotechnology Inc,
Santa Cruz, CA), and CD105 (1:100) (Abcam, Cambridge, UK). After
3 washes with PBS, the cells were incubated in the dark for 1 hour
with anti-mouse Alexa Fluor 488–conjugated secondary antibody
(1:500) (Molecular Probes, Invitrogen, Eugene, OR). Microscope
slides were mounted with anti-fade solution (Vecta shield mounting me-
dium; Vector Laboratories, Hercules, CA) containing 40 ,6-diamidino-
2-phenylindole (DAPI) (Molecular Probes; 0.2 mg/mL in PBS) and
examined in a fluorescence microscope (Leica DMI 4000B, Wetzlar,
Germany). The expression of these markers is recommended by the In-
ternational Society of Cellular Therapy as essential to confirm the
mesenchymal phenotype of the cells (22).
MTT Assay
The proliferation rate of hPDLSCs growing in the presence of the
different endodontic sealer eluates was evaluated by using the MTT assay
(MTT Cell Growth Kit; Chemicon, Rosemont, IL) after 24, 48, 72, and
168 hours of culture by using hPDLCs cultured in complete medium
as controls. Cells were seeded at density equal to 4000 cells/cm2 in
96 wells/plate (well surface is 0.32 cm2) in 200 mL DMEM. To avoid
effects caused by evaporation of the media, no cells were seeded in
the wells next to borders. Instead, sterile Milli-Q water (Millipore Cor-
poration, Billerica, MA) was placed in the wells next to the border. At
indicated times complete culture medium was replaced by serum-
free culture medium. MTT was added at a final concentration equal
to 1 mg/mL, and cells were incubated for 4 hours of the experiment.
Then, culture medium containing MTT was removed, and 100 mL
dimethyl sulfoxide was added to release formazan. The absorbance at
570 nm (Abs570) was measured by using an automatic microplate
reader (ELx800; Bio-Tek Instruments, Winooski, VT) with Abs690 as
the reference wavelength. Each condition was analyzed in quintuplicate.
TABLE 2. Assessment of pH, Osmotic Pressure, and ICP-MS of Sealer Extracts
Sealer Extracts pH Osmotic pressure
HBSS 7.98  0.15 287  2
GuttaFlow Bioseal 8.40  0.04 311  1
GuttaFlow2 8.35  0.04 288  1
MTA Fillapex 7.95  0.18 288  1
AH Plus 8.04  0.03 305  1
818 Collado-Gonz
alez et al.
Scanning Electronic Microscopy and Energy-dispersive
X-ray Analysis
Different samples of GuttaFlow Bioseal, GuttaFlow2, MTA Fillapex,
and AH Plus were shaped into 1.6-mm-thick disks of 5-mm diameter by
using rubber molds. Fifteen disks of each material were prepared and
subdivided into 3 groups, each containing 5 parallel samples. The
hPDLSCs were directly seeded onto each disk at a density of 5 
104 cells/mL. After 168 hours of culture, the samples seeded with
hPDLSCs were removed from the culture wells and fixed with 3% glutar-
aldehyde for 4 hours. Then, samples were dehydrated in a graded series
of ethanol concentrations through 100%, immersed in 100% hexame-
thyldisilazane, air dried, mounted on aluminum stubs, and sputter-
coated with gold/palladium. Finally, gold/palladium-coated specimens
were examined by scanning electron microscopy (SEM) by using a
magnification of 100. Samples of the 4 sealers cell-free were
immersed in HBSS in a ratio 6 cm2/mL and stored at 37
C for 24 hours.
Disks were coated with carbon in a CC7650 SEM Carbon Coater unit
(Quorum Technologies Ltd, East Sussex, UK), and each sample was
examined by using a scanning electron microscope (SEM Jeol 6100
EDAX, Peabody, MA) connected to a secondary electron detector for
energy-dispersive x-ray analysis (EDX) (Oxford INCA 350 EDX, Abing-
don, UK) by using computer-controlled software (INCA energy version
18) with an accelerating voltage of 2 kV, and the full scale for quanti-
fication was 4908 cts.
Statistical Analysis
Data from the MTT assay were analyzed by using SPSS version 22.0
statistical software (SPSS, Inc, Chicago, IL) and Microsoft Office Excel
2013 (Redmond, WA). Each experiment was performed with 5 repli-
cates and carried out at least twice. Absorbance value readings are pre-
sented as the mean  standard deviation (SD). Statistical differences
between control and proliferation in presence of different sealer ex-
tracts were analyzed. Also, statistical differences between proliferation
in presence of different extracts among themselves were analyzed. In
both cases, analyses were assessed by analysis of variance and Tukey
test (*<.05, **<.01, ***<.001). A P value <.05 was considered signif-
icant.
Results
pH, Osmotic Pressure, and ICP-MS: Mass of Sealer
Extracts
Variations in pH and osmolality of Ca2+/Mg2+-free HBSS because
of the incubation of different sealers are shown in Table 2. Extracts of
GuttaFlow Bioseal exhibited a slightly higher pH than GuttaFlow2. In
both cases the pH was higher than the other sealers. Regarding osmo-
lality, the highest value corresponds to GuttaFlow Bioseal. The results
from ICP-MS of the same samples are shown in Table 2.
Isolation and Characterization of hPDLSCs. Before evalu-
ating the behavior of hPDLSCs cultured on endodontic sealer disks
Concentration of element (mg/L solution)
Silicon Phosphorus Calcium Strontium
62.38 27,314.22 36.44 5.73
35,240.93 29,397.02 405.97 9.18
2040.89 27,110.24 225.60 16.15
16,232.27 3192.37 7818.45 500.52
940.09 27,033.42 19.09 8.81
JOE — Volume 43, Number 5, May 2017
 Basic Research—Technology

Figure 1. Representative immunofluorescent images of stained hPDLSCs by using (A) CD90, (B) CD73, (C) CD10, and (D) negative control. Cell nuclei (blue)
accompanied by the respective cytoplasm (green) characterize stem cells (original magnification, 20).

or extracts, hPDLSCs were characterized by immunofluorescence
with specific antibodies for CD73 and CD90, for which they showed
a positive expression higher than 95%. Specific antibodies for CD105
also were used. In this case, the positive expression was at least 75%
(Fig. 1).
Cell Viability. Cell viability results are shown in Figure 2. The
hPDLSCs cultured on endodontic sealer eluates for 24, 48, 72, and
168 hours were measured by MTT assay, considering hPDLSCs cultured
in the absence of the endodontic sealer eulates as control. At 168 hours,
cell viability in the presence of GuttaFlow Bioseal at concentrations of 1,
1/2, and 1/4 was significantly higher than it was with Guttaflow2, AH
Plus, MTA Fillapex, or the control (P < .001), whereas MTA Fillapex
eluates significantly reduced cell viability compared with the control
at 24, 48, 72, and 168 hours (P < .001) (Fig. 2). GuttaFlow2 eluates

Figure 2. Cell viability was determined by using the MTT assay; at 168 hours, MTA Fillapex or AH Plus allowed significantly lower level of viability compared with
control (***P < .001). Also, level of hPDLSC viability in presence of GuttaFlow Bioseal was significantly higher than that observed by using complete medium
(control) at 168 hours, whereas cell viability with GuttaFlow2 was similar to that of the control at the same time. Asterisks indicate significant differences from
control group (*P < .05, **P < .01, ***P < .001).

JOE — Volume 43, Number 5, May 2017 Calcium Silicate Sealers and Human Periodontal Stem Cells 819
Basic Research—Technology

Figure 3. SEM of cells attached on sealers, SEM-EDX evaluating the chemical composition (spectra) and the element distribution (elemental mapping) of Gutta-
Flow Bioseal (column A), GuttaFlow2 (column B), AH Plus (column C), and MTA Fillapex (column D). Morphology of the cells seeded on MTA Fillapex and AH
Plus had similar characteristics, with low cell attachment rates. Using GuttaFlow2 disks, cell attachment was modest, with abundant round cells appearing on surface
of the material. Finally, GuttaFlow Bioseal allowed well-adhered cells with high degree of cell spreading and production of extracellular matrix. Scale bar: 100.

led to similar cell viability results as GuttaFlow Bioseal except at
168 hours.
Cell Attachment on Endodontic Sealer Disks and Charac-
terization of Sealers. The morphology and adhesion of hPDLSCs
on the surface of GuttaFlow Bioseal, GuttaFlow2, MTA Fillapex, and AH
Plus disks after culture for 168 hours are shown in Figure 3. The
morphology of the cells seeded on MTA Fillapex and AH Plus was
similar, and both resulted in a low rate of cell attachment. By using Gut-
taFlow2 disks, cell attachment was modest, with abundant round cells
appearing on the surface of the material. Finally, GuttaFlow Bioseal ex-
hibited well-adhered cells with a high degree of cell spreading and pro-
duction of extracellular matrix. These SEM findings are coherent with
the cellular viability results. EDX analysis yielded the qualitative semi-
quantitative elemental composition of the endodontic sealers, repre-
sented in Figure 3. GuttaFlow Bioseal showed a smooth surface covered
with particles. EDX revealed C, O, Zn, and Zr together with traces of Ca
and Si. GuttaFlow2 showed a finely granular uniform surface displaying
C, O, Si, Fe (from coloring), Zn, and Zr. Neither GuttaFlow Bioseal nor
GuttaFlow2 showed presence of Pt (from platinum catalyst) or Ag (from
microsilver). AH Plus showed an external surface that appeared to be
mostly homogeneous rough surfaces. EDX analysis of the area displayed
in decreasing order of C, O, W, Zr, Si, and Ca peaks. MTA Fillapex
showed a homogeneous irregular surface with different size particles.
EDX showed peaks in a decreasing order of W, C, Ca, O, S, Re, Si,
and Zr.
Discussion
Because of the presence of osteoblast and the MSCs in periapical
tissues (14, 20, 23) and their involvement in the complete healing and
regeneration of the periapical bone defects, cell viability in the presence
of new endodontic sealers such as GuttaFlow Bioseal needs to be tested.
The present study revealed that GuttaFlow Bioseal displays better
cytocompatibility than GuttaFlow2, MTA Fillapex, and AH Plus
(P < .05), leading us to reject the null hypothesis.
Methods for testing the potential adverse effects of materials
include cell cultures, animal experiments, and clinical studies. Howev-
er, novel cell culture methods have been developed in an attempt to
replace animal models, with the aim of improving the comparability
of data generated in different test laboratories as well as to save costs
and the use of test animals by avoiding duplicate testing (24).
A key step when developing new endodontic materials for clinical
use is an evaluation of their possible cytotoxicity. The classic cellular
models used in cytotoxicity assays are primary cell cultures and
transformed strains using traditional two-dimensional or recently

820 Collado-Gonz
alez et al. JOE — Volume 43, Number 5, May 2017


three-dimensional cultures (8, 25). Each of these cell models has its
own advantages and disadvantages. The disadvantages are mainly
related to the high cost and variability of primary cultures and the genetic
instability or physiological responses of transformed strains (26, 27).
On the other hand, stem cells are a good model for predicting toxicity
because they can be used to evaluate cytotoxic effects on cell viability
as well as cell differentiation processes (28) and are probably more
relevant for human toxicity prediction (29). Moreover, they represent
a well-established in vitro model for human multipotent cell popula-
tions, and importantly, they are available from commercial sources
or can be isolated in-house. Moreover, fibroblasts are the major con-
stituents of connective tissue, the predominant cell type of periodontal
ligament, and are the most important collagen producers in this tissue
(30). For these reasons, hPDLSCs were used in the present study.
Cell viability was determined by MTT assay in this study because it
is a commonly used method for cytotoxicity testing (8, 31, 32). The
advantages of the MTT procedure are its simplicity, accuracy,
reliability, and time-saving potential. Cultivation in small volumes and
the sensitive evaluation possible with the MTT assay allow the screening
and testing of many different substances and fractions for the determi-
nation of cytotoxicity. However, there is not always a positive correlation
between cell numbers and MTT reduction. Most importantly, the MTT
assay measures metabolic activity, not necessarily cell viability, allowing
for the presence of viable cells with low metabolic activity (12). Thus, it
is necessary to validate the MTT results by using other methods. Thus,
both the MTT colorimetric assay and SEM observation of cell
morphology were used to evaluate the cytocompatibility of the endodon-
tic sealers under scrutiny.
In this study, MSCs from periodontal ligament cultured in the pres-
ence of GuttaFlow Bioseal extracts showed higher viability than when
GuttaFlow2, AH Plus, or MTA Fillapex was used. These results suggest
that components other than calcium silicate could directly affect any
sealer-induced cytotoxicity. In fact, the significantly higher cytotoxicity
of MTA Fillapex may be a result of the resins it contains or other com-
ponents of the sealer. In this respect, other studies have reported that
MTA Fillapex reduces cell survival rates (12, 25). By contrast,
silicone-based endodontic sealers have shown good biocompatibility
in recent studies. For example, Silva et al (30) found no cytotoxic effects
of the silicon-based sealer GuttaFlow2 on 3T3 fibroblast cells. In
another study, GuttaFlow exhibited lower cytotoxicity than the Re-
silon/Epiphany system or AH Plus (17). As regards epoxy resin sealers,
studies have reported little or no cytotoxicity of AH Plus, although these
studies used different experimental conditions (33, 34) including
different cell types (11, 35), contact areas (15, 25), and direct or
indirect contact of the material with the cells (12, 16).
Laboratory studies on the physicochemical properties of new ma-
terials could contribute to a better understanding of their potential clin-
ical behavior and performance. When the physicochemical properties
of GuttaFlow Bioseal, GuttaFlow2, and MTA Fillapex were analyzed
(20), GuttaFlow2 and MTA Fillapex showed little or no ability to release
ions involved in apatite formation, whereas GuttaFlow Bioseal exhibited
an ability to nucleate deposits of hydroxyapatite and Ca-poor apatite
precursors. These previous findings seem to agree with our results,
which showed GuttaFlow Bioseal to be more cytocompatible than other
endodontic sealers. In our study, no traces of platinum (Pt) or silver
(Ag) were found in GuttaFlow Bioseal or GuttaFlow2. We found an
important amount of tungsten (W) in AH Plus and MTA Fillapex that
might be related to the sealer’s cytotoxicity. However, the limitation of
our study was the lack of previous reports evaluating the cytocompati-
bility of GuttaFlow Bioseal.
On the other hand, adequate contact between cells and test mate-
rials is also crucial in cell cytotoxicity testing. Direct cell-material con-
JOE — Volume 43, Number 5, May 2017
Basic Research—Technology
tact is a clinically relevant in vitro test model considering the exposure
pattern between the root-end filling materials and periapical tissues
(14, 36, 37). The biological response depends on the molecular
interactions that result from the interface between the sealers placed
in contact with the tissues, and a biocompatible sealer should not
retard or arrest the process of tissue repair (32). SEM is a useful
tool for examining the morphology of primary cultured cells seeded
on certain dental materials (34). In the present study, hPDLSCs cultured
on GuttaFlow Bioseal appeared to be elongated and show multiple cyto-
plasmic extensions that projected from the cells to the surrounding sur-
face or adjacent cells. Moreover, hPDLSCs cultured on MTA Fillapex
seemed to be less flattened and exhibited worse spreading than cells
cultured on GuttaFlow Bioseal. Such differences in cellular attachment
and spreading on different materials may be attributed to various factors
such as chemical composition, particle size, surface topography, and
the bioactivity of the samples (20, 38). G€uven et al (34) observed
low cell attachment rates on MTA Fillapex disks, with cells appearing
round in shape on the first day, indicating the toxic potential of the ma-
terial. Moreover, Accardo et al (18) found that AH Plus led to signifi-
cantly lower cell attachment rates than GuttaFlow2, suggesting that
GuttaFlow sealers were more biocompatible than AH Plus, at least
in vitro. Our findings were consistent with these previous studies.
Conclusions
Within the limitations of this in vitro study, clinicians may find the
following conclusion to be of interest: GuttaFlow Bioseal exhibited bet-
ter cytocompatibility than GuttaFlow2, AH Plus, and MTA Fillapex.
Future evaluations, both in vitro and in vivo, are required to confirm
the suitability of GuttaFlow Bioseal for clinical application.
Acknowledgments
Mar Collado-Gonz
alez and Christopher J. Tom
as-Catal
a
contributed equally to this study.
This work was supported by the Spanish Net of Cell Therapy
(TerCel) provided by Carlos III Institute of Health (ISCiii).
The authors deny any conflicts of interest related to this study.
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JOE — Volume 43, Number 5, May 2017