Key words:
Cell morphology - cells viability - sealers
INTRODUCTION
During the final stage of endodontic treatment, root canal filling materials and especially
endodontic sealers may contact periradicular tissues and potentially toxic components
may leach from the canal space over time. Endodontic materials should stimulate repair
or be biologically neutral to promote healing.[1] Therefore, they should not be cytotoxic, so as not to negatively influence the viability
of cells and cause cell death by apoptosis or necrosis.[2] The study of sealer biocompatibility is very important to help the clinician select
sealers that would minimize the potential incidence of unwanted local and/or systemic
side effects.[3]
Recently, four novel endodontic sealers were introduced, namely, mineral trioxide
aggregate (MTA) Fillapex (Angelus Soluções Odontológicas, Londrina, PR, Brazil), GuttaFlow
2 (Coltène/Whaledent, Langenau, Germany), TotalFill BC Sealer (FKG, Dentaire SA, La
Chaux-de-Fonds, Switzerland), and Bioroot™ RCS (BR, Septodont, France). MTA has attracted
considerable attention because of its excellent biocompatibility, sealing ability,
and antimicrobial properties.[4]
[5] For these reasons, newer formulations of MTA as a root canal sealer, such as MTA-Fillapex,
have been developed in an attempt to expand its range of applications. The chemical
composition of MTA-Fillapex consists of two main components: MTA and salicylate resin.[6] Due to its MTA content, MTA-Fillapex is biocompatible and stimulates mineralization.[7]
GuttaFlow 2 is a new formulation of GuttaFlow. It consists of similar components but
in altered proportions. It is a cold flowable silicone-based sealer that is triturated
and consists of guttapercha powder into a silicone matrix (polydimethylsiloxane) and
nanosilver particles. It comes in a unidose capsule and is injected after mixing.[8]
[9] GuttaFlow 2 demonstrates very promising properties due to its insolubility, biocompatibility,
postsetting expansion, great fluidity, and ability for providing a thin film of sealer,
and hence greater adhesion with the dentinal wall.[10]
[11]
[12] However, GuttaFlow, the previous version of this sealer, is reported to display
low contact angle.[11] Furthermore, the previous studies of GuttaFlow exhibited low toxicity profiles when
compared with other sealers both in vitro and in vivo.[8]
[10]
[12]
[13]
EndoSequence BC Sealer, also known as TotalFill BC Sealer in Europe, is a new premixed,
injectable bioceramic root canal sealer. Its major components include tricalcium silicate,
dicalcium silicate, calcium phosphates, colloidal silica, calcium hydroxide, zirconium
oxide, and thickening agents. It utilizes the moisture within the dentinal tubules
following canal irrigation to initiate and complete the setting reaction.[12] Moreover, it demonstrates one of the strongest antimicrobial activities among endodontic
sealers.[14]
BioRoot™ RCS is a bioactive mineral root canal sealer based on innovative mineral
microaggregate chemistry named “active biosilicate technology.”[15] It is a sealer without any sign of resin or eugenol, which makes it different from
conventional root canal sealers. An aqueous solution of calcium chloride and excipients
is mixed with a powder based on tricalcium silicate and zirconium oxide. It allows
pH values to increase over 11 and has hydrophilic properties.[15] According to some studies Bioroot™ RCS has higher bioactivity in comparison to conventional
zinc-oxide-eugenol sealers on human periodontal ligament (PDL) cells[16] and is also less cytotoxic at pulpal stem cells.[15]
In vitro analysis regarding the probable cytotoxic effects of new endodontic sealers is essential
to determine the potential for an adverse event of a new sealer compared with previously
studied endodontic sealers. Based on the current literature, there are few studies
evaluating the cytotoxicity of MTA-Fillapex, GuttaFlow 2, BC sealers, and Bioroot™
RCS.[15]
[16]
[17]
[18] The purpose of this study was to evaluate the viability of PDLs cells on MTA-Fillapex,
GuttaFlow 2, TotalFill Sealer, and BioRoot™ RCS in comparison to conventional epoxy
resin-based (AH Plus) and zinc-oxide-eugenol-based (Roth’s 801) sealers.
MATERIALS AND METHODS
Cell culture
PDL cells were provided by ProCell, Biotechnological Application SA (Athens, Greece).
The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Glasgow,
UK), supplemented with streptomycin, penicillin, and L-glutamine and complemented
with 10% heat-inactivated fetal bovine serum (FBS, Biowest) at 37°C in a 5% CO2 humidified incubator. Cells were used between third and fifth passage. Five independent
experiments were performed to ensure reproducibility.
Cell viability assays
Cell viability assays were performed using transwells (Costar Transwell; Corning Inc.,
Corning, NY, USA). The materials tested in this study were MTA-Fillapex, GuttaFlow
2, BC sealer, Bioroot™ RCS, AHPlus (Dentsply DeTrey, Konstanz, Germany) and Roth’s
801(Roth’s Pharmacy-Chicago). The sealers were prepared under aseptic conditions according
to the manufacturer’s instructions and were immediately placed on a 13 mm coverslip.
These sealers were divided into two groups. In the first group, PDL cells were added
immediately after the preparation of sealers (Fresh Group). In the second group, PDL
cells were added after 24 h, so that sealers were set (24 h Group). Five coverslips
for each material per group were prepared. Each coverslip was placed on the bottom
of a 24-well plate, 200 μl of DMEM media was added in each well, and 2 × 104 PDL cells were seeded on the polycarbonate filter with 30-µm pores in the upper chamber
of the transwells. A control medium was prepared in a similar manner but without materials.
The cells were cultured for 72 h at 37°C in a 5% CO2 humidified incubator. Finally, the cells were collected, trypsinized, stained with
trypan blue, and counted using a hemocytometer.
Statistical analysis
Proliferation rate was not normally distributed. Consequently, Mann–Whitney U-test
was used to compare the means of proliferation between Fresh and 24 h time points
per sealer. In addition, Kruskal–Wallis test was applied for comparison between the
measures of each material in Fresh and 24 h time points, and post hoc analysis with the use of Mann–Whitney test was performed for assessing the mean differences
between each potential pair of materials. All reported probability values (P values) were compared to a significant level of 5%. The analyses of coded data were
carried out using IBM SPSS software version 21.0 (IBM Corporation, New York, USA).
Cell morphology
Different samples of the materials were shaped into 1 mm thick discs of 5 mm in diameter.
Each specimen was placed in the bottom of the well of a 24-well culture plate. A volume
of 300 µl DMEM and 2.5 × 104 cells were added in each well. The cells were cultured for 72 h at 37°C in a 5% CO2 humidified incubator.
The cells cultured on the surface of the samples were fixed in 4% PFA for 20min at
room temperature, followed by PBS washing. The samples were then stained using Phalloidin
Rhodamin solution (Molecular Probes) to reveal the cytoskeleton and more specifically
the actin filaments. 4’,6-diamidino-2-phenylindole was used for the detection of the
nucleus. To assess cell morphology related to the different endodontic sealers, samples
were placed on top of a microscope slide with Vectashield (VECTOR Laboratories, Peterborough,
UK) and observed under the confocal microscope Leica TCS SP5 (Leica, Wetzlar, Germany)
in a ×20 resolution and analyzed using Leica software, LAS AF.
RESULTS
The mean values and standard deviation of viable cells for freshly mixed and 24 h-set
sealers are presented in [Figure 1]. The statistically significant differences among the groups are shown in [Tables 1] and [2]. Roth’s 801, GuttaFlow 2, and Totalfill showed statistically significant differences
(P = 0.008) in the number of viable cells, between freshly and 24 h-set sealers [Table 1]. GuttaFlow 2, for both time points, presented the highest number of viable cells.
The number of viable cells for MTA-Fillapex and BioRoot for the 24 h-set groups was
higher than the freshly mixed, but not statistically significant [Table 1]. In addition, the comparison of sealers after their setting (24 h) shows statistically
significant differences between them [Table 2]. All four novel endodontic sealers presented statistically significant higher number
of viable cells in comparison to the convectional sealers for both time points, except
for MTA-Fillapex and BioRoot, where in the freshly mixed group, there was no statistical
significant difference.
Figure 1: Mean values and standard deviations of periodontal ligament cells at freshly and
24 h-set sealers
Table 1:
Differences in cells proliferation between freshly and 24 h-setpoint time per sealer
|
Sealer
|
P
|
|
Mann-Whitney U-test. NS: Not significant, MTA: Mineral trioxide aggregate
|
|
AH Plus
|
NS
|
|
Roth’s 801
|
0.008
|
|
GuttaFlow 2
|
0.008
|
|
Totafill
|
0.008
|
|
MTA-Fillapex
|
NS
|
|
BioRoot
|
NS
|
Table 2
Differences in cells proliferation between sealers per time point
|
Sealer
|
Fresh
|
|
NS: Not significant, MTA: Mineral trioxide aggregate
|
|
AH Plus versus Roth’s 801
|
0.005
|
|
AH Plus versus GuttaFlow 2
|
0.005
|
|
AH Plus versus Totafill
|
0.008
|
|
AH Plus versus MTA-Fillapex
|
0.008
|
|
AH Plus versus BioRoot
|
0.008
|
|
Roth’s 801 versus GuttaFlow 2
|
0.008
|
|
Roth’s 801 versus Totafill
|
0.008
|
|
Roth’s 801 versus MTA-Fillapex
|
NS
|
|
Roth’s 801 versus BioRoot
|
NS
|
|
GuttaFlow 2 versus Totafill
|
0.008
|
|
GuttaFlow 2 versus MTA-Fillapex
|
0.008
|
|
GuttaFlow 2 versus BioRoot
|
0.008
|
|
Totalfill versus MTA-Filapex
|
0.008
|
|
Totalfill versus BioRoot
|
NS
|
|
MTA-Fillapex versus BioRoot
|
NS
|
Regarding the cell morphology alterations, cells grown on GuttaFlow 2 were observed
to be well-formed, demonstrating a typical fibroblast-like, spindle-shaped, polarized
morphology with elongated cytoskeleton with multiple thin processes [Figure 2a]. Similar morphology was also observed for Totalfill [Figure 2b], as well as BioRoot [Figure 2c], where stress fibers could be detected, and the cytoplasm was adequately stressed.
On the contrary, MTA-Fillapex seemed to impinge on the cell distribution and morphology,
as the cells appeared to abolish their spindle-shape appearance, becoming smaller
and rounder in shape [Figure 2d]. No cells were detected on the surfaces of AH Plus, as well as Roth’s 801.
Figure 2: Cell morphology on the different endodontic sealers. (a) GuttaFlow 2, (b) TotalFill,
(c) BioRoot, (d) mineral trioxide aggregate-Fillapex. No cells were detected on the
surfaces of AH Plus, as well as Roth’s 801
DISCUSSION
In vitro investigations can provide useful information regarding the biological properties
of new dental materials intended for clinical use. Such an experimental approach simplifies
the system under study, so the researcher can focus on a small number of components
and the evaluation of the cytotoxic potential of the materials may give an indication
of the toxicity in the clinical setting.[19] On the other hand, culture conditions are not homeostatic, and there is no eradication
of toxic substances as there would be in vivo. Dissimilarly, the human body possesses a lymphatic system and periapical defenses
such as polymorphonuclear leukocytes and macrophages to help remove toxic substances.[20] These mechanisms do not exist in a culture plate and should be a consideration for
interpretations of the outcome of cell culture-based cytotoxicity studies reported
in the literature.
In this study, an evaluation of cells viability in four novel endodontic sealers in
comparison to two conventional sealers was performed, to simulate the root-end environment.
PDL cells were exposed to sealers, immediately after their preparation as well as
after 24 h. These two-time points were selected because all conventional sealers reveal
toxic effects when freshly mixed; but their toxicity is significantly reduced on setting.[21]
[22] In our study, the aforementioned state was confirmed at almost all sealers. The
number of cells that survived was higher at 24 h-set materials than the freshly mixed
in every sealer except Roth’s 801 [Table 1]. When PDL cells were exposed to fresh Roth’s 801 some of them, manage to survive,
but when they were exposed to Roth’s 801 after 24 h, no vital cells were detected.
This is in accordance with the literature as previous studies have demonstrated that
zinc-oxide–eugenol sealers have presented noticeable cytotoxic and tissue-irrigating
potencies in previous ex vivo cell culture studies.[22] Some reports support that breakdown products from the sealers may have an adverse
outcome on the proliferative capacity of periradicular cell populations.[23]
In our experimental setting, the most biocompatible sealer is GuttaFlow 2 in both
time points. This sealer was also the only one that increased the initial cell number
[Figure 1]. These findings correlate with the results of Willershausen et al. and Bouillaguet et al., that determine that the silicone-based sealer GuttaFlow is not cytotoxic and is
considered to be biocompatible.[24]
[25] Furthermore, according to Accardo et al., GuttaFlow 2 shows a similar biocompatibility profile as GuttaFlow.[8]
TotalFill BC Sealer presented the second highest number of viable cells at both time
points [Figure 1]. This finding is in agreement with previous studies reporting that TotalFill BC
Sealer displayed high biocompatibility with human fibroblasts after 24 h[26] and is more biocompatible than MTA-Fillapex and AH Plus.[17]
[27]
Regarding BioRoot, one of the newest sealers available in the dental market, it seems
that it is more biocompatible than zinc-oxide eugenol sealers.[15]
[16] In our study, BioRoot presented a statistically significant higher number of viable
cells than Roth’s 801 among the 24 h group. MTA-Fillapex and BioRoot seem to demonstrate
similar behavior, when they were freshly mixed or after 24 h [Table 2]. This is in agreement with a previous study, reporting that the cytotoxicity of
MTA-Fillapex decreases after setting.[28] Finally, AH Plus and Roth’s 801 do not exhibit the biological properties of the
others sealers which is in accordance with other studies.[29]
Confocal imaging revealed that PDL cells maintain their initial morphology when in
contact with GuttaFlow 2, TotalFill, and BioRoot. This is an additional factor indicating
their good biological properties. On the other hand, MTA-Fillapex seemed to impinge
on the cell distribution and morphology, which is in accordance with its cells’ proliferation
rate. The low survivability of cells indicates that their morphology is also a result
of sealer cytotoxicity. Furthermore, the loss of spindle shaped appearance and their
rounder shape probably burden their potential adhesion.
CONCLUSIONS
According to the results of this study, all novel endodontic sealers presented increased
cell viability in comparison to conventional sealers such as AH plus and Roth’s 801.
GuttaFlow 2 exhibited the highest cell viability. Further investigations are required
to validate the potential biological responses of those novel endodontic sealers.
Financial support and sponsorship
Nil.
