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materials
Article
Biocompatibility of a High-Plasticity,
Calcium Silicate-Based, Ready-to-Use Material
Tomoharu Okamura 1, Liji Chen 2, Nobuhito Tsumano 3, Chihoko Ikeda 1, Satoshi Komasa 4,
Kazuya Tominaga 1and Yoshiya Hashimoto 5,*
1Department of Oral Pathology, Osaka Dental University, 8-1, Kuzuhahanazonocho, Hirakata,
Osaka 573-1121, Japan; okamu-t@cc.osaka-dent.ac.jp (T.O.); ikeda-c@cc.osaka-dent.ac.jp (C.I.);
tominaga@cc.osaka-dent.ac.jp (K.T.)
2Department of Orthodontics, Osaka Dental University, 8-1, Kuzuhahanazonocho, Hirakata,
Osaka 573-1121, Japan; chen-li@cc.osaka-dent.ac.jp
3Department of Oral and Maxillofacial Surgery II, Osaka Dental University, 8-1, Kuzuhahanazonocho,
Hirakata, Osaka 573-1121, Japan; tsumano@cc.osaka-dent.ac.jp
4Department of Removable Prosthodontics and Occlusion, Osaka Dental University, 8-1,
Kuzuhahanazonocho, Hirakata, Osaka 573-1121, Japan; komasa-s@cc.osaka-dent.ac.jp
5Department of Biomaterials, Osaka Dental University, 8-1, Kuzuhahanazonocho, Hirakata,
Osaka 573-1121, Japan
*Correspondence: yoshiya@cc.osaka-dent.ac.jp; Tel.: +81-72-643106
Received: 1 September 2020; Accepted: 21 October 2020; Published: 26 October 2020
Abstract:
The Bio-C Sealer is a recently developed high-plasticity, calcium-silicate-based, ready-to-use
material. In the present study, chemical elements of the materials were characterized using scanning
electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy
(FTIR). The biocompatibility of the Bio-C Sealer was investigated using cytotoxicity tests and
histological responses in the roots of dogs’ teeth. XRD, SEM, and FTIR produced hydrated calcium
silicate in the presence of water molecules. In addition, FTIR showed the formation of calcium
hydroxide and polyethylene glycol, a dispersing agent. The 1:4 dilutions of Bio-C Sealer presented
weaker cytotoxicity than the Calcipex II in an
in vitro
system using the V-79 cell line. After 90 d,
the periradicular tissue response of beagle dog roots was histologically evaluated. Absence of
periradicular inflammation was reported in 17 of the 18 roots assessed with the Bio-C Sealer,
whereas mature vertical periodontal ligament fibers were observed in the apical root ends filled with
the Bio-C Sealer. Based on these results and previous investigations, the Bio-C Sealer is recommended
as an effective root-end filling material. These results are relevant for clinicians considering the use of
Bio-C Sealer for treating their patients.
Keywords:
calcium-silicate; Bio-C Sealer; root-end filling material; mineral trioxide aggregate (MTA)
cement; biocompatibility; beagle dog
1. Introduction
Calcium hydroxide (Ca(OH)
2
) is an inorganic compound commonly used in dentistry; it has
become one of the most widely accepted materials in dental clinics [
1
]. However, although permanent
root canal sealers containing Ca(OH)
2
have been introduced into the market [
2
], their disadvantages
have also been reported, including (a) lack of adhesion to dentin and other filling materials, (b) lack
of proper sealing, and (c) presence of micropores, which form tunnel defects that promote bacterial
infiltration in the pulp. Therefore, more biocompatible and complex materials are now being produced
to solve such issues [
3
]. Calcium silicate-based cement, conventionally known as mineral trioxide
aggregate (MTA) cement, is biocompatible, prevents microleakage, and promotes the regeneration of
Materials 2020,13, 4770; doi:10.3390/ma13214770 www.mdpi.com/journal/materials
Materials 2020,13, 4770 2 of 12
original tissues when in contact with periradicular tissues [
4
]. In addition, it is mainly composed of
dicalcium and tricalcium silicates with unique properties and a hydraulic self-curing material that
makes it suitable for clinical use [5–9].
Therefore, a single-cone technique with a hydraulic calcium silicate-based sealer may represent
a good tool given the superior flowability of the material and its ability to slightly expand upon
setting [
10
,
11
]. Elizabeth et al. [
10
] evaluated the outcome of nonsurgical root canal treatments using
a single-cone technique and a hydraulic calcium silicate-based sealer. Recently, the Bio-C Sealer
(endodontic sealer, Angelus, Londrina, Paran
á
, Brazil) was introduced in the market in the form of
a premixed bioceramic material, providing the same biological interactions as MTA, although with
improved manipulation and insertion [
7
]. The Bio-C Sealer contains calcium silicates, which become
hydrated on contact with local humidity and produce a hydrated calcium silicate structure. In turn,
the formed Ca(OH)
2
dissociates rapidly into Ca
2+
and OH
−
ions, thereby increasing the pH of the
medium and making the environment inhospitable for bacterial growth [12]. Furthermore, the Bio-C
Sealer also contains zirconium oxide as a radiopacifier [13].
Given that achieving proper root canal filling is important for the long-term success of
endodontically treated teeth, root canal sealers must have appropriate physical and chemical
properties [
14
]. Zordan-Bronzel et al. [
15
] evaluated the physicochemical properties of the new
Bio-C Sealer in comparison with the TotalFill BC calcium silicate-based sealer and the AH Plus epoxy
resin-based sealer using conventional and micro-computed tomography (CT) tests. They concluded
that the Bio-C Sealer has a short setting time, alkalinization ability, and adequate flow and radiopacity.
In endodontic studies, knowing the toxicity and biocompatibility of new materials before applying
them clinically is essential, as some constituent compounds can damage the surrounding tissues [
16
,
17
].
More recently, researchers evaluated the superior cytocompatibility of the Bio-C Sealer in terms
of cytotoxicity in comparison with AH Plus and demonstrated that the composition of a filling
material plays an important role in its biological properties [
18
]. However, the cytotoxicity of root
canal sealers containing Ca(OH)
2
has not been evaluated. In addition, there appears to be little
histopathological information available concerning the use of a high-plasticity calcium-silicate-based
material in root canals.
Therefore, the aim of this study was to address this gap in the current literature by investigating
the biocompatibility of a high-plasticity calcium-silicate-based material, the Bio-C Sealer, in the roots of
dogs’ teeth.
2. Materials and Methods
2.1. Preparation and Characterization
For materials that required moisture for setting, the mold was placed on a glass plate. Briefly,
2 g of Bio-C Sealer (Angelus, Londrina, Paran
á
, Brazil) was mixed according to the manufacturer’s
instructions with 0.02 mL of water and the mold was filled to a slight excess. Another glass plate lined
with a plastic sheet on top of the sealer was pressed and the mold was placed in the cabinet for 24 h.
Thereafter, the specimens were carefully removed from the mold and the periphery of the specimen
was finished to remove flash and irregularities. Successively, the crystal phase of the clinker and setting
material was identified using an XRD system (XRD-6100, Shimadzu, Kyoto, Japan) and characterized
using a database from the International Center for Diffraction Data. Patterns were obtained under the
following conditions: 30.0 mA, 40.0 kV, scan rate: 2
◦
/min, and 10
◦
–70
◦
. Scanning electron microscopy
images were obtained using parameters of 5.0 kV and 10
µ
A (SEM; S-4100, Hitachi High-Technologies
Corporation, Tokyo, Japan). Before observation, the samples were coated with platinum-palladium
alloy using E-1030 (Hitachi High-Technologies Corporation, Tokyo, Japan). Then, attenuated total
reflection Fourier transform infrared spectroscopy (IRAffinity-1S, Shimadzu, Kyoto, Japan) was used
to evaluate the clinker and setting material over a range of 4000–400 cm
−1
with a resolution of 2 cm
−1
.
Materials 2020,13, 4770 3 of 12
2.2. Cytotoxicity Test
This experiment involved comparisons between the Bio-C Sealer and Calcipex II (Nippon Shika
Yakuhin Co., Ltd. Shimonoseki, Japan). There are very few premixed materials available for use in
endodontic treatment, and we considered it reasonable to compare Bio-C Sealer and Calcipex II because
both are premixed, Ca(OH)
2
-based endodontic materials. Briefly, the cytotoxic potential of these dental
materials was evaluated in a 2–(4,5–dimethyl–2–thiazolyl)–3,5–diphenyl–2H–tetrazolium bromide
(MTT) reduction assay using the V-79 cell line. The V-79 cell line was composed of fibroblasts isolated
from the lung tissue of young Chinese male hamsters. The procedure was carried out according to the
ISO guidelines 10993-12 and the ratio of material surface area to extraction vehicle volume was calculated
as 1.5 cm
2
/mL. Furthermore, the extraction medium (Dulbecco’s Modified Eagle medium-high glucose
plus 10% bovine fetal serum and 1% penicillin/streptomycin solution; 10,000 penicillin units/10 mg/mL
streptomycin) was filtered with a 0.22
µ
m filter. Thereafter, the test item extract remained directly in
contact with the cells for 24 h at 37
◦
C and 5% CO
2
at the following concentrations: 12.5%, 25%, 50%,
and 100%. The V-79 cells were maintained in a culture medium (composition the same as extraction
medium) at 37
◦
C and 5% CO
2
for approximately 48 h to promote cell proliferation and to achieve
the desired confluency (approximately 80%). Successively, the fibroblasts from the V-79 cell line were
cultured in 96-well cell culture plates (1
×
10
4
cells per well) and incubated in the same culture medium
for 24 h to facilitate cell monolayer formation. The culture medium was then replaced with a fresh
medium either containing test substance (n=6) or no test substance (n=6). Different concentrations
of the test substance used for the treatment were incubated for 24 h. At the end of the exposure period,
the culture medium with the treatments was discarded and 50
µ
L of the MTT solution (1 mg/mL) was
added to each well. Thereafter, the cells were incubated for 2 h, the MTT solution was discarded, 100
µ
L
of isopropanol was added to each well, and the plate was shaken for 5 min. Finally, the absorbance of the
test substance was measured at a wavelength of 570 nm (reference wavelength: 650 nm). This experiment
was repeated at least three times. The data obtained were analyzed using Statcel, version 4 (OMS, Tokyo,
Japan), and the Students’ t-test was applied. The level of significance was set to p<0.05.
2.3. Animal and Tooth Preparation
Twenty teeth from the second and third maxillary premolars, as well as the second, third,
and fourth mandibular premolars of four 12-month-old beagle dogs were evaluated. All animal
procedures were performed according to the Animal Research Ethics Committee of the Osaka Dental
University (19-02012a). Briefly, the animals were anesthetized 15 min before the operation and received
inhalational anesthesia with isoflurane. Before the treatment, their teeth were radiographed and
cleaned with pumice (Meiji Co. Ltd., Tokyo, Japan), while their surfaces were thoroughly wiped
with a 10% povidone-iodine solution (Meiji Co. Ltd., Tokyo, Japan) to ensure aseptic conditions.
The access cavity was prepared with a sterile, diamond point FG (Shofu Inc., Kyoto, Japan) under
saline irrigation. In addition, the canals were prepared using Tri Auto ZX2 (J. Morita Corp., Kyoto,
Japan) and apically enlarged to the working length with K-type hand files of sizes 30–50 based on
the canal’s size. Furthermore, the canals were intermittently irrigated with 10 mL of 2.5% NaOCl,
rinsed for 30 s with 3% EDTA (Nippon Shika Yakuhin Co., Ltd. Shimonoseki, Japan), flushed with
5 mL of sterile saline, and dried with paper points (Ci Medical Co., Ltd., Ishikawa, Japan).
Thereafter, the prepared roots were assigned to either the Bio-C Sealer or the Calcipex II group.
Given that gutta-percha points were used to condense the sealers on the root canal walls, a decision
was made to investigate the reactions caused by both the points and the sealers. In the Bio-C Sealer
group (n=36 roots), the roots were filled using the Bio-C Sealer and gutta-percha points by lateral
condensation and the teeth were restored with a light-cured, glass-ionomer cement base. A similar
procedure was followed for the Calcipex II group (n=20 roots); however, Calcipex II was used for the
filling. All the animals were euthanized with an overdose of pentobarbital after 28 days and 90 days.
The specimens were removed immediately, each with one root and the surrounding alveolar bone,
and placed in 10% formalin. Successively, they were demineralized in 10% formic acid, processed and
Materials 2020,13, 4770 4 of 12
embedded in paraffin, cut into approximately 4–6
µ
m sections, and stained with hematoxylin and
eosin. Finally, the serial sections were microscopically examined and the periapical inflammation
(PI) was semi-quantitatively graded according to the following scale: 0, none; 1, mild; 2, moderate;
3, severe; 4, tissue necrosis.
3. Results
3.1. Characterization of Bio-C Sealer
The results of the XRD analysis of the clinker and setting material are shown in Figures 1and 2.
The XRD patterns of the clinker suggested that the mineral phase was composed largely of dicalcium
silicate (C
2
S), tricalcium silicate (C
3
S), tricalcium aluminate (C
3
A), and zirconium oxide (ZrO
2
) as the
contrast agent (Table 1) [
19
–
21
]. In contrast, the setting material showed large peaks that represented
calcium silicate hydrate (CSH) (Table 2) [
19
,
20
,
22
]. Initially, stereomicroscopy and SEM were performed
on both the clinker and the material discs (Figure 3), which showed a lump of clinker sized 300–1000
µ
m
(Figure 3). In the SEM observation (Figure 3), the size of the calcium silicate grains in the clinker
before hydration was around 2
µ
m. Images of the setting material indicated the formation of the
CSH phase after the hydration process (average size of these grains was approximately 5–10
µ
m)
(Figure 3). Therefore, the materials were analyzed using FTIR to determine changes between the
clinker and the setting materials (Figure 4, Tables 3and 4). The clinker showed C
3
S and C
2
S bands of
573 cm
−1
, 1112 cm
−1
, 494 cm
−1
, and 912 cm
−1
, respectively [
23
]. The CSH bands were visible in the
setting material at 494 cm
−1
, 575 cm
−1
, 881 cm
−1
, 1647 cm
−1
, and 3431 cm
−1
, whereas a Ca(OH)
2
band
was seen at 3637 cm
−1
[
24
]. Polyethylene glycol, in other words, a dispersing agent, showed bands at
1101 cm−1, 1246 cm−1, 1348 cm−1, 1456 cm−1, 1647 cm−1, and 2868 cm−1[25].
Figure 1.
X-ray diffraction patterns for the clinker. Abbreviations: C
3
S, tricalcium silicate; C
2
S, dicalcium
silicate; ZrO2, zirconium oxide.
Table 1. X-ray diffraction information for the clinker.
Material 2 θIntensity Crystal System Crystal Planes
C3S 28.3 74 Hexagonal 2, 0, −2, 0
ZrO230.2 525 Tetragonal 1, 0, 1
C3S/
C2S31.4 59 Hexagonal
Hexagonal 0, 0, 0, 2
1, 0, −1, 2
C3S/
C2S32.5 43 Hexagonal
Hexagonal 2, 0, −2, 1
2, −1, −1, 0
ZrO235.2 90 Tetragonal 1, 1, 0
ZrO250.3 187 Tetragonal 1, 1, 2
ZrO259.4 72 Tetragonal 1, 0, 3
ZrO260.0 116 Tetragonal 2, 1, 1
Abbreviations: C3S, tricalcium silicate; C2S, dicalcium silicate; ZrO2, zirconium oxide.
Materials 2020,13, 4770 5 of 12
Figure 2.
X-ray diffraction patterns for the setting materials. Abbreviations: ZrO
2,
zirconium oxide;
CSH, calcium silicate hydrate.
Table 2. X-ray diffraction information for the setting materials.
Material 2 θIntensity Crystal System Crystal Planes
ZrO2/CSH 29.6 282 Tetragonal
Orthorhombic
1, 0, 1
0, 2, 0
−2, 2, 0
CSH 31.8 63 Orthorhombic 2, −2, 2
0, 2, 2
ZrO234.4 90 Tetragonal 1, 1, 0
ZrO2/CSH 49.8 213 Tetragonal
Orthorhombic 1, 1, 2
2, 4, 0
ZrO258.7 72 Tetragonal 1, 0, 3
ZrO259.6 147 Tetragonal 2, 1, 1
Abbreviations: ZrO2, zirconium oxide, CSH, calcium silicate hydrate.
Figure 3.
Stereomicroscope and scanning electron micrographs of the (
a
) Bio-C Sealer; (
b
) clinker;
and (c) setting material. The bars are (a) 1 mm and (b,c) 10 µm.
Materials 2020,13, 4770 6 of 12
Figure 4. Fourier transform infrared spectroscopy (FTIR) for the clinker and setting material.
Table 3. Fourier transform infrared spectroscopy (FTIR) information for the clinker.
Material Vibrational
Mode
Wavenumber (cm−1)
Experimental
Wavenumber (cm−1)
(Reference)
C3S
vSi–O stretching vibrations
1112 ∼1060 [23]
C2S
vSi–O stretching vibrations
912 ∼925 [23]
C3SδSiO4bending 573 ∼522 [23]
C2SδSiO4bending 494 ∼452 [23]
Abbreviations: C3S, tricalcium silicate; C2S, dicalcium silicate.
Table 4. Fourier transform infrared spectroscopy (FTIR) information for the setting materials.
Material Vibrational
Mode
Wavenumber (cm−1)
Experimental
Wavenumber (cm−1)
(Reference)
Ca(OH)2vO–H stretching (portlandite) 3637 ∼3644 [24]
CSH vO–H stretching 3431 3300 to 3600 [24]
PEG vC–H stretching mode 2868 ∼2878 [25]
CHS vH–O–H stretching vibrations 1647 ∼1640 [24]
PEG δC–H bending 1456 ∼1464 [25]
PEG δC–H bending 1348 ∼1343 [25]
PEG vC–O stretching vibrations 1246 1000 to 1300 [25]
PEG vC–O stretching vibrations 1101 1000 to 1300 [25]
CSH vSi–O stretching vibrations 925 ∼1060 [24]
CSH vSi–O stretching vibrations 881 ∼900 [24]
CSH δSiO4bending 577 ∼400 to 500 [24]
CSH δSiO4bending 494 ∼400 to 500 [24]
Abbreviations: CSH, calcium silicate hydrate; PEG, polyethylene glycol.
3.2. Cytotoxicity Test for the Bio-C Sealer and Calcipex II
A reduction in cell viability (below 30%) was observed for the Bio-C Sealer test substance compared
to the negative control at the following concentrations: 12.5%, 25%, and 50%. The same observations
were valid at a concentration of 100%. With regards to Calcipex II, a reduction in cell viability
(below 30%) was observed as compared to the negative control at the 12.5% and 25% concentrations.
Similar observations were made at concentrations of 50% and 100% (Figure 5).
Materials 2020,13, 4770 7 of 12
Figure 5.
Cell viability (%) in terms of mean replicate percentages of the test substance concentrations
of the Bio-C Sealer and Calcipex II.
3.3. Histochemical Analysis
Clinically, evidence of swelling associated with the teeth treated with the Bio-C Sealer and
Calcipex II was not reported at 28 days and 90 days from the procedure. Similarly, periapical lesions were
not observed following radiographic assessments (Figures 6and 7). After 28 days and 90 days, both the
Bio-C Sealer- and Calcipex II-treated teeth showed immature periodontal ligament fibers and a thick
layer of cementum at the apex (Figure 6b,c,e,f). In addition, infiltration of round cells (i.e., mesenchymal
cells involved in tissue repair) was seen in the same area (Figure 6b,c,e,f). Although the capillaries in
the surrounding tissues were hyperemic, they did not show infiltration of lymphocytes or plasma cells
(Figure 6b,c,e,f). Overall, evidence of an inflammatory response associated with any of the treated
teeth at 28 days was not present. At 90 days, characteristic findings of healing were seen at the apical
foramen of Bio-C Sealer-treated teeth, whereas the original apical hole was reported to be closed by the
growth of a bone-like hard tissue (Figure 7b). In addition, mature vertical periodontal ligament fibers
were observed at the apical root portion (Figure 7c) from the alveolar bone to the apical cementum.
Conversely, although mature periodontal ligament fibers were observed at the apical root portion of
Calcipex II-treated teeth at 90 days (Figure 7f), they did not grow vertically.
Figure 6.
Histopathological events were observed after the evaluation of the periapical tissue response
to the proposed endodontic treatments. (
a
) Radiographic image and (
b
) histological section of a Bio-C
Sealer-treated tooth after 28 days; (
c
) highest magnification of the same section. (
d
) Radiographic image
and (
e
) histological section of a Calcipex II-treated tooth after 28 days; (
f
) highest magnification of the
same section. The bars of (
b
,
e
) are 100
µ
m, whereas those of (
c
,
f
) are 200
µ
m. Black arrows indicate
cementum, whereas red asterisks indicate immature periodontal ligament fibers.
Materials 2020,13, 4770 8 of 12
Figure 7.
Histopathological events were observed after the evaluation of the periapical tissue response
to the proposed endodontic treatments. (
a
) Radiographic image and (
b
) histological section of a Bio-C
Sealer-treated tooth after 90 days; and (
c
) highest magnification of the same section. (
d
) Radiographic
image and (
e
) histological section of a Calcipex II-treated tooth after 90 days; (
f
) highest magnification of
the same section. The bars of (
b
,
e
) are 100
µ
m, whereas those of (
c
,
f
) are 200
µ
m. Black arrows indicate
cementum, red asterisks indicate mature periodontal ligament fibers, and black triangles represent
bone-like hard tissue.
Table 5shows the scores of periapical inflammation (PI) assessed histologically at 28 days and
90 days after the experimental treatment procedures were completed. At 28 days and 90 days, none of
the roots showed moderate or severe PI or tissue necrosis in both the Bio-C Sealer and control groups.
Mild PI was reported in only 1 of the 18 roots (6%) in the Bio-C Sealer group and 2 of the 10 roots (20%)
in the Calcipex II group. Such PI measures remained constant for both groups at 90 days.
Table 5.
Scores of periapical inflammations in dogs’ premolars following histological assessment at
28 days and 90 days from the experimental treatment procedures.
Groups Roots
Assessed (n)
Level of PI *
None (%) Mild (%) Moderate Severe Tissue Necrosis
Bio-C Sealer Group 28 days 18 17 (94) 1 (6) 0 0 0
Control group (Calcipex II) 28 days
10 8 (80) 2 (20) 0 0 0
Bio-C Sealer Group 90 days 18 17 (94) 1 (6) 0 0 0
Control Group (Calcipex II) 90 days
10 8 (80) 2 (20) 0 0 0
* PI, periapical inflammation.
4. Discussion
In the present study, roots filled with the Bio-C Sealer demonstrated outstanding healing of
periapical tissue, which was evaluated using histological methods. The current study is the first
in vivo
experiment to test this new bioceramic material.
Characterization is an important step in the search for optimal materials given that changes
may occur in the substance, such as hydration of calcium silicate compounds in calcium-based
materials. In the present experiment, XRD was used for both the compositional and crystal phase
characterization of the Bio-C Sealer and the presence of the following mineral phases was indicated:
Ca
2
SiO
5
, Ca
3
SiO
5
, and Ca
3
Al
2
O
3
(i.e., the main constituents of Portland cement). Although the
addition of ZrO
2
provided radiopacity—a basic requirement of any dental material—the peak intensity
remained high. An important point is that ZrO
2
has much higher crystallinity and peak intensity
than clinker. This fact can be explained by the amount of radiopacifier, which is much higher than
Materials 2020,13, 4770 9 of 12
the amount of clinker, and the size of the particles. Marked intensity peaks assigned to CSH in the
setting materials were observed. In addition, water molecules came in contact with the Bio-C Sealer
clinker, causing hydration and setting of the cement. These chemical reactions involve the hydration
of calcium silicate compounds to produce a CSH gel, which is responsible for setting and forming
Ca(OH)
2
[
26
–
28
]. Furthermore, SEM images showed the deposition of the hydro compounds resulting
from the setting reaction on the surface of the cement, as these grains grow and unite. FTIR showed
the Ca(OH)2band at 3640 cm−1gradually sets with time and forms Ca(OH)2in the setting materials.
The MTT assay is a classic test used to evaluate the possible cytotoxic effects of materials and is
employed after 24 h of cell exposure as per the International Organization for Standardization (ISO)
10993 guidelines [
29
]. The V-79 cell line was used in the ISO 10993 guidelines and is easily maintained
in culture. Furthermore, donor biopsy variability was eliminated and greater reproducibility was
possible [
30
]. MTT assays in the present study revealed the Bio-C Sealer to have high cell viability
rates at all the dilutions tested (undiluted, 1/2, 1/4), whereas strong cytotoxicity was observed with
1/2 dilutions of Calcipex II at the same time points. Calcipex II is a Ca(OH)
2
-based sealer and the
cytotoxicity of such material is known to be due to the Ca(OH)
2
, which produces a high pH [
31
].
Given that the samples in the present study were uncured, both components might have been involved
in cytotoxicity in this experiment. Nonetheless, the components might elute even in cured samples
given their high solubility, resulting in cytotoxicity. Sergio L
ó
pez-Garc
í
a et al. [
7
] showed that the
Bio-C-Sealer had higher cell viability than AH Plus containing formaldehyde and bisphenol-A, both of
which are known for their toxicity. In the present study, cytotoxicity tests were conducted using cell
lines. Much like target cells, primary cells may have specific metabolic potentials; therefore, they may
be more consistent with
in vivo
conditions [
32
,
33
]. Meanwhile, cytotoxicity tests for periodontal
ligament cells or dental pulp cells will be needed in the future.
The
in vivo
response of periradicular tissues to various root canal sealers or root filling materials
has been previously investigated [
12
,
34
–
36
]. Bernab
é
et al. [
34
] demonstrated that the periradicular
tissues of roots filled with a zinc oxide eugenol sealer had more severe inflammation than those filled
with gray MTA. Furthermore, Torabinejad et al. [
37
] reported that all root-ends of monkeys that were
filled with amalgam had periradicular inflammation. Supporting the present results, Chene et al. [
38
]
showed minimal or no inflammatory reaction in the tissues adjacent to the gray MTA and proved that
healing took place at all the surgical sites. In the present study, the use of the Bio-C Sealer resulted in
a more effective healing process and showed no inflammation in the periapical tissue. In addition,
the bone-like hard tissue was in close contact with the filled root end.
Bio-C Sealer is the only ready-to-use cement with tricalcium aluminate, which is known to induce
osteo-promotive and bone-remembering and is thought to contribute to the mineralization process of
the periapical tissue [
39
]. In the present study, Calcipex II also allowed a satisfactory healing process and
minimal or no inflammatory reactionl although it showed a PI score similar to that of the Bio-C Sealer.
Economiades et al. [35] demonstrated that the inflammatory reaction of Calcipex II was more intense
than that of MTA, probably due to its composition. In contrast, Snot et al. [
36
] used a Ca(OH)
2
-based
sealer in the treatment of root canal fillings in dogs’ teeth and reported a less severe irritation in 30-day
specimens, as well as areas of newly formed cementum. This observation was supported by their
results obtained from 90-day specimens, which also supported the results of the present study.
Another important feature of MTA is its ability to promote the formation of cementum directly
on the surface of the root tip, as reported in several animal experiments [
37
,
38
,
40
]. The formation
of cementum adjacent to MTA might be a result of the reaction between calcium oxide in MTA and
water or tissue fluids. This results in the formation of Ca(OH)
2
, which may stimulate hard tissue
deposition [
41
]. In the present study, areas of newly formed cementum were observed; this observation
was supported by the results obtained with 28-day specimens. After 90 days, some cementum surfaces
were characterized by mature periodontal ligament fibers that mimicked Sharpey’s fibers. The source
of new cementum is either derived from the remaining periodontal ligament (which grows from the
sides) or from the ingrowing connective tissue of the bone [37].
Materials 2020,13, 4770 10 of 12
5. Conclusions
The Bio-C Sealer presented weaker cytotoxicity than the Calcipex II sealer containing Ca(OH)
2
in
an
in vitro
system. In addition, the Bio-C sealer is biocompatible and safe to use in close contact with
the periapical tissue. Although the induction of periradicular inflammation was not reported, the Bio-C
sealer was shown to favor tissue repair. Furthermore, the sealer may contribute to the mineralization
process of the periapical tissue, which demonstrates its bioactive potential. The findings of the present
study are relevant for clinicians looking to explore the use of the Bio-C Sealer in clinical practice to
achieve better outcomes in their patients.
Author Contributions:
Conceptualization, Y.H. and T.O.; methodology, T.O.; formal analysis, T.O.; investigation,
T.O. and K.T.; data curation, T.O., L.C., and N.T.; writing—original draft preparation; writing—review and editing,
Y.H., K.T., S.K., and C.I.; funding acquisition, Y.H. All authors have read and agreed to the published version of
the manuscript.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflict of interest.
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