Basic Research—Technology
Cell Viability and Tissue Reaction of NeoMTA
Plus: An In Vitro and In Vivo Study
Lucas Siqueira Pinheiro, DDS, MSc,* J
ulia Eick Iglesias, DDS, MSc,*
Daiana Boijink, DDS, MSc,* Let
ıcia Boldrin Mestieri, DDS, MSc,*
Patr
ıcia Maria Poli Kopper, DDS, MSc, PhD,† Jos
e Ant^
onio de Poli Figueiredo, DDS, MSc, PhD,‡
†
and Fabiana Soares Grecca, DDS, MSc, PhD
Abstract
Introduction: The aim of this study was to evaluate the
cell viability and tissue reaction of NeoMTA Plus (NMP;
Avalon Biomed Inc, Houston, TX) compared with min-
eral trioxide aggregate (MTA; Angelus, Londrina, PR,
Brazil) and Biodentine (BD; Septodont, Saint-Maur-de-
Foss
es, France). Methods: Fibroblasts (3T3) were
plated and exposed to 1% extract from the test material
before and after setting. Cytotoxicity assessment was
performed using the 3-(4,5-dimethyl-thiazoyl)-2,5-
diphenyl-tetrazolium bromide and sulforhodamine B as-
says. In vivo evaluation consisted of polyethylene tube
implantation of the materials in rat subcutaneous tissue.
Histologic analysis occurred at 7, 30, and 90 days,
scoring inflammatory events and collagen fiber forma-
tion. Analysis of variance and the Tukey and t tests
were used for cytocompatibility assays, and the
Kruskal-Wallis test followed by the Dunn test were
used for biocompatibility assays (P # .05). Results:
The materials in the cytotoxicity assays presented
greater viability after setting (P # .05). NMP and MTA
presented higher viability than the control (Dulbecco
modified Eagle medium) on the 3-(4,5-dimethyl-thia-
zoyl)-2,5-diphenyl-tetrazolium bromide assay before
and after setting (P # .05). The sulforhodamine B assay
showed that MTA and BD presented less viability
than NMP and the control, and NMP was similar to
the control before setting. After setting, MTA and BD
presented higher viability when compared with the
control group (P # .05), and NMP was similar to control.
Inflammatory infiltrate reduction occurred throughout
the test periods for all materials. At 7 days, neutrophils
were present in BD (P # .05), and granuloma and giant
cells were present in BD and MTA. At 30 days, BD
showed intense inflammatory infiltrates and a large
number of macrophages when compared with NMP,
MTA, and the control (P # .05). At 90 days, BD
presented a thick fiber layer compared with NMP
From the *Postgraduate Program in Dentistry and †Department of Conservative Dentistry, Dental School and ‡Department of Morphology, Institute of Basic Health
Sciences, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil.
Address requests for reprints to Dr Fabiana Soares Grecca, Universidade Federal do Rio Grande do Sul, Faculdade de Odontologia, Rua Ramiro Barcelos, 2492 –
Bairro Santana, Porto Alegre, RS, Brazil, 90035-003. E-mail address: fabiana.grecca@ufrgs.br
0099-2399/$ - see front matter
Copyright ª 2018 American Association of Endodontists.
https://doi.org/10.1016/j.joen.2018.03.007
1140 Pinheiro et al.
(P # .05). Conclusions: NMP showed similar biocompatible behavior to MTA and
BD. (J Endod 2018;44:1140–1145)
Key Words
Biocompatibility, calcium silicate materials, cytotoxicity, endodontics, mineral trioxide
aggregate, subcutaneous connective tissue
T he primary purpose of
conservative treatment
is maintaining normal his-
Significance
A material is considered biocompatible when it
tologic characteristics of promotes cell viability, and the tissue inflammatory
response becomes insignificant over time. Neo-
the remaining pulp tissue
MTA Plus, MTA, and Biodentine can be considered
by using biocompatible
options when used as repair materials.
materials to protect the
exposed area and form a
complete barrier of calcified tissue (1, 2). In cases of an incomplete apex, it also
allows for continuation of root formation, which leads to apical physiological
closure with a strengthened root structure (3).
Moreover, in cases of root perforation, resorptions, and retrograde fillings, a ma-
terial with good sealing ability and the capacity to promote periradicular tissue regen-
eration can be used to prevent continuous exposure to a contaminating environment
(4). These materials come into direct contact with periapical tissues; hence, the cyto-
toxicity of these materials may cause cellular degeneration and delayed wound healing.
Known as bioactive endodontic cements (5), calcium silicate–based materials
have emerged as an option for these treatments to present low cytotoxicity and good
biocompatibility, antibacterial activity, and bioactivity (6–9). The industry has
produced various bioactive endodontic materials, each claiming the best
performance, including mineral trioxide aggregate (MTA; Angelus, Londrina, PR,
Brazil), Biodentine (BD; Septodont, Saint-Maur-de-Foss
es, France), and NeoMTA
Plus (NMP; Avalon Biomed Inc, Houston, TX). Although these materials contain
calcium silicate, they present different components in their formulas that may
interfere with the biological properties.
NMP is a new tricalcium silicate material of very fine powder, similar in chemistry
to MTA Plus (Avalon Biomed Inc, Bradenton, FL), that avoids discoloration by using
tantalum oxide as a radiopacifier instead of bismuth oxide. It is mixed with a water-
based gel that has good handling properties (10). The powder-to-gel mixing ratio
can vary, and a thin consistency can be used as a sealer or thick mixture for perforation
JOE — Volume 44, Number 7, July 2018

repair, root-end filling, and other treatment modalities. NMP presents
adequate radiopacity and prolongs setting time (10). Calcium and hy-
droxyl ion release is greater and more prolonged with NMP than MTA
Plus. In addition, NMP increases the stability of the root filling and pro-
motes endodontic and periodontal tissue regeneration, enhancing
bioactivity and biocompatibility (10).
There are no available data concerning cytotoxicity and tissue re-
action to NMP. Therefore, we aimed to evaluate the cytotoxicity and
biocompatibility of NMP compared with MTA and BD.
Material and Methods
The present study was approved by the Research Committee of the
School of Dentistry of the Federal University of Rio Grande do Sul, Porto
Alegre, RS, Brazil.
Cytocompatibility Assays
The repair materials were prepared according to the manufac-
turers’ instructions. Immediately before setting, 1 mL of each material
was distributed to wells of two 12-well plates (314.0 mm2 area and
3.0 mm height, n = 8 wells per sealer) (TPP Techno Plastic Products,
Trasadingen, Switzerland).
Fresh extracts were obtained by filling 1 of the plates with 5 mL Dul-
becco modified Eagle medium (DMEM; Sigma-Aldrich, St Louis, MO)
supplemented with 10% fetal bovine serum (FBS; Gibco Life Technolo-
gies, Grand Island, NY) and 1% penicillin and streptomycin (P/S, Gibco
Life Technologies) per well. The plate was incubated at 37 C with 100%
humidity for 24 hours. To acquire the extracts after setting, the other
plate was kept in an incubator for 24 hours to allow the materials to
set. Next, the plate was exposed to ultraviolet light in a laminar flow cham-
ber for 30 minutes to prevent contamination, and 5 mL DMEM culture
medium supplemented with 10% FBS and 1% P/S was placed in each
well. This plate was incubated at 37 C with 100% humidity for 24 hours.
Extraction media were collected and passed through a 0.22-mm
filter (Merck Millipore, Billerica, MA). Subsequently, 1% diluted extrac-
tion media were prepared with fresh DMEM.
3T3 fibroblasts (ATCC CRL-1658; American Type Culture Collec-
tion, Manassas, VA) were cultured at 37 C with 100% humidity, and
the cells were maintained in DMEM supplemented with 10% FBS and
1% P/S. At 80% confluence, the cells were treated with 0.25%
trypsin-EDTA solution (Sigma-Aldrich) in the well plates. Cells were
seeded into 96-well plates (TPP Techno Plastic Products) at a concen-
tration of 1  104 cells/well. The culture medium was removed after
24 hours, and the cells were incubated in the extracts for 24 hours at
37 C in 100% humidity and 5% CO2. DMEM was used as the control.
Cytotoxicity was assessed using 3-(4,5-dimethyl-thiazoyl)-2,5-
diphenyl-tetrazolium bromide (MTT, Sigma-Aldrich) and sulforhod-
amine B (SRB, Sigma-Aldrich). For the MTT assay, 10 mL of a 5%
dye solution was added to each well, and the plates were incubated
for approximately 3 hours. After incubation, the well content was
removed, and 100 mL acidified isopropanol (Sigma-Aldrich) was added
to each well to dissolve the formazan crystals. Cell viability was deter-
mined using a spectrophotometer (Thermo Fisher Scientific Inc, Wal-
tham, MA) at a wavelength of 570 nm.
For the SRB assay, 25 mL 50% trichloroacetic acid solution
(Sigma-Aldrich) was added to each well and was then incubated for
1 hour at 4 C. After this period, the well content was removed, and
100 mL SRB dye (0.4%) (Sigma-Aldrich) was added to each well for
30 minutes. The plate was washed with 1% acetic acid solution and
100 mL Trizma base (10 mmol/L, Sigma-Aldrich) to solubilize the
colorimetric product. A spectrophotometer was used to measure the
optical densities of the solutions at a 560-nm wavelength. For MTT
JOE — Volume 44, Number 7, July 2018
Basic Research—Technology
and SRB, cell viability was expressed as a percentage of the mean of
DMEM controls, which was considered to represent 100% cell viability.
Biocompatibility Assay
A total of 18 Wistar rats (Rattus norvegicus) weighing between
180 and 220 g at 4 weeks of age were used. They were divided into 3
experimental periods (7, 30, and 90 days) (n = 6).
The animals were anesthetized with 0.008 mL/100 g ketamine and
0.004 mL/100 g 2% xylazine hydrochloride (Virbac do Brasil Ind
ustria
e Com
ercio Ltda, S~ao Paulo, SP, Brazil). The area was disinfected with
alcohol-iodine solution after the dorsal skin was shaved. Four incisions
measuring 1.0-cm long were made in the animals’ backs. Using blunt-
tipped scissors, lateral tearing of the subcutaneous tissue provided 4
surgical cavities in quadrants equidistant from the center of the animals’
backs.
Polyethylene tubes approximately 10-mm long and 1.3 mm in
diameter (Abbott Laboratories do Brasil, S~ao Paulo, SP, Brazil) were
filled with the experimental materials, which were prepared according
to the manufacturers’ instructions. The filled tubes were inserted into the
surgical cavities parallel to the incision. An empty tube was used as the
control. The incisions were closed using 3-0 silk thread (Johnson &
Johnson Produtos Profissionais Ltda, S~ao Jos
e dos Campos, SP, Brazil).
After each experimental period, euthanasia was performed with
intraperitoneal thiopental 0.012 mL/100 g in a local anesthetic solution
(lidocaine 0.001 mL/100 g). Trichotomy and excisional biopsy of the
implant area were performed, and the resulting specimens were placed
on paper discs and fixed in 10% formalin for 24 hours.
After fixation, the polyethylene tubes containing the test materials
were removed, and the tissue was embedded in paraffin. Sections 5- to
6-mm thick were taken along the axis of the tube, mounted on slides,
and stained with hematoxylin-eosin. Slices were examined under a light
microscope by a single-blinded and calibrated examiner (kappa >7 for
all evaluated variables).
Cellular and inflammatory events were determined by the presence
of neutrophils, lymphocytes/plasmacytes, eosinophils, macrophages,
giant cells, and granuloma scored according to the following scale:
1. Absent, no inflammation
2. Mild, cells were present but sparse or in reduced clusters
3. Moderate, cells were present but did not dominate the microscopic
field
4. Intense, cells were present in the form of an infiltrate similar to the
material used
Fiber condensation was classified according to the following scale:
1. Absence
2. Presence of a thin layer
3. Presence of a thick layer of collagen fibers
Abscess formation was classified as follows:
1. Absence
2. Presence of an abscess in contact with the surgical cavity where the
material was inserted
3. Presence of an abscess far from the surgical cavity.
Statistical Analysis
Differences among the materials were subjected to analysis of vari-
ance followed by the Tukey post hoc test for cytocompatibility, and dif-
ferences before and after setting were evaluated using the t test. The
Kruskal-Wallis test followed by the Dunn post hoc test was performed
for biocompatibility. The significance level was set at .05.
Cell Viability and Tissue Reaction of NeoMTA Plus 1141
Basic Research—Technology

Figure 1. Cytocompatibility evaluation (MTT and SRB assays) of repair material extracts at 1% concentration before and after setting. Different letters indicate a
significant difference among repair materials in the same experimental period (P # .05). *A significant difference between the same repair material before and after
setting (P # .05).

TABLE 1. Absolute and Relative Frequencies for Observed Histologic Features According to Periods and Groups
NeoMTA Plus, n (%) MTA, n (%) Biodentine, n (%) Control, n (%)
Scores 7 30 90 7 30 90 7 30 90 7 30 90
Inflammatory infiltrate intensity
1 0 (0) 1 (16.6) 6 (100) 0 (0) 3 (50) 2 (40) 0 (0) 0 (0) 2 (33.3) 0 (0) 2 (40) 6 (100)
2 1 (20) 5 (83.3) 0 (0) 1 (20) 1 (16.6) 1 (20) 0 (0) 0 (0) 1 (16.6) 0 (0) 4 (60) 0 (0)
3 4 (80) 0 (0) 0 (0) 2 (40) 2 (33.3) 2 (40) 1 (20) 6 (100) 3 (50) 4 (80) 0 (0) 0 (0)
4 0 (0) 0 (0) 0 (0) 2 (40) 0 (0) 0 (0) 4 (80) 0 (0) 0 (0) 1 (20) 0 (0) 0 (0)
Neutrophils
1 4 (80) 6 (100) 6 (100) 4 (80) 6 (100) 6 (100) 0 (0) 6 (100) 6 (100) 4 (80) 6 (100) 6 (100)
2 1 (20) 0 (0) 0 (0) 1 (20) 0 (0) 0 (0) 1 (20) 0 (0) 0 (0) 1 (20) 0 (0) 0 (0)
3 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 4 (80) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
4 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
Lymphocytes/plasmacytes
1 0 (0) 4 (66.6) 6 (100) 0 (0) 3 (50) 3 (50) 0 (0) 1 (16.6) 4 (66.6) 0 (0) 6 (100) 6 (100)
2 2 (40) 2 (33.3) 0 (0) 1 (20) 2 (33.3) 3 (50) 0 (0) 4 (66.6) 2 (33.3) 3 (60) 0 (0) 0 (0)
3 2 (40) 0 (0) 0 (0) 2 (40) 1 (16.6) 0 (0) 3 (60) 1 (16.6) 0 (0) 1 (20) 0 (0) 0 (0)
4 1 (20) 0 (0) 0 (0) 2 (40) 0 (0) 0 (0) 2 (40) 0 (0) 0 (0) 1 (20) 0 (0) 0 (0)
Giant cells
1 5 (100) 6 (100) 6 (100) 4 (80) 6 (100) 6 (100) 0 (0) 6 (100) 6 (100) 5 (100) 6 (100) 6 (100)
2 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
3 0 (0) 0 (0) 0 (0) 1 (20) 0 (0) 0 (0) 3 (60) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
4 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 2 (40) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
Eosinophils
1 5 (100) 6 (100) 6 (100) 5 (100) 6 (100) 5 (100) 5 (100) 6 (100) 6 (100) 5 (100) 6 (100) 6 (100)
2 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
3 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
4 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
Macrophages
1 0 (0) 1 (16.6) 6 (100) 2 (40) 3 (50) 2 (40) 0 (0) 0 (0) 2 (33.3) 0 (0) 3 (50) 6 (100)
2 4 (80) 5 (83.3) 0 (0) 2 (40) 2 (33.3) 1 (20) 0 (0) 0 (0) 1 (16.6) 1 (20) 3 (50) 0 (0)
3 1 (20) 0 (0) 0 (0) 1 (20) 1 (16.6) 2 (40) 1 (20) 2 (33.3) 3 (50) 3 (60) 0 (0) 0 (0)
4 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 4 (80) 4 (66.6) 0 (0) 1 (20) 0 (0) 0 (0)
Granuloma
1 5 (100) 6 (100) 6 (100) 0 (0) 6 (100) 6 (100) 0 (0) 6 (100) 6 (100) 5 (100) 6 (100) 6 (100)
2 0 (0) 0 (0) 0 (0) 4 (80) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
3 0 (0) 0 (0) 0 (0) 1 (20) 0 (0) 0 (0) 2 (40) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
4 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 3 (60) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
Abscess
1 5 (100) 6 (100) 6 (100) 5 (100) 6 (100) 5 (100) 5 (100) 6 (100) 6 (100) 5 (100) 6 (100) 6 (100)
2 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
3 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
Fiber condensation
1 3 (60) 0 (0) 0 (0) 5 (100) 0 (0) 0 (0) 5 (100) 0 (0) 0 (0) 5 (100) 0 (0) 0 (0)
2 2 (40) 6 (100) 6 (100) 0 (0) 5 (83.3) 4 (66.6) 0 (0) 6 (100) 2 (33.3) 0 (0) 4 (66.6) 4 (66.6)
3 0 (0) 0 (0) 0 (0) 0 (0) 1 (16.6) 2 (33.3) 0 (0) 0 (0) 4 (66.6) 0 (0) 2 (33.3) 2 (33.3)
MTA, mineral trioxide aggregate.

1142 Pinheiro et al. JOE — Volume 44, Number 7, July 2018

 Basic Research—Technology

Figure 2. Comparison among materials and experimental periods for each variable including inflammatory infiltrate intensity, lymphocytes/plasmacytes, neutro-
phils, giant cells, granuloma, fiber condensation, and macrophages. No eosinophils or abscesses were found; thus, it was not classified in the figure. Different
uppercase letters indicate significant differences among different repair materials in the same period (P # .05). Different lowercase letters indicate a significant
difference in the same material in the different experimental periods (P # .05).

Results Biocompatibility Assay

Cytocompatibility Assays
For the MTT and SRB assays, repair materials presented greater
viability after setting when compared with before setting (P # .05).
For MTT before setting, NMP and MTA showed higher viability
compared with the control group (P # .05), which was similar to
BD. NMP, MTA, and BD were more viable than the control after
setting (P # .05). For the SRB assay, MTA and BD presented less
viability than the control before setting (P # .05). No statistical dif-
ference was observed between NMP and the control group
(P $ .05). NMP differed from BD and MTA (P # .05) after setting
and was similar to the control group (P $ .05). MTA and BD pre-
sented higher viability when compared with the control group
(P # .05) (Fig. 1).
The results are summarized in Table 1 and Figures 2 and 3A and B.
No eosinophils or abscesses were found in any of the groups. At 7 days,
neutrophils were present in BD (P # .05), and granuloma and giant
cells were found in BD and MTA. In addition, BD presented the highest
scores for giant cells (P # .05), but there was no difference between the
repair materials and the control at 30 and 90 days.
Reductions of inflammatory and lymphoplasmacytic infiltrates
were observed for all materials throughout the experimental periods.
In addition, BD showed the most intense inflammatory infiltrate and
a greater amount of lymphoplasmacytic infiltrate and macrophages at
30 days (P # .05). These events decreased in intensity at 90 days, being
similar to MTA (P $ .05) and different from NMP and the control
(P $ .05).

JOE — Volume 44, Number 7, July 2018 Cell Viability and Tissue Reaction of NeoMTA Plus 1143
Basic Research—Technology
Figure 3. The connective tissue response to the different repair materials. (A) BD, 7 days (*intense inflammatory infiltrate) (40); (B) MTA, 7 days (*moderate
inflammatory infiltrate) (40); (C) NMP, 7 days (*moderate inflammatory response) (300); and (D) BD, 90 days (*thick fibrous condensation) (300).
An increase in collagen fibers was seen from 7 to 30 and 90 days
for all groups. At 90 days, BD presented a thick layer of fibers when
compared with NMP (P # .05).
Discussion
A material must exhibit low cell toxicity without promoting an in-
flammatory reaction to be considered biocompatible or, at least, should
reduce over time to insignificant or mild (11). Furthermore, fibrous tis-
sue capsule formation around the material indicates that it is being
tolerated by the tissues (12). In this regard, it is recommended that
initial tests be performed (ie, cytotoxicity, mutagenicity, and systemic
toxicity) followed by other preliminary tests (ie, subcutaneous,
muscular, and osseous implants tests) and then preclinical animal us-
age studies; only then should tests with humans be performed (11).
In addition to satisfactory biological characteristics, the physico-
chemical properties of materials should be considered, and bioceramic
repair materials present different components for improving these
physicochemical properties, which may interfere with the tissue inflam-
matory response and cell viability.
In this study, the 3T3 fibroblast mouse embryo cell line was cho-
sen to perform the assays. Fibroblasts are the major constituents of con-
nective tissue, and the predominant cell type of the periodontal ligament
and pulp tissue that will be in contact with endodontic materials (13). It
1144 Pinheiro et al.
is necessary to analyze the different cell parameters to evaluate the po-
tential toxicity of the materials (14). MTT evaluates mitochondrial suc-
cinate dehydrogenase, one of the enzymes responsible for cellular
respiration, and SRB evaluates protein synthesis in cells.
After setting, all materials showed higher cell viability compared
with the control group for MTT and SRB. Before setting, the SRB assay
showed that MTA and BD had less viability than the control group and
NMP, and for the MTT assay, only BD showed less viability than the con-
trol group and was not statistically different. In 2017, Tanomaru-Filho
et al (14) also observed different results for MTT and neutral red cell
viability methodologies. After setting, NMP cytotoxicity was compared
with an experimental tricalcium silicate cement and MTA on human os-
teoblastlike cells (Saos-2). The neutral red assay results did not show a
difference among the materials. The MTT assay revealed different results
depending on the materials and extract dilutions. Lower cytotoxicity was
observed for NMP and MTA and was higher for experimental cement at
1:1 and 1:2 dilutions.
For both assays, the cellular viability was lower before the set. Bio-
ceramic materials have a high initial pH, which may have negatively
influenced cellular metabolism (15). Because ISO 10993-5:2009
(16) accepts cytotoxic materials presenting viability values below
70%, it is possible to affirm that the tested materials provided positive
outcomes.
JOE — Volume 44, Number 7, July 2018

Regarding biocompatibility results, NMP presented a moderate
initial inflammatory response, which was similar to MTA and less
than BD; besides the reduction of this inflammation over time, no gran-
ulomas, giant cells, eosinophils, or abscesses were observed, which was
similar to the control group and showed biocompatibility. The inflam-
matory response decreased for all materials during the 90-day period
with no statistical differences among them.
The predominance of inflammatory infiltrate in the early periods
could be explained by the release of calcium ions from calcium
silicate–based materials. The increase in pH values during setting, in
addition to the heat released by this reaction, illustrates inflammatory
cell recruitment and the production and release of proinflammatory cy-
tokines (17–19). On the other hand, the release of calcium ions and the
alkalinity of the medium stimulate hydroxyl apatite formation and the
release of alkaline phosphatase and bone morphogenetic protein 2,
which is important in the mineralization process (20).
BD presented the highest inflammatory response at 7 days with the
presence of neutrophils, giant cells, and granuloma. At 30 days, it
showed an intense presence of macrophages, which demonstrates
the body’s attempt to eliminate the foreign material via phagocytosis.
A significant reduction of the inflammatory response was observed at
90 days because this reaction was gradually replaced by a fibrous
capsule composed of bundles of collagen and fibroblasts.
An intense inflammatory response in the early period for BD was
also reported by Mori et al (21) in rat’s connective tissue. They
observed a moderate reaction to BD and a mild reaction to MTA at
7 days; however, the inflammatory process was mild at 14 and
30 days. Calcium chloride, present in BD liquid to reduce the setting
time, also reduces viability and cell binding when compared with orig-
inal MTA (22). Another component, the water-soluble carboxylate-
based polymer, was able to induce a mild inflammatory reaction in
the dental pulp but was gradually replaced by healthy tissue (23). More-
over, inflammatory cell recruitment is associated with high pH (20). On
the other hand, BD presents zirconia oxide as a radiopacifier, and its
main advantages are reduced setting time and better mechanical prop-
erties (24); it presented low cytotoxicity when tested on fibroblasts in an
experimental sealer (25).
NMP was developed with a similar composition to MTA Plus, but
bismuth oxide was replaced by tantalum oxide, avoiding discoloration
(26). This radiopacifier, when in contact with rabbit condyle, was
considered biocompatible (27), and it has low cytotoxicity (28, 29).
NMP was able to stimulate mineralized nodule formation when
evaluated by alizarin red staining on human osteoblastlike cells,
highlighting its bioactivity (14).
In conclusion, a material is considered biocompatible when it
promotes cell viability, and the tissue inflammatory response becomes
insignificant over time. Considering the limits of the methods used here-
in, NMP, MTA, and BD can be considered options in clinical situations in
which a repair material is required.
Acknowledgments
Supported by grants from Coordenaç~ao de Aperfeiçoamento de
Pessoal de N
ıvel Superior - CAPES.
The authors deny any conflicts of interest related to this study.
References
1. Nair PN, Duncan HF, Pitt Ford TR, et al. Histological, ultrastructural and quantitative
investigations on the response of healthy human pulps to experimental capping with
JOE — Volume 44, Number 7, July 2018
Basic Research—Technology
mineral trioxide aggregate: a randomized controlled trial. Int Endod J 2009;42:
422–44.
2. Lee H, Shin Y, Kim S, et al. Comparative study of pulpal responses to pulpotomy with
ProRoot MTA, RetroMTA, and TheraCal in dogs’ teeth. J Endod 2015;41:1318–24.
3. Katebzadeh N, Dalton BC, Trope M. Strengthening immature teeth during and after
apexification. J Endod 1998;24:256–9.
4. Rathinam E, Rajasekharan S, Chitturi RT, et al. Gene expression profiling and mo-
lecular signaling of various cells in response to tricalcium silicate cements: a sys-
tematic review. J Endod 2016;41:56–81.
5. Parirokh M, Torabinejad M, Dummer PMH. Mineral trioxide aggregate and other
bioactive endodontic cements: an updated overview- part I: vital pulp therapy.
J Endod 2018;51:177–205.
6. Liu S, Wang S, Dong Y. Evaluation of a bioceramic as a pulp capping agent in vitro
and in vivo. J Endod 2015;41:652–7.
7. Torabinejad M, Parirokh M, Dummer PM. Mineral trioxide aggregate and other
bioactive endodontic cements: an updated overview- part II: other clinical applica-
tions and complications. Int Endod J 2018;51:284–317.
8. Camilleri J. Mineral trioxide aggregate: present and future developments. Endod
Topics 2015;32:31–46.
9. Gandolfi MG, Taddei P, Modena E, et al. Biointeractivity-related versus chemi/
physisorption-related apatite precursor-forming ability of current root end filling
materials. J Biomed Mater Res B Appl Biomater 2013;101:1107–23.
10. Siboni F, Taddei P, Prati C, Gandolfi F. Properties of Neo MTA Plus and MTA Plus
cements for endodontics. Int Endod J 2017;50:83–94.
11. Hauman CH, Love RM. Biocompatibility of dental materials used in contemporary
endodontic therapy: a review. Part 2. Root-canal-filling materials. Int Endod J
2003;36:147–60.
12. Yaltirik M, Ozbas H, Bilgic B, et al. Reactions of connective tissue to mineral trioxide
aggregate and amalgam. J Endod 2004;30:95–9.
13. Silva EJ, Senna PM, De-Deus G, et al. Cytocompatibility of Biodentine using a three-
dimensional cell culture model. Int Endod J 2016;49:574–80.
14. Tanomaru-Filho M, Andrade AS, Rodrigues EM, et al. Biocompatibility and miner-
alized nodule formation of Neo MTA Plus and an experimental tricalcium silicate
cement containing tantalum oxide. Int Endod J 2017;50:e31–9.
15. Lucas CP, Viapiana R, Bosso-Martelo R, et al. Physicochemical properties and dentin
bond strength of a tricalcium silicate-based retrograde material. Braz Dent J 2017;
28:51–6.
16. International Organization for Standardization. Biological evaluation of medical
devices: part 5—tests for in vitro cytotoxicity. ISO 10993. Geneva, Switzerland:
International Organization for Standardization; 2009.
17. Koh ET, Torabinejad M, Pitt Ford TR, et al. Mineral trioxide aggregate stimulates a
biological response in human osteoblasts. J Biomed Mater Res 1997;37:432–9.
18. Vosoughhosseini S, Lotfi M, Shahi S, et al. Influence of white versus gray mineral
trioxide aggregate on inflammatory cells. J Endod 2008;34:715–7.
19. Shahi S, Rahimi S, Yavari HR, et al. Effect of mineral trioxide aggregates and Portland
cements on inflammatory cells. J Endod 2010;36:899–903.
20. Gandolfi MG, Siboni F, Primus CM, et al. Ion release, porosity, solubility, and bioac-
tivity of MTA Plus tricalcium silicate. J Endod 2014;40:1632–7.
21. Mori GG, Teixeira LM, Oliveira DL, et al. Biocompatibility evaluation of biodentine in
subcutaneous tissue of rats. J Endod 2014;40:1485–8.
22. Kang J, Lee B, Son H, et al. Biocompatibility of mineral trioxide aggregate mixed with
hydration accelerators. J Endod 2013;39:497–500.
23. Hill EE. Dental cements for definitive luting: a review and practical clinical consid-
erations. Dent Clin North Am 2007;51:643–58.
24. Camilleri J, Sorrentino F, Damidot D. Investigation of the hydration and bioactivity of
radiopacified tricalcium silicate cement, Biodentine and MTA Angelus. Dent Mater
2013;29:580–93.
25. Slompo C, Peres-Buzalaf C, Gasque KCS, et al. Experimental calcium silicate-based
cement with and without zirconium oxide modulates fibroblasts viability. Braz Dent J
2015;26:587–91.
26. Camilleri J. Staining potential of Neo MTA Plus, MTA Plus, and Biodentine used for
pulpotomy procedures. J Endod 2015;41:1139–45.
27. Hoekstra JW, Beucken JJ, Leeuwenburgh SC, et al. Tantalum oxide and barium sul-
fate as radiopacifiers in injectable calcium phosphate-poly (lactic-co-glycolic acid)
cements for monitoring in vivo degradation. J Biomed Mater Res A 2014;102:
141–9.
28. Oh MH, Lee N, Kim H, et al. Large-scale synthesis of bioinert tantalum oxide nano-
particles for X-ray computed tomography imaging and bimodal image-guided
sentinel lymph node mapping. J Am Chem Soc 2011;133:5508–15.
29. Mohandas G, Oskolkov N, McMahon MT, et al. Porous tantalum and tantalum oxide
nanoparticles for regenerative medicine. Acta Neurobiol Exp (Wars) 2014;74:
188–96.
Cell Viability and Tissue Reaction of NeoMTA Plus 1145