Basic ResearchTechnology

Tissue Reactions to a New Mineral Trioxide

Aggregatecontaining Endodontic Sealer
Cauana Oliva Tavares, DDS,* Daiana Elisabeth B ottcher, DDS, MSc, Elosa Assmann, DDS,

onio Poli de Figueiredo, PhD, DDS, MSc,*
Patrcia Maria Poli Kopper, PhD, DDS, MSc, Jose Ant^
Fabiana Soares Grecca, PhD, DDS, MSc, and Roberta Kochenborger Scarparo, DDS, MSc, PhD*

Abstract
Introduction: The purpose of this study was to analyze
^
the connective tissue reactions to MTA Fillapex (Angelus
Industria de Produtos Odontlogicos Ltda, Londrina,
Brazil) compared with a zinc oxidebased sealer
(EndoFill; Dentsply Industria e Comerico Ltda, Petropolis,
Brazil) and an epoxy resinbased material (AH Plus;
Dentsply DeTrey GmbH, Konstanz, Germany) in Wistar
rats. Methods: Polyethylene tubes containing the test
materials and empty polyethylene tubes (control) were
implanted in the subcutaneous tissue of 12 rats. Empty
tubes were used as a negative control. After 7 and 60
days (n = 6 per period), observations were made for
cellular inflammatory components, fibrous condensa-
tion, and abscess formation. Comparisons among the
groups and between the experimental periods were
made using 2-way analysis of variance and the Bonfer-
roni post hoc test (P < .05). Results: At the end of the
7-day experimental period, all sealers scored higher
than the control group for the variable lymphocytes,
and MTA Fillapex presented lower fiber condensation
compared with empty tubes. After 60 days, macro-
phages and lymphocytes scored significantly higher for
MTA Fillapex and EndoFill compared with the negative
control, and AH Plus showed similar results related to
the empty tubes. Comparing the materials responses
at the end of the 2 evaluated periods, for EndoFill
samples the variable neutrophils was detected less after
60 days. Both EndoFill and MTA Fillapex presented
increased fiber condensation after 60 days. Conclu-
sions: Although none of the sealers promoted ideal
tissue responses, AH Plus presented the best outcomes.
Although MTA Fillapex contains MTA powder, it pre-
sented no biocompatibility advantages when compared
with AH Plus and EndoFill. (J Endod 2013;39:653657)
Key Words
Biocompatibility, endodontics, endodontic sealers, MTA
Fillapex, root canal filling
I t is well documented that obturation procedures have a role in favoring and maintain-
ing adequate conditions for endodontic repair (1, 2). In this regard, biocompatibility
(35) and sealing ability (1) are of paramount importance, and several endodontic
sealers have been constantly developed with the aim of improving these features (6).
MTA Fillapex (^Angelus Industria de Produtos Odontlogicos Ltda, Londrina, Brazil)
is an endodontic sealer that claims to take advantage of the biological and physical prop-
erties of mineral trioxide aggregate (MTA) (68). Nevertheless, apart from containing
MTA compounds, this sealer is comprised of resins (ie, salicylate, diluting, and
natural), radiopaque bismuth, nanoparticulated silica, and pigments (6, 7).
Although some authors describe a similar tissue response when compared with the
original MTA formulations (8), the biological responses to MTA Fillapex compared
with the more commonly used endodontic sealers have not been explored yet. More-
over, a severe reduction of cell survival rates has been reported to be associated
with MTA Fillapex (9), leading to uncertainties regarding its biological properties.
Because of these conflicting results, the aim of the present study was to analyze
the connective tissue reactions to MTA Fillapex compared with a zinc oxidebased
sealer (EndoFill; Dentsply Industria e Comerico Ltda, Petropolis, Brazil) and an
epoxy resinbased material (AH Plus; Dentsply DeTrey GmbH, Konstanz, Germany)
in Wistar rats.
Materials and Methods
The present study was approved by the Research and Ethics Committee of the
School of Dentistry of the Federal University of Rio Grande do Sul, Porto Alegre, Brazil
(no. 13534). The inflammatory reactions of 12 animals to MTA Fillapex, AH Plus, and
EndoFill were evaluated after 2 experimental periods (ie, 7 and 60 days, n = 6 per
period). The sealers were inserted into autoclaved polyethylene tubes approximately
10 mm long and 1.5 mm in diameter (Abbott Laboratorios do Brasil, S~ao Paulo, Brazil)
to be implanted in the animals dorsal subcutaneous tissue. An empty tube was used as
the negative control.
The animals were anesthetized with 0.008 mL/100 g ketamine and 0.004 mL/100 g
2% xylazine hydrochloride (Virbac do Brasil Industria e Comercio Ltda, S~ao Paulo,
Brazil). Dorsal trichotomy was manually performed in an area comprising approxi-
mately 10 cm2. Disinfection with an alcohol-iodine solution was performed. Four
0.5-cm-long incisions were made in the animals backs 2 cm from the spine and at least
2 cm apart from one another. Using blunt-tipped scissors, the lateral tearing of the
subcutaneous tissue provided 4 surgical cavities displayed in quadrants equidistant
from the center of the animals backs. The materials were prepared according to the
manufacturers instructions and introduced into their respective tubes using sterile

From the *Clinical Department, School of Dentistry, Pontifical Catholic University of Rio Grande do Sul (PUCRS), Porto Alegre, Rio Grande do Sul, Brazil; and

Conservative Dentistry Department, School of Dentistry, Federal University of Rio Grande do Sul (UFRGS), Porto Alegre, Rio Grande do Sul, Brazil.
Address requests for reprints to Dr Daiana Elisabeth Bottcher, Av Ramiro Barcelos, 2492, Cep: 90035-003, Porto Alegre, RS, Brazil. E-mail address: daibottcher@
hotmail.com
0099-2399/$ - see front matter
Copyright 2013 American Association of Endodontists.
http://dx.doi.org/10.1016/j.joen.2012.10.009

JOE Volume 39, Number 5, May 2013 Tissue Reactions to MTA Fillapex 653
Basic ResearchTechnology
insulin syringes (Injex Industria Cirurgica Ltda, Ourinhos, Brazil). The
tubes filled with the materials were immediately inserted into the
surgical cavities parallel to the incision. The position in which each
sealer was implanted was standardized. The incisions were closed using
a 3-0 silk thread (Johnson & Johnson Produtos Profissionais Ltda, S~ao
Jose dos Campos, Brazil).
At the end of each experimental period, the animals were
euthanized by CO2 inhalation. Trichotomy was performed once
more, and an excisional biopsy of the implant area was performed
with a safety margin of 1 cm. The samples were placed on paper
discs and fixed in 10% formalin for 24 hours. Then, the polyeth-
ylene tubes were removed from the samples, set in paraffin blocks,
and processed for histologic analysis. Sections with a thickness of 3
to 4 mm were cut along the axis of each tube, mounted on slides,
and stained with hematoxylin-eosin. Three sections were obtained
per sample. The slices were examined under a light microscope
by a single-blinded, calibrated examiner (k > 6 for all evaluated
variables).

Figure 1. A comparison among the study groups and between the 7- and 60-day experimental periods regarding the following variables: neutrophils, lymphocytes,
macrophages, giant cells, eosinophils, fibers, and abscesses. Statistically significant differences were detected in the study groups compared with the control
(*P < .05, **P < .01). The same materials also showed different scores between the 2 experimental periods (+P < .05, ++++P < .0001).

654 Tavares et al. JOE Volume 39, Number 5, May 2013


Slides were analyzed qualitatively according to the criteria
described by Figueiredo et al (10) and adapted for other studies
(3, 4). The cellular inflammatory component was determined by the
presence of neutrophils, lymphocytes, eosinophils, macrophages, and
giant cells. Cellular events were classified according to the following
scale:
1. Absent: Inflammation was either absent or within blood vessels.
2. Mild: Cells were present although 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 close to the
material used.
Fiber condensation was classified according to the following scale:
1. The absence of collagen fibers
2. The presence of a thin layer of collagen fibers
3. The presence of a thick layer of collagen fibers
Abscess formation was classified as follows:
1. The absence of an abscess
2. The presence of an abscess in contact with the surgical cavity in
which the material had been inserted
3. The presence of abscess areas far from the surgical cavity in which
the material had been inserted
Differences among the groups and between the 2 experimental
periods were evaluated by using 2-way analysis of variance in conjunction
with the Bonferroni post hoc test. The significance level was set at .05.
Results
The study results are summarized in Figure 1. The histologic
parameters used in assessing the results over the experimental periods
Figure 2. Features observed 7 days after the surgical procedure: connective tissue responses to (A) the control and (B and C) MTA Fillapex (100). Empty tube
implants (ET) promoted the formation of a vascularized tissue presenting few inflammatory cells (*) and fibrous condensation (arrows). On the contrary, MTA
Fillapex (FL) produced an intense and extended inflammatory response, mainly comprised of lymphocytes (arrows) and not limited by fibrous condensation. In
some samples, an abscess formation could also be observed next to the experimental material (>).
JOE Volume 39, Number 5, May 2013
Basic ResearchTechnology
are shown in Figures 2 and 3. After 7 days, statistically significant
differences were detected among the groups. All sealers scored
higher than the control group for the variable lymphocytes. MTA
Fillapex presented lower fiber condensation compared with the other
groups responses (Fig. 2A and B). Although significant differences
were not observed for the variables macrophages and neutrophils, at
the end of the 7-day experimental period, these cells were more
intensely detected in the reaction to the sealers than in the control
group. Similarly, EndoFill and MTA Fillapex were likely to present
higher scores for giant cells and abscesses (Fig. 2C), whereas eosino-
phils were more frequently detected in the EndoFill samples.
At the end of the 60-day experimental period, significant differ-
ences were observed for macrophages and lymphocytes in the MTA
Fillapex (Fig. 3A) and EndoFill (Fig. 3B) groups compared with the
negative control. In contrast to the results obtained after the 7-day
experimental period, AH Plus showed similar results related to the
empty polyethylene tubes after 60 days (Fig. 3C and D).
Also, at the end of the 60-day experimental period, there was
a decrease in the number of neutrophils for EndoFill samples compared
with the number of neutrophils at the end of the 7-day experimental
period. EndoFill and MTA Fillapex produced increased fiber condensa-
tion at the end of the 7-day experimental period (Figs. 2 and 3).
Discussion
The present investigation showed that although it was expected that
a sealer containing MTA acted as a repairer, no advantage could be ob-
tained regarding the tissue responses to MTA Fillapex compared with
the epoxy resinbased sealer AH Plus or the zinc oxidebased sealer
EndoFill. In this regard, significant differences were observed when
MTA Fillapex and AH Plus were compared, and the MTA-containing
material scores of inflammatory cells and abscesses were only slightly
lower compared with EndoFill. Moreover, after the 7-day experimental
Tissue Reactions to MTA Fillapex 655
Basic ResearchTechnology
Figure 3. Tissue responses to the sealers. (A) After 60 days, MTA Fillapex (FL) showed inflammatory infiltrate (*), and giant cells (arrows) were identified in
some of the samples (200). (B) At the same period, Endofill promoted the maintenance of an inflammatory response, mainly comprised of lymphocytes and
macrophages (arrows) (400). AH Plus (AHP) showed a moderate inflammatory response (*) (C) after 7 days (100), which became mild and well limited for
fibrous condensation (D) after 60 days (40).
period, MTA Fillapex showed limited fiber condensation (which had an
influence on the extension of the inflammatory reaction), and the vari-
ables lymphocytes and macrophages scored higher than the control
group throughout the experiment. Although not statistically significant,
taken together with the presence of macrophages for long periods, the
detection of giant cells emphasizes MTA Fillapexs potential for tissue
irritation. Therefore, MTA Fillapex does not have the claimed biological
advantages over other available products.
These results probably are associated with MTA Fillapexs chem-
ical composition, which is mainly resin based but also contains MTA
compounds. A previous in vitro study (9) showed that white MTA
was able to keep cell viability and increase its proliferation, whereas
MTA Fillapex was severely cytotoxic. The current results showing intense
and extensive inflammation in response to MTA Fillapex corroborate
these findings.
On the contrary, in earlier studies (4, 11) Endo-CPM sealer,
which also attempts to maintain the MTA biological properties, showed
a more favorable connective tissue response (4, 11), which reflects the
materials composition. Apart from containing the setting accelerator
calcium chloride, Endo-CPM sealer basically presents the same
compounds of the original MTA formulation (7, 12). On the other
hand, although reformulated to improve handling characteristics,
Endo-CPM sealer has some disadvantages regarding its physical prop-
erties, leading to failures in root canal fillings radiographically detected
as voids and gaps (7). For this reason, the attempt to combine physical
properties from resin-based sealers with the MTA biological and sealing
features is fully justified.
The results of this study point out that the more commonly used
epoxy resinbased sealer AH Plus promoted more favorable results
than MTA Fillapex and EndoFill; this is also corroborated by other inves-
tigations comparing endodontic sealers (13, 14). In a previous study
(3), AH Plus was the only sealer that exhibited similar inflammatory
infiltration compared with the control group after longer periods.
656 Tavares et al.
The differences between MTA Fillapex and AH Plus suggest that some
of the compounds of the first material have the potential to cause tissue
aggression, impairing MTA biological properties. Thus, although not
presenting ideal tissue responses at short experimental periods, AH
Plus should still be considered the gold standard because of its biocom-
patibility. Accordingly, the response to MTA Fillapex was similar to that
observed with zinc oxidebased sealers, which are recognized for
their potential to bring about greater and longer-lasting inflammatory
reactions (315).
Unlike the results of this study, MTA Fillapex promoted favorable
reactions of the connective tissue in a previous investigation (8). Apart
from the differences in the study groups, the conflicting results are
probably related to details in the experimental procedures and the eval-
uation methodology. In the present study, after the preparation of the
sealers, they were immediately inserted into the implants and placed
into the surgical cavities in such a way that allowed their contact with
the rat connective tissue to set. Moreover, the scores adopted herein
made it possible to supplement an objective analysis of the results
with the description of the inflammatory events, considering details
regarding the predominant cell type and its role in the different stages
of tissue reaction (3, 10). On the contrary, the study by Gomes-Filho
et al (8) used scores that considered inflammatory cell counts, regard-
less of their type, in conjunction with a less detailed classification of the
host response intensity. Taking into account that several studies empha-
size that the quantification of the tissue response is only possible in
materials with very distinct degrees of irritability (3, 10, 16), this
evaluation criterion could have masked more subtle differences.
Conclusion
The current results showed that although none of the sealers
promoted ideal tissue responses, AH Plus presented the best outcomes,
which was evidenced by the similarities between this sealer and the
JOE Volume 39, Number 5, May 2013

control group after longer periods of time. Despite containing MTA,
MTA Fillapex presented no biocompatibility advantages when compared
with AH Plus and EndoFill.
Acknowledgments
The authors deny any conflicts of interest related to this study.
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