Review Article
Mineral Trioxide Aggregate: A Comprehensive Literature
Review—Part II: Leakage and Biocompatibility
Investigations
Mahmoud Torabinejad, DMD, MSD, PhD,* and Masoud Parirokh, DMD, MS†
Abstract
Introduction: Mineral trioxide aggregate (MTA) was
developed because existing materials did not have the
ideal characteristics for orthograde or retrograde root-
end fillings. MTA has been recommended primarily as
a root-end filling material, but it has also been used in
pulp capping, pulpotomy, apical barrier formation in
teeth with open apexes, repair of root perforations,
and root canal filling. Part I of this literature review pre-
sented a comprehensive list of articles regarding the
chemical and physical properties as well as the antibac-
terial activity of MTA. The purpose of part II of this
review is to present a comprehensive list of articles
regarding the sealing ability and biocompatibility of
this material. Methods: A review of the literature was
performed by using electronic and hand-searching
methods for the sealing ability and biocompatibility of
MTA from November 1993–September 2009. Results:
Numerous studies have investigated the sealing ability
and biocompatibility of MTA. Conclusions: On the basis
of available evidence it appears that MTA seals well and
is a biocompatible material. (J Endod 2010;36:190–202)
Key Words
Apical plug, biocompatibility, cytokine production,
marginal adaptation, mineral trioxide aggregate, MTA,
root-end filling, sealing ability, signaling molecule
From the *Department of Endodontics, School of Dentistry,
Loma Linda University, Loma Linda, California; and
†
Department of Endodontics, School of Dentistry, Oral and
Dental Diseases Research Center, Neuroscience Research
Center, Iranian Center for Endodontic Research, Kerman
University of Medical Sciences, Kerman, Iran.
Address requests for reprints to Mahmoud Torabinejad,
DMD, MSD, PhD, Professor and Director, Endodontic Residency
Program, Department of Endodontics, School of Dentistry,
Loma Linda University, Loma Linda, CA 92350. E-mail address:
mtorabinejad@llu.edu.
0099-2399/$0 - see front matter
ª 2010 Published by Elsevier Inc. on behalf of the American
Association of Endodontists.
doi:10.1016/j.joen.2009.09.010
190 Torabinejad and Parirokh
M ost endodontic failures occur as a result of leakage of irritants from pathologically
involved root canals into the periradicular tissues; therefore, a repair material
should provide a good seal to an otherwise unobturated root canal or improve the
seal of an existing filling material. An adequate apical seal is a major factor for improving
endodontic success (1). One of the most important criteria for an ideal endodontic
material is its sealing ability and marginal adaptation (2). The sealing ability of original
mineral trioxide aggregate (MTA), other types of MTA, and its new compositions has
been tested by leakage studies (dye, fluid filtration, bacteria and bacterial by-products)
and scanning electron microscopy (SEM). Because materials used in endodontics are
frequently placed in close contact with the periodontium, they also must be biocompat-
ible with host tissues. The purpose of this literature review is to present a comprehensive
list of articles from November 1993–September 2009 regarding the sealing ability and
biocompatibility of MTA.
Inclusion and Exclusion Criteria
The inclusion and exclusion criteria for this literature review were identical to
those used for Part I of the review (3).
Search Methodology
An electronic search was conducted in the PubMed and Cochrane databases with
appropriate MeSH headings and key words related to the sealing ability and biocom-
patibility of MTA. The hand-searching methodology used in this literature review was
identical to that used for Part I of the review (3).
Leakage Studies
Leakage investigations on MTA have evaluated the sealing ability of the material as
a root-end filling material, perforation repair material, root canal filling, coronal and
apical barrier material (4, 5–85).
Leakage of MTA as a Root-end Filling Material and Other Root-end
Filling Materials to Materials Tested
The sealing ability of MTA and other root-end filling material has been tested by
using dye, fluid filtration, protein leakage, and bacterial leakage methods.
Dye Leakage. Numerous dye leakage studies have been performed on MTA when
used as a root-end filling material (4–20). Various types of dyes have been used to eval-
uate MTA’s sealing ability, including methylene blue, fuchsin, rhodamine B, silver
nitrate, India ink, and Pelikan ink.
Results from most of these investigations indicated that MTA exhibits significantly
less dye leakage in comparison with Super EBA, amalgam (4–7, 9, 10), and interme-
diate restorative material (IRM) (5, 7, 11). An investigation compared dye penetration
in roots filled with MTA as a root canal filling material (in an orthograde manner) with
fresh MTA as a root-end filling material (in a retrograde manner). Results showed no
significant difference between the 2 groups (8).
Gondim et al (17) investigated microleakage of three root-end filling materials
with or without finishing. Their results revealed that Pro Root MTA displays significantly
less mean dye microleakage as a root-end filling material than Super EBA and IRM.
JOE — Volume 36, Number 2, February 2010

Variation in the finishing techniques does not significantly affect dye
leakage of the tested materials (17).
In contrast to these studies, 2 investigations showed the inferiority
of MTA in terms of dye (India ink) penetration when compared with
Super EBA and Geristore (15, 16). Tobón-Arroyave et al (16) attributed
more leakage in MTA specimens to placing the white MTA (WMTA)
samples in dye before complete setting.
Rahimi et al (85) compared dye leakage of different depths of MTA
when used as a root-end filling material. They placed their samples in
phosphate-buffered saline (PBS) for 48 hours before dye exposure. Dye
penetration was not significantly different between 1-mm, 2-mm, or
3-mm thicknesses of MTA.
One investigation reported that the amounts of dye leakage of MTA
without calcium phosphate cement (CPC) matrix samples was greater in
acidic pH than in a neutral environment (19).
A dye leakage study testing different dental materials showed a de-
coloration effect of MTA with methylene blue (20). The formation of
calcium hydroxide (CH) after the reaction of MTA with moisture (86,
87) might be the reason for methylene blue decoloration when the
dye is used for leakage evaluation. The same finding was observed
when the roots were filled with CH (20). A comparison of WMTA and
a glass ionomer cement (GIC) by using rhodamine B (with varying
pH values) showed that WMTA is affected less by changes in dye pH;
however, more dye extension was observed through the material rather
than at the tooth-to-material interface (21). Another dye leakage study
with India ink showed penetration of dye through WMTA (16).
Storage of the teeth in formalin before a dye study might also affect
leakage results. An investigation on various root-end filling materials re-
vealed that storing the teeth in formalin for 4 weeks significantly
decreases the amount of dye leakage in comparison to that observed
with freshly extracted teeth (15). A recent investigation evaluated the
effect of the presence of cracks on sealing ability and quality of filling
with a GIC or WMTA as root-end filling materials. Both materials showed
similar sealing ability, but WMTA had better obturation quality in
comparison to Fuji IX (88).
On the basis of available information, it appears that MTA is one of
the most resistant root-end filling materials to dye penetration. Different
factors influence MTA leakage. They include thickness of the dentinal
wall (18), the dye pH (19), the type of dye (12, 14, 20), pretreatment
with chelating agents (13), the tooth storage environment before the
experiment (15), and the setting status of MTA before its placement
in the dye (16).
Fluid Filtration. Fluid filtration investigations showed the superi-
ority of MTA compared with amalgam (22–25) and Super EBA (24).
In an investigation with 2 different cavity preparation techniques
(erbium:YAG laser and ultrasonic), IRM, Super EBA, and MTA were
used as root-end filling materials. MTA showed significantly less leakage
than the other test materials when a laser was used for cavity preparation
(26). Another fluid filtration study evaluated the sealing ability of MTA
when used as a root canal filling material followed by root-end resec-
tion. No significant leakage was observed when at least 3 mm of MTA
remained after root-end resection. However, the authors reported
significantly more leakage when 2 mm or less thickness of MTA re-
mained after root-end resection (27).
De Bruyne et al (28) used fluid filtration and capillary flow poro-
metry for comparing leakage of WMTA with Fuji IX and IRM. After 6
months, Fuji IX leaked significantly less than IRM and MTA. The
same investigators repeated the study with capillary flow porometry
and reported that IRM and WMTA leak significantly less than Fuji IX
(29). The authors attributed the conflicting results to the size of the
root-end cavity preparations and the use of human teeth in the first study
JOE — Volume 36, Number 2, February 2010
Review Article
in contrast with bovine teeth in their second study (29). Nakamichi et al
(30) compared the adhesive strength of 5 dental materials to bovine and
human teeth. They found no statistically significant difference between
human and bovine teeth, although the mean values were always slightly
lower for bovine teeth.
Most fluid filtration investigations have shown the superiority of
MTA as a root-end filling material in comparison to other currently
used root-end filling materials.
Protein Leakage. Valois and Costa (31) used bovine albumin for
evaluating the influence of the thickness of MTA on the sealing ability
of this material as a root-end filling material. A 4-mm thickness of MTA
leaks significantly less than lesser thicknesses of the material. Another
study evaluated the effect of pH on protein leakage of WMTA (32). Teeth
that were stored in acidic environments had significantly lower resistance
to leakage than teeth that were stored under high pH conditions.
On the basis of limited available data and using protein as a tracer,
it appears that a thin layer of MTA and acidic conditions adversely influ-
ence the leakage of MTA to protein leakage.
Bacterial Penetration. A large number of bacterial penetration
investigations have been performed on MTA, comparing it with other
currently used root-end filling materials (33–40). These studies have
used different species of microorganisms to test bacterial penetration.
The majority of the studies have compared amalgam and MTA (33–35,
40); most of them showed that MTA is more resistant to bacterial pene-
tration than amalgam (33, 34, 40). In contrast, one investigation re-
ported no significant difference in bacterial penetration between the
2 materials (35). Many bacterial penetration studies demonstrated
the superiority of MTA to Super EBA (33, 34, 38). Conversely, others
have shown no significant difference between the 2 materials (36, 37).
In an endotoxin leakage study, MTA was more resistant to leakage
than amalgam, Super EBA, and IRM in most time intervals tested (41).
Two investigations compared the bacterial penetration of IRM with
MTA, showing the superiority of MTA (33, 34). Other studies showed
no significant difference between MTA and Geristore (36), hydroxyap-
atite (HA) (37), a composite, amalgam with Pro-Bond dentin bonding
agent (35), and Resilon (38).
In an investigation of WMTA as a root-end filling material, the
material was contaminated with blood, saline, and saliva. The saliva-
contaminated samples showed significantly more bacterial leakage in
comparison with uncontaminated WMTA (39).
In an investigation simulating an environment similar to that of the
human body, researchers immersed teeth with MTA as a root-end filling
either in PBS or in normal saline for 1 month and showed formation of
HA over MTA in all teeth placed in PBS and significantly higher resis-
tance to Enterococcus faecalis penetration in this group in comparison
to teeth placed in normal saline (43).
Available data showed that the type of microorganism and the
length of leakage evaluation directly influence the results of bacterial
leakage investigations. A meta-analysis of the results of current studies
showed that MTA prevents bacterial and dye penetration better than
Super EBA, amalgam, or IRM (44).
Leakage of MTA as a Perforation Repair Material
Many restorative materials have been used for repairing furcation
perforations (45). Various in vitro methods have been used to
compare MTA with these materials (46–55).
Dye Leakage Investigations. The superiority of MTA over IRM
and amalgam was initially established in a study that used MTA as an
endodontic filling material for repairing lateral root perforations
(46). Gray MTA (GMTA) has superior sealing ability as a perforation
MTA: Leakage and Biocompatibility Investigations 191
Review Article
repair material compared with Vitrebond (a modified GIC) (48), Cavit-
W, and tricalcium phosphate in conjunction with AH26 (50).
Hamad et al (53) used a dye extract method (using a spectropho-
tometer) and compared WMTA and GMTA as furcation perforation
repair materials; no significant difference was found between the 2
materials. Greater dye penetration was observed in an orthograde direc-
tion (from coronal to apical) than in a retrograde direction (from
furcation to coronal portion).
One of the drawbacks of MTA is its long setting time. For this
reason, many investigations are searching for permanent filling mate-
rials that can be placed over MTA without interfering with its setting
properties for 1-visit furcal perforation repair. Nandini et al (89) re-
ported that placement of GIC 45 minutes after placing WMTA does
not interfere with MTA setting, and calcium salt can be formed in the
interface of both materials.
In another dye extraction investigation, an internal matrix was
used before placing various furcation repair materials (55). The pres-
ence or absence of an internal matrix has no significant effect on the
leakage of GMTA, whereas it does reduce leakage of IRM. MTA samples
leak significantly less than IRM, regardless of the use of an internal
matrix. Hamad et al (53) have shown that using a collagen matrix
does not prevent MTA overextension as a perforation repair material.
Most studies showed that MTA is resistant to dye penetration when
used as a perforation repair material. In addition, the presence of
a matrix beneath the material has no significant effect on its resistance
to dye penetration.
Fluid Filtration. An investigation compared Super EBA, MTA, and
a combination of both to repair furcation perforation (49). All test
materials sealed the perforations well. The Super EBA group showed
significantly less leakage than the other groups only at the 24-hour
observation period. Another investigation compared sealing saucer-
shaped furcation perforations by using MTA, One-Up Bond, and MTA
with a secondary seal of One-Up Bond or Super EBA (50). Fluid filtra-
tion was used for leakage evaluation. After 24 hours, MTA demonstrated
significantly more leakage than the other groups. However, after 1
month there was no significant difference between the test materials.
These investigations showed that when MTA is used as a perfora-
tion repair material, it has no superiority over other materials when
tested with the fluid filtration technique.
A recent fluid filtration investigation examined the effect of
different irrigation regimens on the sealing ability of WMTA and showed
that using sodium hypochlorite with a mixture of tetracycline, an acid,
and a detergent (MTAD) or ethylenediaminetetraacetic acid (EDTA)
significantly increases leakage in comparison to using only NaOCl or
no irrigation before placing WMTA for perforation repair (90). Another
study showed that WMTA partially dissolves when it remains in contact
with BioPure MTAD for 5 minutes (91).
Bacterial Leakage Studies. MTA has been shown to be superior
to amalgam when challenged by a bacterial leakage model using
Fusobacterium nucleatum (47). Another investigation showed no
significant difference between GMTA and WMTA after 60 days when
they were used as furcation repair materials (51). De-Deus et al
(54) compared MTA and PC as perforation repair materials and found
no significant difference between the two.
On the basis of limited available data, it can be concluded that MTA
is superior to amalgam as a perforation repair material, and that there is
no significant difference between MTA and Portland cement (PC).
Other Leakage Methods. Zou et al (56) used a glucose leakage
test for evaluating GMTA as a perforation repair material. Calcium sulfate
and CollaPlug were used as a barrier (matrix) beneath GMTA. Neither of
the barrier materials simultaneously improved the seal and prevented
192 Torabinejad and Parirokh
overextension of GMTA. The reliability of this method is questionable
because MTA produces CH after hydration, which can react with glucose.
The results of a recent study confirmed that the glucose model should not
be used for evaluating leakage of MTA, PC, and CH (57).
Leakage of MTA as a Coronal Plug Material
Two bacterial leakage investigations compared WMTA and GMTA
with Fuji II and Fuji Triage glass ionomer as a coronal plug and reported
no significant difference among the tested materials (58, 59). However,
a dye comparison of GIC and GMTA as an orifice plug showed that GMTA
is more resistant to dye penetration than GIC (60).
In a dye leakage study using India ink, Jenkins et al (61) compared
various depths of Cavit, MTA, and Tetric composite as orifice plugs. The
results of this study showed that Tetric composite is significantly more
resistant to dye penetration, irrespective of orifice depth. In a saliva
leakage penetration study comparing MTA, Geristore, Fuji Plus, and
amalgam as orifice plugs, all test materials exhibited leakage in some
specimens at 90 days (62).
Data regarding the effectiveness of MTA as a coronal plug are
limited, and more studies are needed.
Leakage of MTA as an Apical Plug Material
Traditionally, apexification with CH has been the treatment of
choice for teeth with necrotic pulps and open apexes (92). Generally,
it is accepted that treatment time is too long, takes more visits, costs
more, and increases the susceptibility of the tooth to fracture (92–
94). In addition, the patient might experience periradicular necrosis
after CH therapy (95). Shabahang and Torabinejad (96) described
the use of MTA as an apical barrier in teeth with open apexes.
Dye Leakage. The results of dye leakage studies with MTA as an
apical barrier are in favor of GMTA (63, 97). In a dye leakage investi-
gation comparing WMTA and GMTA as an apical barrier, the latter mate-
rial showed significantly less leakage (97). Gutta-percha obturation
after MTA has set showed less dye leakage in comparison with 1-step
MTA insertion and immediate gutta-percha obturation. The drawback
of this study (97) is the fact that MTA decolors methylene blue dye (20).
Another experiment comparing WMTA and GMTA as an apical
barrier used CH medication before MTA placement (63). Results
showed that pretreatment with CH adversely affects the seal of WMTA.
A recent investigation reported unfavorable effect of a high alkaline envi-
ronment on the MTA surface (98). In contrast to this finding , the results
of a bacterial leakage study showed that pretreatment with CH has no
significant effect on sealing ability of MTA as an apical barrier (99).
Available data suggested that inserting GMTA as an apical barrier
and obturating the rest of the canal space later with gutta-percha
improve resistance to dye penetration.
Fluid Filtration. Martin et al (64) compared leakage resistance of
various thicknesses of WMTA by using a fluid filtration technique. Teeth
whose root canals were filled completely with WMTA leaked signifi-
cantly less after 48 hours. However, no significant difference was
observed among the groups after 4 weeks. Immersion of the teeth in
PBS during the study time (4 weeks) might have contributed to
decreased leakage over time, along with the formation of HA crystals
over the MTA samples (100, 101).
Bacterial Penetration. Hachmeister et al (99) investigated CH
pretreatment in advance of MTA plug insertion and reported that
most of the teeth with or without CH pretreatment showed bacterial
penetration during the first 10 days of the study. By day 70, all MTA
barriers showed bacterial penetration. In contrast, only 20% of control
samples that had been filled in a retrograde manner revealed bacterial
leakage at the same time. On the basis of these findings, the authors
JOE — Volume 36, Number 2, February 2010

concluded that the method of insertion might play an important role in
bacterial penetration when MTA is used as an apical barrier.
The use of ultrasonic to increase the sealing ability of MTA is
a matter of debate among investigators. An investigation compared
using ultrasonic and non-ultrasonic placement of MTA as an apical
barrier (102). Ultrasonic placement resulted in a significant reduction
of bacterial penetration after 45 days. However, after 90 days no signif-
icant difference was observed between the groups (102). In contrast,
a recent bacterial leakage investigation showed significantly lower E.
faecalis penetration after use of MTA as an apical barrier with ultra-
sonic activation (103).
de Leimburg et al (65) used a polymerase chain reaction (PCR)
for bacterial leakage detection. They showed no significant difference
between 1-mm, 2-mm, and 3-mm thicknesses of MTA as an apical
barrier in teeth with open apexes. In contrast, one investigation demon-
strated that only a 5-mm thickness of MTA adequately prevents bacterial
penetration when used as an apical barrier (66).
Many factors influence bacterial penetration of MTA when it is
used as an apical barrier in teeth with open apexes (42, 65, 66, 101,
102), including the type of bacteria, methods of MTA insertion and
compaction, the bacterial leakage evaluation method, thickness of the
MTA, and length of study time.
Leakage of MTA as a Root Canal Filling Material
Vizgirda et al (67) compared lateral condensation, thermoplasti-
cized gutta-percha, and MTA as root canal filling materials in bovine
teeth. Both gutta-percha obturation techniques showed less methylene
blue penetration than MTA. In contrast, another experiment comparing
GMTA, WMTA, and gutta-percha with sealer as root canal filling
materials by using a saliva leakage model on human teeth showed signif-
icantly less leakage in GMTA samples than in those filled with gutta-
percha and sealer (68).
There are 3 important differences between the methods of these
studies. First, Vizgirda et al (67) used a lentulo spiral for MTA place-
ment, which has not been previously recommended for MTA placement
(69, 101, 104). Second, there are many investigations that have ques-
tioned the similarity of human and bovine teeth in terms of dye leakage
(70), dentin tubule size (71), and diffusion of cations and liquids (76,
77). Third, these studies used different methods (dye, bacteria, saliva)
for leakage evaluation (67, 68).
A fluid filtration study reported significantly less leakage after 48
hours when WMTA was used as a root canal filling material compared
with an apical plug and gutta-percha insertion. However, no significant
difference was reported after 4 weeks (64).
Chogle et al (105) evaluated the sealing ability of WMTA as a root
canal filling material. After a 3-mm root-end resection, the apical 9 mm
of each root was filled with WMTA. The samples that were incubated for
2 and 7 days demonstrated significantly less bacterial leakage than those
incubated for 4 hours.
MTA can be used as a root canal filling material, although clini-
cians should be aware of some of its limitations such as difficulty in
controlling the length of the filling, the chance of producing voids,
and the absence of a known solvent for MTA removal.
Marginal Adaptation of MTA
In several investigations, MTA has shown better marginal adapta-
tion than IRM, Super EBA (74, 75), amalgam (74, 76), and GIC (21). In
one investigation comparing marginal adaptation of MTA and Super EBA
as root-end filling materials (77), samples were subjected to in vitro
chewing cycles in a computer-controlled chewing simulator. Marginal
adaptation was controlled before and after testing. Both materials
JOE — Volume 36, Number 2, February 2010
Review Article
showed no significant change after occlusal loading, although margin
continuity was slightly decreased, particularly for Super EBA. Although
bur-finishing techniques do not significantly improve MTA marginal
adaptation after root-end filling (75), MTA has better marginal adapta-
tion than amalgam when observed under both high- and low-vacuum
SEM (76).
Gandolfi et al (78) compared 2 new cements with WMTA by using
SEM. Their evaluation of WMTA samples showed limited marginal gaps.
Another recent study compared the marginal adaptation of WMTA,
GMTA, and PC (106). Results showed no significant difference between
the tested materials, although GMTA showed better marginal adaptation.
In contrast to these findings, another investigation compared marginal
adaptation of WMTA, IRM, and Super EBA (16) and reported that IRM
has the best marginal adaptation, whereas WMTA is prone to washing
out and exhibits a higher number of underfilled specimens.
The type of SEM used for evaluation of marginal adaptation and
methods for preparation of the samples are very important in SEM
studies. In an SEM study, Shipper et al (76) compared MTA with amalgam
by using high- and low-vacuum conditions. The results of this study
demonstrated that the size of the gap between the root-end filling material
and the margin of the root-end cavity is smaller under the low-vacuum
microscope. They attributed this finding to examination of the samples
without standardized preparations and in moist conditions.
A number of investigations have shown that MTA has better adap-
tation to tooth structure than Super EBA, IRM, and amalgam. Test condi-
tions can influence the results of these investigations.
Leakage Studies and Marginal Adaptation of Other Types
of MTA
A dye leakage study compared Angelus MTA (AMTA), zinc-free
amalgam, Vitremer (a resin-modified GIC), and Super EBA (10). The
results showed that AMTA presented the best seal among all of the tested
materials. Use of 1% methylene blue was a shortcoming of this study
because of the decoloration effect of MTA on the dye (20). Another
dye and marginal adaptation investigation compared AMTA, Vitremer,
and Super EBA as root-end filling materials (79). The authors also
prepared 3 transverse slices from each sample and assessed the amount
of dye leakage in each slice separately. Super EBA showed significantly
less leakage than AMTA and Vitremer in most apical sections. However,
in other sections, no significant difference was found between AMTA
and Super EBA. AMTA showed significantly smaller gaps between the
material and the root-end cavity than Vitremer and Super EBA when
examined under SEM. Statistical analysis revealed no correlation
between the dye penetration and the marginal adaptation (79). An
SEM investigation has reported similar marginal adaptation when white
Portland cement (WPC) was compared with white AMTA (AWMTA) as
root-end filling materials (107). Another investigation showed no
significant difference between AMTA and a GIC as root-end filling mate-
rials in a dye leakage model (108).
Another investigation compared AWMTA and WMTA as apical
barrier for open apex teeth. Results showed no significant difference
in dye penetration (109). However, the major drawback for this study
was use of methylene blue dye for assessing leakage of the materials
(20, 82).
Another study evaluated the effect of calcium chloride (CC) on the
sealing ability of WMTA, AWMTA, and a modified WPC as root-end filling
materials. Results showed that the addition of 10% CC improves the seal-
ing ability of all 3 tested materials (110).
Two experiments evaluated the effect of a laser on AMTA as a root-
end filling material (80, 81). The first study showed that diode laser
irradiation does not improve the apical seal of AMTA (80). A dye
MTA: Leakage and Biocompatibility Investigations 193
Review Article
leakage study compared a combination of using Er,Cr:YSGG laser and
an AMTA root-end filling with laser and cyanoacrylate (81). The results
of this study revealed that using the laser adversely affects leakage of
AMTA as a root-end filling material.
Hashem and Hassanien (55) recently compared AMTA with IRM
and GMTA as perforation repair materials. GMTA samples showed the
least amount of dye leakage. In addition, their results showed that the
presence of an internal matrix had no influence on the amount of
dye leakage of GMTA. However, a gray AMTA (AGMTA) demonstrated
a better seal when an internal matrix was used for this material.
A fluid filtration investigation compared PC, AWMTA, and MTA-Bio
as a perforation repair material (111). The results of this study showed
no significant difference between the tested materials, and none of them
could develop a fluid-tight seal. A dye extraction study testing perfora-
tion repair materials demonstrated that AMTA without an internal
barrier matrix absorbs significantly more dye than GMTA with or
without an internal matrix (55). The methods and materials of this study
have been questioned because they used 2% methylene blue, which
interacts with MTA during the experiment (20, 82).
Compared with MTA, there are a limited number of studies related
to the sealing ability of AMTA as a root-end filling or perforation repair
material.
A recent investigation reported that various types of MTA and WPC,
with or without CC, form an interfacial layer between the material and
dentin, which might affect their sealing ability (112).
Leakage Studies and Marginal Adaptation of New
Compositions of MTA
Pelliccioni et al (83) compared WMTA mixed with sterile water as
recommended or dry MTA for filling root-end preparations. They used
the fluid filtration method for measuring microleakage and reported
no significant difference between the 2 groups, except for the 1-week
samples. Nonetheless, it has been shown that when MTA is placed under
dry conditions, a complete set can be delayed (113), compromising the
biocompatibility and bioactive properties of MTA, including the release of
the calcium ions, production of CH, and a delayed increase in pH value.
Two separate dye and fluid filtration investigations evaluated the
sealing ability of MTA mixed with 10% CC when used either as an apical
barrier or as a root-end filling material (110, 114). Both investigations
reported significantly better results when MTA is mixed with 10% CC
rather than with sterile water.
A dye leakage investigation compared chlorhexidine (CHX) mixed
with water as a vehicle for WMTA and GMTA (84). Results revealed no
significant difference between the 2 groups. However, MTA’s compressive
strength decreased after mixture with CHX (115). Peters and Peters (77)
used SEM and showed that the marginal adaptation of MTA used as a root-
end filling material might be slightly affected after occlusal loading.
There are limited data available regarding leakage and marginal
adaptation of the new versions of MTA.
MTA as Root Filling Material and Root Canal Sealer
A recent form of MTA was introduced as ProRoot Endo Sealer
(Dentsply Tulsa Dental Specialties), which is a calcium silicate–based
endodontic sealer. The liquid component consists of a viscous aqueous
solution of a water-soluble polymer (116). An investigation comparing
ProRoot Endo sealer with AH Plus and pulp canal sealer either stored in
tissue synthetic fluid or without storing reported higher push-out
strength for the former material (117).
A fluid filtration investigation on sealing ability of a new form of
MTA, namely ProRoot Endo Sealer, as root canal sealer showed signif-
icantly less leakage in ProRoot Endo Sealer samples in comparison to
194 Torabinejad and Parirokh
pulp canal sealer but no significant difference between the pulp canal
sealer and AH Plus root canal sealer (116).
Removing smear layer always is a matter of debate among
endodontists. Results of a meta-analysis based on in vitro investigations
showed superior apical seal after removal of smear layer before root
canal obturation (118). However, a recent long-term fluid filtration
investigation on MTA as root canal filling material showed significantly
higher leakage when smear layer was removed before placing the mate-
rial (119). An investigation has reported EDTA might influence MTA
setting (120). The higher leakage in smear removed group might be
because of reaction between MTA and residual EDTA inside the root
canal (119).
A coronal bacterial leakage investigation reported no E. faecalis
penetration in teeth that had AWMTA as a root canal filling material.
A major drawback of this particular study, however, was the duration
of the investigation, which was too short (30 days) (121).
A recent dye leakage investigation used AGMTA and CPM sealer
(Egeo S.R.L., Buenos Aires, Argentina) and MBPc sealer (School of
Dentistry, University of São Paulo, São Paulo, Brazil) as an apical
plug and reported no significant difference between these materials
(122). The major drawback for this study was using EDTA for removing
the smear layer before MTA placement (119, 120).
Biocompatibility
Materials used in endodontics are frequently placed in intimate
contact with the periodontium and thus must be nontoxic and biocom-
patible with host tissues. There are several in vitro and in vivo tests to
evaluate the biocompatibility of dental materials. They include testing
the general toxicity profile of potential materials in a cell culture,
implantation tests, and usage tests in experimental animals according
to accepted clinical protocols. A number of biocompatibility and muta-
genicity studies have shown that MTA is a biocompatible material (123–
164). In fact, the results of a meta-analysis on MTA biocompatibility
showed that MTA is more biocompatible than Super EBA, IRM, and
silver amalgam (44).
Mutagenicity
The Ames mutagenicity test was used to assess the mutagenicity of
MTA (129). This test uses strains of Salmonella typhimurium LT-2,
which are sensitive to various classes of mutagens. Results of this study
determined that MTA is not mutagenic.
Neurotoxicity and Neurologic Effects
With murine cerebral cortical cells (130), neurotoxic effects of
MTA, Diaket, amalgam, and Super EBA were compared on both glial
and neuronal cultures. Results showed that all of the materials except
MTA are toxic in either freshly mixed or set conditions.
Nociceptive and anti-nociceptive effects of WMTA were evaluated
in a rat animal model. WMTA did not irritate nerve tissues and was
more effective in relieving orofacial nociceptive pain of formalin injec-
tion in comparison to eugenol (131).
Vascular Effect
Two different models have evaluated the effect of MTA on vascular
tissues (132, 133). One investigation on rabbit ear chambers evaluated
the effect of MTA on microcirculation (132). The results of this inves-
tigation showed that 4 weeks after MTA placement, microcirculation
was completely restored, and new vessels were formed. Another exper-
iment used a rat aortic ring model to simulate the pulpal vessels’ smooth
muscle contraction (133). The results of this investigation showed that
MTA induces vessel contraction in a dose-dependent manner.
JOE — Volume 36, Number 2, February 2010

On the basis of current information, it can be concluded that MTA
is nonmutagenic and non-neurotoxic and does not produce a side effect
on microcirculation, despite the fact that it can influence a vessel’s
contraction.
Cell Cultures
Cell culture studies evaluate cytotoxicity through morphologic
observation of the cultured cells in the vicinity of the test materials or
their extracts, recording the number of detached cells, alkaline phos-
phatase activity, SEM observation, fluorescent measurements, and cell
viability (123–127, 134–166).
By using various cell culture systems, a number of investigations
have shown that MTA is one of the least cytotoxic dental materials
(123–164). Torabinejad et al (134) have shown that freshly mixed
and set MTA and amalgam are less cytotoxic than Super EBA or IRM.
Another study on human periodontal ligament (PDL) cell cultures
compared MTA with amalgam and Super EBA (136). Results indicated
that freshly mixed MTA exhibits lower cytotoxicity than Super EBA or
amalgam. Twenty-four hours after mixing at a higher extract concentra-
tion, MTA showed the least amount of cytotoxicity of all the tested mate-
rials; at a lower extract concentration, Super EBA showed higher
cytotoxicity than amalgam or MTA.
Cytotoxicity and cell attachment investigations with various cell
cultures showed better results with MTA in comparison to amalgam
(135, 137, 138, 140, 144), gallium GF2 (135), Super EBA (135,
144, 147, 151, 155), IRM (137, 138, 156, 161), various types of glass
ionomers (135, 149, 155, 157), gutta-percha (155), N-Rickerts (155),
Diaket (157), a cyanoacrylate-based adhesive dental cement (157), Life
(147, 151, 162), and Dycal (163).
In contrast to these studies, an experiment with mouse fibroblast
and macrophage cells on freshly mixed and set MTA, IRM, amalgam and
Retroplast (166) found no significant difference between amalgam and
MTA. The total cell number in freshly mixed or set Retroplast was signif-
icantly less than both MTA and amalgam. The cytotoxicity of MTA is
similar to chemically inert titanium alloy (140). In another study on
murine pre-osteoblastic clone cell cultures, MTA and Super Bond resin
showed no significant difference in cell adhesion and proliferation
(156). Results of an observational study examining various root-end
filling materials on gingival fibroblast cells showed greater cell attach-
ment to Geristore in comparison to MTA (165). In a study on rat bone
marrow cells, MTA did not inhibit cell growth but suppressed osteo-
blast-like cell proliferation (146).
Because of differences in the chemical composition of WMTA and
GMTA (167, 168), several investigations have been performed on the
cytotoxicity of both types of MTA (124, 139, 150, 154). There is
disagreement among investigators regarding the cytotoxicity of WMTA
and GMTA (124, 139, 150, 154). One study using osteoblast and
MG-63 osteosarcoma cells determined that after initial cell attachment
to the WMTA surface after 13 days, the former cell type could not survive
over WMTA, whereas they did grow over GMTA during the same period
of time (139). In contrast, an investigation comparing the incubation of
WMTA and GMTA with oral keratinocyte and cementoblast cell cultures
showed greater proliferation of both cell types on the WMTA surface in
comparison with GMTA (154).
Another investigation on osteoblast cell cultures compared 1-
day and 28-day cured samples of WMTA and GMTA (124). Overall,
1-day cured samples of both GMTA and WMTA showed more
biocompatibility than the 28-day cured samples. In contrast to these
results, another experiment found that 12-day cured GMTA signifi-
cantly increases cell proliferation in comparison with 1-day cured
GMTA (154). Two other studies with human alveolar bone, fibro-
blast, and macrophage cell cultures showed no difference between
JOE — Volume 36, Number 2, February 2010
Review Article
GMTA and WMTA in terms of cytotoxicity and cell proliferation
(123, 150).
A recent investigation has shown that the survival rate and morpho-
logic appearance of cementoblasts are not affected by low concentration
of WMTA (169). However, a high concentration of the material (20 mg/
mL) decreased cell viability and inhibited mineralization as well as bone
sialoprotein production by cementoblasts.
MTA releases significantly more calcium ions than Dycal (152). In
the absence of MTA, when 0.3 mmol/L of CC is added to the cell culture,
the same pattern of cell proliferation is observed. On the basis of these
results, the authors concluded that the continuous release of ions from
MTA provides an optimum amount of calcium for cell proliferation.
A recent investigation examined the effect of MTA and CH on 3T3
fibroblast cells and showed that MTA has a significantly shorter duration
of cytotoxicity in comparison to CH (170).
In a human PDL cell culture study on root ends that were filled with
MTA or gutta-percha and treated with a CO2 laser, SEM observation indi-
cated no cell attachment to either the laser-irradiated area or gutta-per-
cha (141). In contrast, cell attachment to MTA and the root surface area
without laser irradiation was observed.
PDL fibroblasts show enhanced proliferation on MTA and survival
on amalgam when compared with gingival fibroblasts (142). The authors
also reported that MTA induces an osteogenic phenotype, which reflects
up-regulation of the expression of alkaline phosphatase, osteonidogen,
osteonectin, and osteopontin. Twenty-four–hour cured MTA shows
a more favorable response to PDL fibroblast cell types than freshly mixed
MTA (142). On the other hand, WMTA induces proliferation of murine
odontoblast-like and undifferentiated pulp cells, with no noticeable
difference between fresh and premixed WMTA (148). Investigators re-
ported that WMTA induces more DNA synthesis in both cell types.
In an experiment on the antiproliferative effects of dental materials,
Koulaouzidou et al (145) compared MTA, zinc oxide–eugenol (ZOE)
cement, and glass ionomer on fibroblast cell lines (L929, BHK21/C13,
and RPC-C2A). They determined that MTA exhibits the lowest antiproli-
ferative activity, whereas ZOE has the highest antiproliferative activity.
A study comparing WMTA, Resilon, and gutta-percha on primary
osteoblast and osteoclast cultures showed that none of the materials led
to any significant osteoclast formation (160). Another investigation
compared WMTA, Sealapex, and Roth 801 on macrophages and gingival
fibroblasts (153). Only WMTA showed no adverse effect on the viability
of either cell line, and none of the materials tested increased the release
of prostaglandin E2 (PGE2). A recent experiment comparing the cyto-
toxicity of CH and GMTA (161) demonstrated that CH produces more
cytotoxicity and decreases cell metabolic activity approximately 3 times
more than GMTA.
By using rat dental pulp cell cultures, Yasuda et al (163)
compared MTA, Dycal, and an adhesive resin cement. Their data indi-
cated that MTA has no cytotoxicity after 72 hours, and it not only signif-
icantly increases mineralization by stimulating dental pulp cells but also
increases the amount of bone morphogenetic protein-2 (BMP-2)
production. In contrast, Dycal decreases BMP-2 production and
increases cell death, whereas dentin adhesive cement has no effect
on cytotoxicity and BMP-2 production.
Several cell culture studies have shown production of type I
collagen and osteocalcin expression in the presence of MTA (144,
158, 163, 171).
Two recent studies have confirmed cementoconductivity, cemen-
toinductivity, and osteoconductivity of WMTA (169, 171).
Cell culture studies on MTA showed that the cell response to the
material depends on many factors such as the cell types and the choice
of study duration (139, 142, 154, 155), use of a fresh or cured material
(124), frequency of changing the medium (123), the use of direct
MTA: Leakage and Biocompatibility Investigations 195
Review Article
contact or extract of MTA (123, 162), and the concentration of the
material in the cell culture media (169).
Cell Culture and Genotoxicity Studies of Other Types of
MTA
Results of 3 cell culture studies determined that both AGMTA and
AWMTA exhibit no cytotoxicity and genotoxicity on various cell lines
(172–174). Two other studies confirmed these results and showed
that PC and WPC, as well as AMTA, have no cytotoxicity and genotoxicity
in concentrations of 1 to 1000 mg/mL for 1-hour exposure at 37 C
(175, 176).
De Deus et al (177) showed that both GMTA and AMTA signifi-
cantly inhibit endothelial cell viability during the first 24 hours.
However, at 48 and 72 hours, no significant difference was found
between control cell cultures and the 2 experimental materials. Two
separate investigations indicated that both ProRoot MTA and AMTA
have cytotoxic effects on the cell viability of V79 fibroblast (155) and
human gingival fibroblasts (159).
A recent cell culture investigation showed that both AWMTA and
WMTA are relatively inert and superior in cell viability in comparison
to Vitrebond and Super EBA (178). Another study compared GMTA
with AWMTA in a L929 cell culture and reported that both types of
MTA were superior in cytotoxicity to a CH base cement. The major draw-
back of the study was mixing both types of MTA in a 1:1 ratio of powder
to liquid (179). Another cell culture investigation has shown that AMTA
had the least cytotoxicity compared with some current pulpotomy agents
such as Buckley’s formocresol, CH, and ferric sulfate solution (180).
An investigation that compared AGMTA with AWMTA and castor oil
reported no significant difference among these materials; none showed
a cytotoxic effect on transfected human pulp cells (181).
On the basis of these studies, it appears that both AMTA and MTA
have similar effects on cell cultures.
Cell Culture Studies of New Compositions of MTA
Many investigations have tried to change the MTA powder compo-
sition or substitute distilled water with other liquids in the MTA mixture
(89, 110, 115, 182–188). The rationale for these various combinations
includes a possible increase in antimicrobial activity, a decrease in
setting time, or improved ease of handling of this material. A number
of investigations have been performed to evaluate the cytotoxicity of
the new compositions of MTA (189–193).
Mixing WMTA with 0.12% CHX significantly increases mouse fibro-
blast and macrophage apoptosis in comparison with a mixture of WMTA
and sterile water (192). Karimjee et al (193) mixed K-Y jelly (Johnson
& Johnson) with WMTA powder and examined its cytotoxicity on human
PDL cells. Their results showed no significant difference between WMTA
mixed with water and WMTA mixed with K-Y jelly. A mixture of 15%
Na2HPO4 solution and WMTA cultured on L929 cells showed no signif-
icant difference in cell viability after 1 and 7 days (189).
An L929 cell culture investigation tested the effect of various addi-
tives such as water, saline, 2% lidocaine, 5% CaCl2, 3% NaOCl gel, and K-
Y liquid on set and freshly mixed WMTA and GMTA cytotoxicity. Except
for 3% NaOCl gel, the rest of these additives had no significant effect on
MTA cytotoxicity for time intervals up to 48 hours (190).
A recent investigation reported that a combination of enamel
matrix derivative with MTA improves human dental pulp cell differenti-
ation, alkaline phosphatase activity, dentin sialophosphoprotein, bone
sialoprotein, and mineralization (191). The investigators suggested this
combination as a pulp capping agent (191).
From the results of these studies, it can be concluded that
improving antibacterial properties of MTA when mixed with CHX
196 Torabinejad and Parirokh
adversely affects the material’s biocompatibility. Comprehensive evalu-
ations should be performed before recommending new compositions
for clinical applications. Because responses of various cell types are
different to MTA in various cell culture environments, investigators
should carefully select cell types that would be in contact with MTA
for clinical applications of this material (139, 142, 154, 155).
Production of Cytokine and Signaling Molecules
Cell Culture Studies. Cell response to the presence of MTA or MTA
extracts has been extensively investigated (138, 140, 158, 159, 162, 164–
166, 194–202). Up-regulation of various types of cytokines and biologic
markers has been reported in the presence of MTA in several cell culture
studies when compared with control or other tested materials. These
cytokines and biologic markers include interleukin (IL)-1a (164,
195), IL-1b (164, 194, 195), IL-2 (198), IL-4 (198), IL-6 (164,
194–196, 203), IL-8 (196), IL-10 (198), IL-18 (194), osteocalcin
(138, 158, 171, 194, 195), alkaline phosphatase (138, 171, 191),
bone sialoprotein (171), osteopontin (142, 171), and BMP-2 (159).
In an investigation on cytokine up-regulation, 3 variants of MTA
were compared with Dycal, dental plaster, HA, and Biogran. In contrast
to the findings of Koh et al (164, 195), there was no evidence of IL-1a
expression in the cell culture (196, 203).
Huang et al (198) compared the effect of a CH-based material
(Life), Super EBA, and MTA on inflammatory cytokines. Their results
determined that the amount of IL-4 and IL-10 is significantly greater
in the presence of MTA than in the presence of the other 2 materials.
Separate studies have shown that macrophage colony-stimulating
factor up-regulation is not specific to MTA, and its production occurs in
the presence of other dental materials (164, 196). Guven et al (159)
found no significant difference between the control and GMTA group
when they compared the up-regulation of transforming growth
factor–b1 (TGF-b1) derived from human gingival fibroblasts.
Tomson et al (200) investigated the effect of soluble components of
set and setting WMTA and GMTA on the release of signaling molecules.
Their results showed that both types of MTA liberate glycosaminoglycans
and noncollagenous proteins, adrenomedullin (ADM), and TGF-b1.
GMTA liberates more ADM, whereas WMTA liberates more TGF-b1.
In contrast to the above-mentioned experiments, 2 investigations
revealed that MTA does not influence cytokine production when mouse
fibroblasts and macrophages are used (166, 199). Another experiment
reported that MTA has no influence on M1 and M2 macrophage phago-
cytosis (201).
Pistorius et al (140) used gingival fibroblasts and compared MTA
with amalgam and an inert titanium alloy to measure the amount of
cellular PGE2 synthesis. Their results showed that amalgam down-regu-
lates PGE2 synthesis, whereas titanium alloy and MTA up-regulate PGE2.
In a cell culture investigation, MTA up-regulated PGE2 production
and increased cyclooxygenase-2 and nitric oxide synthase mRNA
expression via activation of the nuclear factor kappa B signaling system
(204). The mitogen-activated protein kinase (MAPK) pathway in part
regulates cellular growth, division, differentiation, and death. By using
human osteosarcoma cell line U2OS, Huang et al (197) determined that
in the presence of MTA, extracellular regulated kinases (ERKs) are
increased in cell culture. Suppression of the ERK pathway by the
addition of an ERK inhibitor suggests that MTA’s effect on osteosarcoma
cells is induced by a cascade of ERK MAPK.
Tomson et al (200) compared the release of dentin components
when in contact with solubilized components of GMTA, WMTA, and CH.
The results of this study indicated that CH and GMTA release more ADM
than WMTA, whereas TGF-b1 is up-regulated in WMTA samples in
comparison to GMTA and CH samples.
JOE — Volume 36, Number 2, February 2010

Recently, Simon et al (202) reported the strong expression of type
I collagen and dentin sialoprotein (DSP) when media containing
extracts of MTA were added to pulp-derived cell cultures. Meanwhile,
Laurent et al (162) observed nestin expression and formation of
a mineralized matrix after adding a medium containing MTA extracts
to cell cultures. The presence of DSP in dentin bridges after pulp
capping has also been confirmed (205).
Another cell culture study on human alveolar osteoblasts reported
the expression of Runt-related transcription factor 2 (essential for oste-
oblast differentiation and bone formation) in the presence of both types
of GMTA and WMTA mixed with either sterile water or anesthetic solu-
tion (206).
An investigation using rat pulp cells and immunohistochemical
method compared the effect of WMTA with Dycal (207). Most of the
cells cultured with WMTA revealed heat-shock protein 25 as a marker
for odontoblast differentiation during pulp healing and higher mRNA
expression for both DSP and heat-shock protein 25.
Another investigation on mouse preosteoblasts exhibited produc-
tion of IL-6 in the presence of both types of GMTA and WMTA (203).
Conflicting results among the studies in regards to the effect of
MTA on cytokine production can be attributed to the type of cell culture,
the type of MTA and the cytokines investigated (140, 164–166, 194–
202). Most in vitro investigations have confirmed MTA as a cytokine
and signaling molecule productive material.
Animal Studies. Andelin et al (205) investigated the presence of
DSP after pulp capping with either MTA or BMP-7. Their results revealed
more staining for DSP in the calcified bridge of teeth that were capped
with MTA than with BMP-7. On the basis of the DSP-positive staining of
MTA-capped pulps, the authors concluded that hard tissue produced by
MTA has a closer similarity to dentin.
Ham et al (208) compared CH and MTA for expression of BMP-2
after apexification of immature teeth in monkeys. Both materials
produced similar BMP-2 expression. Kuratate et al (209) showed up-
regulation of osteopontin, nestin, and 5-bromo-2-deoxyuridine assay
(BrdU) immunopositive cells after pulp capping with WMTA in rats.
Studies showed that an increased concentration of some cytokines
and signaling molecules can cause adverse effects that include cell
apoptosis (209–213). It is therefore important that the release of bioac-
tive signaling molecules from the dentin matrix occur at an appropriate
amount, rate, and ratio (200). Investigations showed that HA forms over
MTA after the contact of this material with a simulated tissue fluid (100,
101). It should be emphasized that the amount of osteopontin (a mole-
cule that might have an important role in the differentiation and migra-
tion of new odontoblasts) should be at a certain level to encourage HA
formation (214, 215).
Human Study
Min et al (216) capped human third molar pulps with MTA or Dy-
cal and examined dentin bridge formation, expression of DSP, and
heme oxygenase-1 in the dental pulp. Results indicated the presence
of significantly more positive immunostaining in the MTA group than
in the Dycal group for DSP and heme oxygenase-1.
Both animal and human investigations have confirmed the encour-
aging role of MTA on the production of signaling molecules.
Production of Cytokines and Signaling Molecules by
Other Types of MTA
Macrophages produce different types of cytokines and inflamma-
tory products. A cell culture study evaluated the response of 2 mouse
macrophage cell lines to GMTA and AMTA (199). Results indicated
the absence of cytokine up-regulation by both cell lines.
JOE — Volume 36, Number 2, February 2010
Review Article
Gomes et al (217) investigated the effect of AMTA on polymorpho-
nuclear (PMN) migration by injection of AMTA suspension into the peri-
toneal cavity of mice. Their results determined that in the presence of MTA,
mast cells and macrophages produce many cytokines such as IL-1b,
macrophage inflammatory protein-2 (MIP-2), and LT-B4 that mediate
PMN migration in a time-dependent and dose-dependent manner.
Several substances have been known as vaccine adjuvants.
Immune adjuvants induce antibody response against protein antigens
(218). Aluminum hydroxide is the only adjuvant approved for human
use by the U.S. Food and Drug Administration. Because aluminum is
one of the elements that is released from MTA in direct contact with
tissue synthetic fluid (100), an animal study evaluated the effect of
AMTA on immunoglobulin G production against F. nucleatum and Pep-
tostreptococcus anaerobius (219). Because the activation of T-helper
cells is necessary for induction of specific responses against protein
antigens, the investigators assessed cytokines released from memory
T cells after a primary challenge with F. nucleatum or P. anaerobius.
They reported that AMTA had little or no effect on anti-inflammatory or
bone-destructive cytokines such as receptor activator for nuclear factor
kappa B ligand (RANKL), IL-10, and tumor necrosis factor–a, whereas
the material exhibited an adjuvant effect against F. nucleatum.
Guven et al (159) compared the effects of GMTA and AMTA on
TGF-b1 and BMP-2 production by human gingival cells; they showed
that both types of MTA stimulate cell growth. The amount of TGF-b1
at 24 and 72 hours was higher in the AMTA group than in the GMTA
group. In contrast, BMP-2 levels were higher in the GMTA group at
24 hours compared with AMTA. As previously indicated, higher levels
of TGF-b1 might have a down-regulatory effect on the proliferation of
some cell types (212).
Silva et al (220) evaluated remaining pulp tissue after partial pul-
potomy in murine teeth for mRNA expression in the presence of various
inflammatory cytokines. Their results indicated that AGMTA down-regu-
lates mRNA expression for CC5, IL-1a, and interferon-g. On the basis of
these results, it appears that AGMTA has anti-inflammatory effects.
Subcutaneous Reaction
Many studies have compared the subcutaneous reaction of experi-
mental animals to MTA and other materials such as amalgam, CH, Super
EBA, various root canal sealers, IRM, ZOE, cold ceramic and ethoxyben-
zoic acid (EBA) (132, 151, 221–229). Some studies reported a calcific
tissue response to MTA specimens (221–226). Most of these studies have
used the Von Kossa technique to detect the presence of calcified struc-
tures (221, 223–225). Their results showed the presence of Von
Kossa–positive structures around MTA after 1 week. Larger calcified
structures were present in MTA specimens at longer time intervals
(221, 224). Both GMTA (221–226) and WMTA (225) show calcified
structures around the implanted tubes. A similar subcutaneous response
is reported for PC, CH, and MTA (223). Holland et al (225) suggested
that all tested materials produce calcified tissue through the same
phenomenon, which is mediated by calcium released from the materials
when carbon dioxide is present in tissues. There are some studies that
have not reported the formation of calcified structures around implanted
MTA (151, 228–230) despite using the Von Kossa technique (231).
Moretton et al (222) determined that GMTA produces significantly
more inflammation in comparison to EBA. One study revealed no signif-
icant difference between MTA and amalgam at early intervals (228), in
contrast with another study that showed less inflammation around
GMTA samples in comparison to amalgam specimens at early time inter-
vals (229). Conflicting reports are available regarding subcutaneous
reactions to WMTA and GMTA (229, 230). In one study, after 3 days,
WMTA caused significantly less inflammation in comparison with
GMTA; in contrast, after 1 week, GMTA samples showed less
MTA: Leakage and Biocompatibility Investigations 197
Review Article
inflammation than WMTA (229). In another study, a group of investiga-
tors from the same institution found no difference between the inflam-
matory response to GMTA and WMTA (230). The authors of these
studies attributed the conflicting results to using different criteria for
histologic evaluation.
A recent investigation showed similar mild to moderate tissue
reaction when PC and WMTA were implanted in the subcutaneous tissue
of rats (232). Another study reported more tissue necrosis and giant
cell formation around MTA at 7 and 14 days after implantation in
comparison to CPM, which showed more eosinophils over the same
time periods (233). Another recent investigation added 2.5 wt%
Na2HPO4 to WMTA and reported a significantly lower inflammatory
reaction in comparison to WMTA when the materials were implanted
subcutaneously (234).
Another investigation compared subcutaneous reaction to AMTA,
Sealapex, and Endo CPM sealer (MTA sealer by an Argentine manufac-
turer). Results showed that except for early interval (7 days), no signif-
icant difference was seen between both types of MTA, which were more
biocompatible than Sealapex. Calcified precipitations were observed
around all implanted MTA samples (235).
These studies showed that subcutaneous responses to MTA range
from necrosis to dystrophic calcification. In addition, at first, MTA
produces a moderate to severe subcutaneous response, which subsides
at longer time intervals.
Intraosseous Implantation
In a preliminary investigation, Torabinejad et al (236) examined
the tissue reaction to implanted Super EBA and MTA in guinea pig
mandibles and reported that bone reaction to MTA is slightly milder
than to Super EBA. In another study, these researchers investigated
the reaction of guinea pig tibias to amalgam, Super EBA, IRM, and
MTA and reported that MTA had the most favorable response among
the tested materials (237).
Sousa et al (238) compared the osseous reaction of guinea pigs to
ZOE, light-cured composite, and MTA. Four weeks after implantation,
whereas the MTA response was rated as none to slight, ZOE showed
necrosis, bone resorption, and infiltration of mononuclear inflamma-
tory and foreign body giant cells. Light-cured composite specimens
showed moderate chronic inflammatory infiltrate near the implanted
material. After 12 weeks, all tested materials exhibited biocompatible
characteristics.
Moretton et al (222) examined subcutaneous and intraosseous
reaction to implantation of MTA and EBA. Although the pattern of osteo-
genesis was similar at early time intervals, at the 60-day interval, they
observed greater osteogenesis around EBA. This study reported that
both materials are osteoconductive. Cintra et al (239) analyzed the alve-
olar bone response of rats to MTA and a new CH-containing sealer and
found no significant difference between the 2 test materials.
Osseous reaction investigations have shown that the bone
response to MTA is relatively mild and with minor inflammation.
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