Basic Research—Technology
Biomineralization Ability and Interaction of Mineral
Trioxide Aggregate and White Portland Cement With
Dentin in a Phosphate-containing Fluid
Jessie F. Reyes-Carmona, MSD,* Mara S. Felippe, PhD,† and Wilson T. Felippe, PhD†
Abtract
Introduction: Mineral trioxide aggregate (MTA) has
been shown to be bioactive because of its ability to
produce biologically compatible carbonated apatite.
This study analyzed the interaction of MTA and white
Portland cement with dentin after immersion in phos-
phate-buffered saline (PBS). Methods: Dentin disks
with standardized cavities were filled with ProRoot
MTA, MTA Branco, MTA BIO, white Portland cement
+ 20% bismuth oxide (PC1), or PC1 + 10% of calcium
chloride (PC2) and immersed in 15 mL of PBS for 2
months. The precipitates were weighed and analyzed
by scanning electron microscopy (SEM) and x-ray diffrac-
tion. The calcium ion release and pH of the solutions
were monitored at 5, 15, 25, and 35 days. The samples
were processed for SEM observations. Data were
analyzed by using analysis of variance or Kruskall-Wallis
tests. Results: Our findings revealed the presence of
amorphous calcium phosphate precipitates with
different morphologies. The apatite formed by the
cement-PBS system was deposited within collagen
fibrils, promoting controlled mineral nucleation on
dentin, observed as the formation of an interfacial layer
with tag-like structures. Conclusions: All the cements
tested were bioactive. The cements release some of
their components in PBS, triggering the initial precipita-
tion of amorphous calcium phosphates, which act as
precursors during the formation of carbonated apatite.
This spontaneous precipitation promotes a biominerali-
zation process that leads to the formation of an interfa-
cial layer with tag-like structures at the cement-dentin
interface. (J Endod 2009;35:731–736)
Key Words
Amorphous calcium phosphate, biomineralization, bioac-
tivity, carbonated apatite, mineral trioxide aggregate
(MTA), Portland cement.
From the *Postgraduate program of the Federal University of Santa Catarina, Florianopolis, Santa Catarina, Brazil, and Department of Restorative Sciences,
University of Costa Rica, San Jose, Costa Rica; and †Department of Endodontics, Federal University of Santa Catarina, Florianopolis, Santa Catarina, Brazil.
Address requests for reprints to Professor Wilson Tadeu Felippe, Department of Endodontics, School of Dentistry, Federal University of Santa Catarina, Florianopolis,
Santa Catarina, Brazil. E-mail address: wtfelippe@hotmail.com.
0099-2399/$0 - see front matter
Copyright ª 2009 American Association of Endodontists.
doi:10.1016/j.joen.2009.02.011
JOE — Volume 35, Number 5, May 2009
M ineral trioxide aggregate (MTA) is the most commonly recommended material for
sealing communications between the root canal system and the periodontium. For
this reason, it is currently being used in several clinical situations such as root-end
filling, repair of root and furcation perforations, apical plugs, and root canal filling
(1, 2).
The bioactivity of MTA has been attributed to its ability to produce hydroxyapatite
(HA) in the presence of phosphate-buffered saline (PBS) (3, 4). Sarkar et al (3) sug-
gested that calcium ions released by MTA react with phosphate in PBS, yielding an HA
interfacial layer in the MTA-dentin interface. However, recently it was shown that hard-
ened Portland cement, an active ingredient in MTA, releases calcium hydroxide, which
interacts with a phosphate-containing fluid to produce calcium-deficient apatite via an
amorphous calcium phosphate phase (5, 6).
Originally, ProRoot MTA was only available as a gray-colored powder, but in 2002
white MTA was introduced to address esthetic concerns (7). Low concentrations of
magnesium, aluminum, and particularly iron are thought to be responsible for the vari-
ation in color (8).
After the commercialization of white MTA, a Brazilian dental company (Angelus,
Londrina, PR, Brazil) developed MTA BIO cement, which is free of toxic substances,
including arsenic (9).
Because MTA is composed primarily of Portland cement (10), some authors have
suggested replacing MTA with Portland cement (11–13).
Researchers have also found that the setting time can be reduced and the handling
properties improved by adding calcium chloride to the MTA or Portland cement
(14–16). Although some studies have demonstrated favorable results with the addition
of this product (14–16), it is not known whether this additive influences the formation
of precipitates.
Sarkar et al (3) found that an interfacial layer of HA formed when gray MTA was
used in vitro. However, the biochemical reactions occurring when MTA Branco, MTA
BIO, and Portland cement are used in simulated clinical environments have yet to be
investigated.
Taking these points into consideration, this study aimed to analyze the interactions
of ProRoot MTA, MTA Branco, MTA BIO, and white Portland cement, with or without
calcium chloride, with dentin after immersion in PBS.
Materials and Methods
The research protocol was approved by the Ethics Committee of the Federal
University of Santa Catarina.
The crowns of 28 single-rooted, extracted human teeth were removed. The
middle third of the roots was sectioned transversely by using a water-cooled low-speed
Biomineralization Ability and Interaction of MTA and White Portland Cement With Dentin 731
Basic Research—Technology

732 Reyes-Carmona et al. JOE — Volume 35, Number 5, May 2009


ISOMET diamond saw (Buehler, Lake Bluff, NY) to obtain 2-mm-thick
sections. In each section, the space of the canal was enlarged with
a spherical diamond bur to obtain 2-mm-diameter standardized cavi-
ties. The sections were immersed in 17% ethylenediaminetetraacetic
acid for 3 minutes followed by 1% sodium hypochlorite for the
same period of time. They were then immediately washed in distilled
water and dried.
The root sections were randomly divided into 5 groups (n = 11),
and the cavities were filled with different cements as described below:
group 1: ProRoot MTA (Dentsply Tulsa Dental, Tulsa, OK); group 2:
MTA Branco (Angelus Soluções Odontológicas, Londrina, PR, Brazil);
group 3: MTA BIO (Angelus); group 4: PC1, white Portland cement (Ira-
jazinho, Votorantim, São Paulo, SP, Brazil) + 20% bismuth oxide
(Seelze, Hanover, Germany); and group 5: PC2, PC1 + 10% calcium
chloride (Labsynth, Diadema, SP, Brazil).
The MTA cements were mixed following the manufacturer’s
recommendations. PC1 was mixed with distilled water at a water-to-
powder ratio of 0.26:1 by weight, and PC2 was mixed at a ratio of
0.18:1, according to the recommendations of Bortoluzzi et al (15, 16).
With the aim of determining the pH and the calcium ion release of
the dentin, a sample (root section) without cement was used as a control.
A calcium-free and magnesium-free PBS solution containing 136.4
mM NaCl, 2.7 mM KCl, 8.2 mM NaH2PO4, and 1.25 mM KH2PO4 in de-
ionized water (pH 7.2) was prepared and filtered. The samples were
suspended individually in sterile plastic vials containing 15 mL of PBS
for 2 months at 37 C. The PBS solution was collected and replaced
every 5 days.
Chemical Composition and Amount of Precipitate
Every 5 days after replacement of the solution and when sufficient
formation of precipitate was noted, the surfaces of 10 samples were har-
vested by gentle scraping with a sterile dental mixing spatula. The
removed precipitates were centrifuged to eliminate the dissolved salts
from the PBS, desiccated in an oven, and weighed with a digital analytical
scale (FA-2104 N; Bioprecisa, Curitiba, PR, Brazil). The weights of the
precipitates (grams) were recorded and statistically analyzed by using
analysis of variance (ANOVA) and least significant difference (LSD) tests.
The dry precipitates were milled with an agate mortar and pestle
and analyzed with x-ray diffraction for identification of phase composi-
tion. X-ray diffraction patterns were recorded with a Philips PW 1830
diffractometer unit (PANalytical, Almelo, Netherlands) by using Ni-
filtered CuKa radiation (40 KeV, 40 mA), in the 2 W range of 20–50
degrees, with a scan rate of 4 degrees/min. Results were analyzed
with automated software for PC-APD Powder diffraction and PC-IDEN-
TIFY software for crystalline phase identification.
One sample from each group was mounted on an aluminum stub
and sputter-coated with a 300-Å gold layer. The elemental composition
of the precipitate was determined by using energy dispersive x-ray anal-
ysis (EDAX) with a scanning electron microscope (SEM) (Philips SEM
XL 30; Philips, Eindhoven, Netherlands) at 15 kV. Three evaluations
were performed for each sample in different areas. Serial SEM photo-
micrographs at different magnifications (125–8000) were taken to
analyze the ultrastructure of the precipitate.
:
Figure 1. Morphologic characterization of precipitates formed by MTA BIO after 2-month immersion in PBS. (A) High-magnification SEM showing the acicular
nature of spherules (original magnification, 8000). (B) EDAX spectrum for precipitates in (A) and semiquantitative chemical composition showing their Ca/P
molar ratio. (C) Photomicrograph showing petal-like precipitates (original magnification, 1000) for which (D) SEM-EDAX revealed a greater Ca/P molar ratio
and lattice substitution of Na and Cl. (E) SEM of compact lath-like precipitates (original magnification, 1000) that demonstrates (F) a Ca/P molar ratio of 1.61
with lattice substitution of Na, Cl, and Mg. (G) X-ray diffraction pattern of the calcium phosphate precipitate obtained after 2 months of immersion in PBS, revealing
the presence of a weakly crystalline apatite.
JOE — Volume 35, Number 5, May 2009 Biomineralization Ability and Interaction of MTA and White Portland Cement With Dentin
Basic Research—Technology
Determination of pH and Calcium Ion Release
The PBS solution, changed every 5, 15, 25, and 35 days, was
filtered and collected individually in sterile specimen vials and pre-
washed with 5% nitric acid for 24 hours followed by a wash with deion-
ized water for the same period of time.
Immediately after collection of the solution (PBS) in contact with
the samples, pH was determined with a pH-meter (INSTRUTHERM, PH-
1700, São Paulo, SP, Brazil) previously calibrated with an alkaline solu-
tion. The readings were taken for 5 samples from each group, selected
randomly. The pH data were analyzed through ANOVA and the LSD test.
Calcium ion release was measured with an atomic absorption
spectrophotometer (model Z-8230; Polarized Zeeman HITACHI,
Tokyo, Japan) for 3 samples from each group, selected randomly.
Data obtained were recorded and submitted to descriptive analysis.
Cement-Dentin Interface Analysis
After 2 months of immersion in PBS, the samples were washed in
distilled water for 2 hours and processed for SEM observation (17). The
specimens were mounted on aluminum stubs and sputter-coated with
a 300-Å gold layer. They were then observed under an SEM (Philips
SEM XL 30) at an accelerating voltage of 15 kV.
The cement-dentin interface was examined at different magnifica-
tions (15–3000) to verify the formation of an interfacial layer
between the cement and the dentinal wall. When a layer was evident,
its elemental composition was investigated by EDAX with an SEM
(Philips SEM XL 30). Data obtained were statistically analyzed by using
ANOVA and the LSD test.
Results
A white precipitate was formed in the first hour after immersion of
the samples in PBS and for up to 25 days. MTA BIO (0.95 g) produced
the greatest amount of precipitate, when compared with MTA Branco
(0.66 g) (P = .007), PC1 (P < .001), and PC2 (P = .001). ProRoot
MTA (0.83 g) produced a greater amount of precipitate than PC1
(0.44 g) (P < .001) or PC2 (0.56 g) (P = .014).
SEM analysis revealed the presence of precipitates with different
morphologies (Fig. 1). The x-ray diffraction pattern of the precipitates
(Fig. 1G) showed broad but clear peaks at about 26 and 32 degrees in
a 2-degree theta position, along with several other small peaks.
Although poorly crystalline, the material seemed to contain an
apatite-like phase. Indeed, the diffraction pattern resembled that of
a poorly crystallized (non-stoichiometric) HA or bone mineral.
SEM-EDAX indicated that the precipitates contained mainly
calcium and phosphorus. Depending on the analyzed area, different
Ca/P molar ratios were found. The spherical precipitates with acicular
crystallites along the periphery (Fig. 1A) were composed mainly of
calcium and phosphorus and other elements in trace amounts,
including carbon and oxygen, with Ca/P molar ratios of 1.40–1.42.
The petal-like precipitates (Fig. 1C) showed a similar chemical compo-
sition, with Ca/P ratios of 1.41–1.55. Compact and agglomerate lath-
like precipitates were also observed (Fig. 1E), with Ca/P ratios of
1.60–1.63. In contrast to the other types of precipitates, small traces
of magnesium were detected. The EDAX spectra of the 3 types of precip-
itates are presented in Fig. 1B, D, and F.
733
Basic Research—Technology
Figure 2. (A) Photomicrograph of ProRoot MTA–dentin interface showing cement (C), interlayer (IL), and dentin (D). (B) Higher magnification showing TS and
lateral branches. Photomicrograph of (C) MTA Branco–dentin interface, (D) MTA BIO–dentin interface, (E) PC1-dentin interface, and (F) PC2-dentin interface.
(E) and (F) show the formation of fewer, short TS.
In pH analyses, regardless of the group, the highest pH value was
recorded on day 5 (9.4–11.0). On day 15, pH decreased in all the
samples (8.8–7.7). On days 25 and 35, pH values remained relatively
stable (7.4–7.7). The pH values recorded for groups 1 (ProRoot MTA)
and 3 (MTA BIO) were slightly higher than those recorded in the other
groups at all time periods. The pH values recorded for the solution in
contact with the control group remained at 7.2 throughout the various
time periods.
The total calcium release values recorded for MTA BIO (36.89 mg/L)
and ProRoot MTA (20.08 mg/L) were higher than those of MTA Branco
(16.45 mg/L), PC1 (15.40 mg/L), and PC2 (15.92 mg/L). Despite
variations in the amount of calcium released into the solution, a similar
trend was observed for the groups. The values were higher for day 5
than for day 15, after which they tended to decrease until day 25,
when they remained stable until the end of the experiment. In compar-
ison to PC1 (1.67 mg/L), PC2 (3.05 mg/L) demonstrated a higher
amount of calcium ion release at day 5. Trace amounts of calcium
were detected in the solution in contact with the control group for
all time periods.
On examination of the cement-dentin interface, it was possible to
distinguish the cement, interfacial layer, and dentin (Fig. 2A). Tag-like
structures (TS) resembling demineralized tubular dentin were
observed in the interfacial layer (Fig. 2B). Depending on the group,
TS presented different morphologies and densities. MTA BIO, ProRoot
734 Reyes-Carmona et al.
MTA, and MTA Branco displayed TS that were much longer and of
higher density than those of the other groups (Fig. 2). Lateral branch
formation was also observed in the samples from groups 1, 2, and 3.
The elemental composition profiles obtained by x-ray analysis of
areas identified as cement, interfacial layer, TS, and dentin are shown
in Table 1.
The composition of the interfacial layer was different from that of
the cements, comprising primarily calcium and phosphorus. The inter-
facial layer, the TS, and the dentin showed similar elemental com-
position, except for the higher amount of phosphorus present in the
TS (P = .008) and the absence of silicon in the dentin (P = .275).
Discussion
Precipitation began in the first hour after immersion. After 5 days,
the surface of the samples was entirely covered. These findings are
consistent with those of Sarkar et al (3), Bozeman et al (4), and Tay
et al (5). Sarkar et al and Bozeman et al characterized the precipitate
as HA, whereas Tay et al reported the formation of carbonated apatite.
The ultrastructural examination allowed verification of the pres-
ence of different precipitates. Higher amounts of spherical precipitates
with acicular crystallites were observed along the periphery (aciculars).
Tay et al (5) also observed this type of formation after immersing white
Portland cement in PBS for 10 days. According to the authors, this type
JOE — Volume 35, Number 5, May 2009
 Basic Research—Technology
TABLE 1. Semiquantitative elemental composition (wt%) of areas identified as cements, interfacial layer (I), TS, and dentin (D)
Ca P Al Si Bi Fe Mg O S C Na
ProRoot MTA 24.40 0.89 7.18 21.97 13.96 9.02 2.90 17.83 1.30 6.34 0.14
MTA Branco 22.99 0.50 4.06 25.94 13.68 0.06 2.38 12.51 1.81 16.49 0.74
MTA BIO 25.07 0.37 6.33 22.88 18.54 0.46 1.01 11.96 0.39 12.35 0.58
PC1 16.88 0.03 7.09 28.40 7.47 1.10 12.65 12.18 5.48 9.93 0.24
PC2 20.99 0.36 7.98 22.87 8.17 1.65 9.43 12.18 5.47 9.93 0.98
I 44.33 8.69 1.04 3.26 1.53 0.25 0.97 14.98 0.00 24.49 0.86
TS 37.81 14.87 0.89 7.86 0.00 0.00 0.58 21.01 0.00 16.00 0.21
D 45.50 12.91 0.26 1.01 0.00 0.00 0.62 19.73 0.00 10.96 0.02

of precipitate is characteristic of the pH-dependent autocatalytic trans-
formation of the metastable amorphous calcium phosphate phase into
an apatite phase. The occurrence of acicular crystallites is a conse-
quence of the superficial activity of apatites, that is, they incorporate
elements into their structure and substitute certain elements for others,
resulting in morphologic alterations (18). The petal-like precipitates
(nano-sized flaky crystallites) and the compact lath-like precipitates
(dune-like layer) have not previously been reported in the literature
for the use of MTA or Portland cement. Petal-like precipitates have
been observed on the surface of HA coatings after 14 days of immersion
in PBS (19–21) and were related to octocalcium phosphate (OCP)
formation (21). OCP and apatite reveal close structural similarities,
because the OCP structure is composed of apatite layers separated by
hydrated layers (21–23). OCP has been proposed frequently as
a mandatory precursor phase for the formation of non-stoichiometric,
biologic apatites (21–24). This gradual phase transformation from OCP
to an apatite phase has been observed in previous in vitro and in vivo
studies (23–26) and was described as a long-term mineralization
process yielding phase mixtures that are richer in apatite (25).
At physiologic pH, the formation of carbonated apatite can be
described as a two-stage process (5). Initially, the Ca/P ratio of the
precipitates is lower than 1.5 and is associated with an increase in
the pH of the solution. During the second stage, there is an increase
in the Ca/P ratio and a drop in pH. As observed at the beginning of
this study, the pH of the solution (11.0–9.4) was highly alkaline. At
day 15, a decrease in the pH was observed (8.8–7.7). The change in
pH can be attributed to the incorporation of OH– ions released from
the cement into the OH– sites of the apatite (5).
On the basis of this information and in our results, it is possible to
deduce that initially an amorphous calcium phosphate phase is formed,
which acts as a precursor to the secondary phase during which carbon-
ated apatite is formed.
Bioactivity is an expression that describes the beneficial effect of
a material implanted in living tissue. The capacity of the material to
interact with the living matter allows the integration of the biomaterial
into the environment. When a bioactive material is implanted, a series
of biochemical and biophysical reactions occurs at the implant-tissue
interface, resulting in the formation of a carbonated apatite layer
(18–24, 27, 28). The ability to induce apatite formation is a common
characteristic for biomaterials (5) and can be reproduced in vitro in
a PBS system (22, 27).
Carbonated apatite is also known as biologic apatite and repre-
sents the mineral phase of hard tissue (bone, dentin, and cementum)
(5, 29). The bioactivity of MTA and Portland cement can thus be attrib-
uted to its capacity to form carbonated apatite, which is important to the
formation and maintenance of the bone tissue biomaterial interface
(27, 28). Previous studies showed the growth of crystalline deposits,
composed mainly of calcium and phosphorus, along the pulp-MTA
interface (30) and in dentinal bridges (31). These observations,
together with our data, can account for the potential of MTA to stimulate
repair and to promote hard tissue deposition. Because the formation of
apatite was confirmed on the surface of the 5 cements tested, they could
all be considered as bioactive.
Besides verifying the presence of the interfacial layer previously
identified by Sarkar et al (3), this study reports the formation of TS ex-
tending from the intermediate layer to the dentinal tubules, similar to
those reported at the resin-dentin interface (32, 33). Thus, it appears
that constant formation of the precipitate contributes not only to the
formation of the interfacial layer but also to the promotion of an intra-
tubular mineralization process.
The mechanisms involved in mineralization are not completely
understood, but there are many studies suggesting the involvement of
noncollagen proteins in this process (6, 34). Recombinant DMP1
molecules have been thought to perform specific molecular recognition
with the apatite surface, guiding calcium phosphate clusters during
recruitment through the collagen matrix (6, 34). This process has
been described as a controlled biomineralization (34).
On the basis of our ultrastructural results, this experiment
provided compelling evidence of the biomineralization process
promoted by the interaction of MTA and Portland cement with dentin.
The apatite formed by the cement-PBS system was deposited within
collagen fibrils, promoting controlled mineral nucleation on dentin.
Because of the methodology used in this study for the cement-dentin
interface analysis, this phenomenon was observed as the formation of
an interfacial layer with TS. This mechanism, theoretically, could
initially involve a diffusion-controlled reaction between the cement,
apatite layer, and dentin, which would be responsible for their chemical
bonding, as suggested by Sarkar et al (3).
Although all cements form an interfacial layer and TS, it is impor-
tant to note that the samples of ProRoot MTA, MTA Branco, and MTA
BIO formed longer TS with lateral branches. However, the Portland
cement with or without calcium chloride presented fewer and shorter
TS. Notably, it is well-known that ionic dissolution is an initial step in
apatite precipitation (19). Weng et al (35) stated that the growth and
nucleation of the apatite layer are proportional to the concentration
of available ions. This information suggests that ProRoot MTA, MTA
Branco, and MTA BIO release a greater amount of calcium ions and,
consequently, produce the highest amount of precipitates, which seems
to have positively influenced the formation of the interfacial layer and
the TS.
Thus it could be hypothesized that the ability to precipitate greater
amounts of carbonated apatite might be significant in minimizing
leakage. As evidenced in this study, the superior performance of the
various commercial forms of MTA in the formation of the precipitates,
interfacial layer, and TS suggests that these cements might offer a supe-
rior sealing ability when compared with the other materials. Studies are
being conducted to evaluate the influence of the interfacial layer and the
TS on the sealing ability and push-out strength of the cements.
Our findings showed that the addition of calcium chloride in PC2
promoted the release of a greater amount of calcium ions during the
first 5 days, contributing to the formation of the highest amount of
precipitates, in comparison to the homologous sample (PC1). In

JOE — Volume 35, Number 5, May 2009 Biomineralization Ability and Interaction of MTA and White Portland Cement With Dentin 735
Basic Research—Technology
a recent study, it was observed that the seal of an MTA orthograde apical
plug improved after the specimens were immersed in PBS for 4 weeks
(36). Thus, we speculate that a higher precipitation of carbonated
apatite could be important in minimizing leakage. This additive should
be useful in situations in which sealing ability is a concern, such as root-
end filling, repair of root and furcation perforations, and apical plugs.
The possible biologic and clinical significance of our findings
includes the following: (1) the interaction of MTA and Portland cement
with dentin in a phosphate-containing fluid promotes a biomineraliza-
tion process, (2) this process could be significant in minimizing
leakage, (3) the formation of the interfacial layer and the intratubular
mineralization process could influence the push-out bond strength, and
(4) the formation of carbonated apatite precipitates could be respon-
sible for the ability of the cements to stimulate repair and dentinogenesis
or cementogenesis.
We concluded that ProRoot MTA, MTA Branco, MTA BIO, and
white Portland cement are bioactive. The cements release some of their
components in PBS, triggering the initial precipitation of amorphous
calcium phosphates, which act as precursors for the formation of
carbonated apatite. This spontaneous precipitation promotes a biomi-
neralization process that leads to the formation of an interfacial layer
with TS at the cement-dentin interface.
Acknowledgments
Dr. Reyes-Carmona is a fellow of University of Costa Rica. This
research was supported in part by Grants in Aid for Scientific
Research from the Federal University of Santa Catarina and
University of Costa Rica.
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JOE — Volume 35, Number 5, May 2009