doi:10.1111/iej.

12683

Human dental pulp cells response to mineral

trioxide aggregate (MTA) and MTA Plus:
cytotoxicity and gene expression analysis

E. M. Rodrigues1, A. L. G. Corne
lio1, L. B. Mestieri1, A. S. C. Fuentes2, L. P. Salles3,

C. Rossa-Junior , G. Faria , J. M. Guerreiro-Tanomaru1 & M. Tanomaru-Filho1
4 1
1
Department of Restorative Dentistry, Araraquara School of Dentistry, UNESP – Univ Estadual Paulista, Araraquara, S~ ao
Paulo; 2Laboratory of Molecular Biology, Department of Genetic and Evolution, Federal University of S~ ao Carlos, S~
ao Carlos,
S~ao Paulo; 3Cellular Biology Department, Institute of Biological Sciences, University of Bras
ılia, Distrito Federal; and
4
Department of Diagnosis and Surgery, Araraquara School of Dentistry, UNESP – Univ Estadual Paulista, Araraquara, S~ ao
Paulo, Brazil

Abstract expression of BMP-2, OC and ALP was quantified

with real-time PCR. Statistical analysis was performed
Rodrigues EM, Corne
lio ALG, Mestieri LB, Fuentes
with analysis of variance and Bonferroni or Tukey
ASC, Salles LP, Rossa-Junior C, Faria G, Guerreiro-
post-test (a = 0.05).
Tanomaru JM, Tanomaru-Filho M. Human dental pulp
Results MTA and MTA P were not cytotoxic and
cells response to mineral trioxide aggregate (MTA) and MTA
did not induce apoptosis. MTA P had significant
Plus: cytotoxicity and gene expression analysis. International
higher ALP activity in relation to MTA and the con-
Endodontic Journal, 50, 780–789, 2017.
trol (P < 0.05). MTA had a significantly higher per-
Aim To investigate the cytotoxicity, osteogenic centage of mineralized area than MTA P (P < 0.05).
bioactivity and mRNA expression of osteogenic mark- The expression of BMP2 and OC mRNA was signifi-
ers of bone morphogenetic protein 2 (BMP-2), osteo- cantly higher in cells exposed to MTA than MTA P
calcin (OC) and alkaline phosphatase (ALP) induced after 1 day (P < 0.05). At day 3, the mRNA expres-
by the extracts of set MTA Plus (MTA P) (Avalon sion of ALP was significantly higher in MTA P com-
Biomed Inc. Bradenton, FL, USA) in comparison with pared with MTA (P < 0.05).
MTA (Angelus, Londrina, PR, Brazil) on human den- Conclusions MTA and MTA Plus were noncyto-
tal pulp cells (hDPCs). toxic, increased mineralization processes in vitro and
Methodology Cell viability was assessed by mito- induced the expression of osteogenic markers.
chondrial dehydrogenase enzymatic (MTT) assay, and
Keywords: cytotoxicity, dental pulp cells, mineral
the mechanism of cell death was evaluated by flow
trioxide aggregate, MTA Angelus, MTA Plus, pulp
cytometry. Bioactivity was evaluated by alkaline
capping.
phosphatase (ALP) assay and detection of calcium
deposits with alizarin red staining (ARS). The gene Received 15 November 2015; accepted 11 August 2016

formation of reparative dentine (Prati & Gandolfi

Introduction
2015). Dental pulp cells are responsible for repair
Direct pulp capping has been used to preserve dental after pulp capping (Ford et al. 1996, Bogen et al.
pulp viability and function and to induce the 2008, Accorinte et al. 2009, Okiji & Yoshiba 2009,
Mente et al. 2010, Lin & Rosenberg 2011). The differ-
entiation of dental pulp cells (DPCs) to specific cell lin-
Correspondence: M
ario Tanomaru-Filho, Department of eage is mainly determined by the components of the
Restorative Dentistry, Araraquara School of Dentistry, S~
ao local microenvironment such as extracellular matrix
Paulo State University – UNESP, Rua Humait
a, 1680, CEP:
proteins, growth factors, receptor molecules and sig-
14801-903, Araraquara, SP, Brazil (Fax: +55 16 3301
6392; e-mail: tanomaru@[Link]). nalling molecules (Estrela et al. 2011). Some

780 International Endodontic Journal, 50, 780–789, 2017 © 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd
 Rodrigues et al. Pulp cells response to MTA and MTA plus

materials such as mineral trioxide aggregate (MTA) BMP-2 messenger RNA (mRNA) expression was
can provide gene activating ions to induce DPCs dif- examined. The null hypothesis was that MTA Plus is
ferentiation in cells that are able to produce mineral- not biocompatible and has no potential to induce
ized tissue (Rashid et al. 2003, Moghaddame-Jafari osteogenic bioactivity compared with the reference
et al. 2005, Takita et al. 2006, Mizuno & Banzai material (MTA).
2008, Paranjpe et al. 2011, Gandolfi et al. 2015a,b).
MTA is able to facilitate the proliferation and differen-
Material and methods
tiation of DPCs into odontoblast-like cells forming
dentine by inducing the secretion of morphogenetic
Cell culture
proteins and growth factors such as BMP-2 and TGF-
b1 (Moghaddame-Jafari et al. 2005, Paranjpe et al. After approval of the Research Ethics Committee of
2011, Asgary et al. 2014). the Dental School – UNESP – Araraquara – SP, Brazil
MTA is a tricalcium silicate-based cement indicated (Protocol 33/10), hDPCs were obtained from the third
for root perforations, root-end filling and filling the molars of donor patients (18–25 years old) at the
apical portion of canal in immature teeth, and as a stage of incomplete root formation. The teeth were
pulp-capping material (Parirokh & Torabinejad 2010, indicated for extraction for orthodontic reasons, as
Prati & Gandolfi 2015). When used as a direct pulp- previously described (Gronthos et al. 2000).Cells were
capping agent, MTA has a higher success rate, pro- cultured in a-MEM nonosteogenic medium (Sigma-
viding less pulp inflammation and more predictable Aldrich, St. Louis, MO, USA), supplemented with 10%
hard dentine bridge formation compared with calcium foetal bovine serum, penicillin (100 IU mL 1), strep-
hydroxide (Li et al. 2015). Among the reasons for tomycin (100 lg mL 1), under 5% CO2 in 95%
MTA success are its cytocompatibility (Takita et al. humidified atmosphere at 37 °C until confluency.
2006), bioactivity (Gandolfi et al. 2010), bacterial After that, hDPCs were plated and allowed to attach
leakage resistance and sealing (Pelliccioni et al. 2007, for 24 h in a 95% humidified atmosphere, 5% CO2
Camilleri et al. 2011) and biomineralization (Reyes- and 37 °C. The cell culture medium was replaced,
Carmona et al. 2009). and the cells were then incubated with cement
MTA Plus (MTA P) was introduced on the market extracts diluted in nonosteogenic serum-free medium
as a calcium silicate material indicated for root filling, at 1 : 2, 1 : 4 and 1 : 8 dilutions to test viability and
root-end filling and for pulp-capping procedures (Gan- 1 : 8 to following experiments. The same medium
dolfi et al. 2014, Prati & Gandolfi 2015). MTA P is a was used for the osteoinductive assays (osteogenic
tricalcium silicate-based cement that has similar medium), supplemented with 50 lg mL 1 L-ascorbic
chemical composition to ProRoot MTA and MTA acid (Sigma-Aldrich) and 5 mmol L 1 b-glyceropho-
Angelus but has been ground finer (Gandolfi et al. sphate (b-GP) (Sigma-Aldrich, St. Louis, MO, USA)
2014). MTA P has demonstrated improved reactivity (Liu et al. 2012).
and higher calcium release (Gandolfi et al. 2014,
2015a). However, it is more porous, water soluble
Preparation of material extracts
and water sorptive compared to ProRoot MTA (Gan-
dolfi et al. 2014). A biological assay indicated both White MTA Angelus (Angelus, Londrina, PR, Brazil,
the grey and white MTA P had negligible in vitro Ref 3040-822, expiry date -11/2017) and MTA Plus
cytotoxic risks on a murine odontoblast-like cell line (Avalon Biomed Inc. Bradenton, FL, USA, Ref 50023,
MDPC-23 (Eid et al. 2014). However, information is expiry date – 03/2014) set cements were tested. The
lacking on the biological effects of MTA P. The cyto- cements were prepared by mixing 1 g of powder with
toxicity of MTA P remains undetermined when in 320 lL of deionized MilliQ water. The materials (1 g)
contact with human dental pulp cells (hDPCs), and were placed in empty wells of 12-well culture plates
there are no studies evaluating whether MTA P has (314.0 mm2 area and 3.0 mm height) and were
the ability to induce mineralized tissue formation. incubated for 15 h to ensure their polymerization at
The purpose of this study was to evaluate the cyto- 95% humidity, 5% CO2, 37 °C and subsequently
toxicity, and osteogenic bioactivity of MTA Plus (MTA exposed to ultraviolet light (UV) with power of 30 W
P), compared with MTA Angelus (MTA) in hDPCs and 253.7 nm total amount of radiation under lami-
culture. Furthermore, the effect of MTA and MTA P nar flow for 30 min for disinfection (Katara et al.
on osteocalcin (OC), alkaline phosphatase (ALP) and 2008). Subsequently, 5 mL of a-MEM medium

© 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd International Endodontic Journal, 50, 780–789, 2017 781
 Pulp cells response to MTA and MTA plus Rodrigues et al.

serum-free was added to each well followed by a new Brazil). A total of 2 9 105 cells mL 1 was plated in
incubation for 24 h to obtain the experimental 96-well culture plates and exposed to the cement
extracts. To observe a dose–response relationship, all extracts (1 : 8 dilution) for 1, 3 and 7 days. Absor-
extracts were further serially diluted at 1 : 2, 1 : 4 bance was measured at 590 nm in a spectrophotome-
and 1 : 8 (v : v) in a-MEM medium serum-free (Lee ter. To verify the possibility of phosphatase activity
et al. 2014). variation, the same cement extract was tested in
nonosteogenic and osteogenic medium (50 lg mL 1
L-ascorbic acid and 5 mmol L 1 b-glycerophosphate)
Cell viability
both serum-free. Three independent experiments were
Cell viability was determined by the mitochondrial performed in sextuplicate for each experimental group
dehydrogenase enzymatic assay (MTT). A total of and outcome. Data were expressed as ALP activity
2 9 105 cells mL 1 were plated in 96-well plates, and normalized with the number of viable cells detected in
the cells exposed to the cement extracts or a-MEM MTT assay at the respective culture period (Salles
nonosteogenic medium serum-free (negative control et al. 2012).
group) and 1 mM hydrogen peroxide (positive control),
for 24 h, as required in ISO 10993 standards. The cul-
Alizarin Red Staining (ARS) for calcium
ture medium was then replaced with a-MEM contain-
compounds
ing 0.55 mg mL 1 MTT. The optical density
(OD = 570 nm) was measured using an automated hDPCs (1 9 104 cells mL 1) were plated in 12-well
microplate reader. Three independent experiments culture plates. After 14 days of cell culture in osteo-
were performed in sextuplicate for each experimental genic medium with and without the cement extracts
group and outcome. According to the MTT results, the (1 : 8 dilution), cells were washed with PBS, fixed
cement dilution selected for the subsequent test (apop- with 10% paraformaldehyde (Sigma) and then stained
tosis and necrosis, ALP, ARS and qPCR) was 1 : 8. with 2% alizarin red S (pH 4.1). The use of osteogenic
medium may have overwhelmed minor effects of the
materials in inducing mineralization. Three indepen-
Detection of apoptosis and necrosis by flow
dent experiments were performed in sextuplicate for
cytometry
each experimental group and outcome. Digital images
To assess the effect of MTA and MTA P extracts on were obtained from the well and analysed as mineral-
hDPCs apoptosis, the percentage distribution of living ized area percentage using the program UTHSCSA
cells, initial apoptosis, late apoptosis or secondary Image Tool for Windows version 3.0 (San Antonio,
necrosis, and necrotic ell populations was determined TX, USA).
by flow cytometry using an Annexin V detection kit
according to the manufacturer’s instructions (FITC
Quantitative real-time PCR (qPCR)
Annexin V Apoptosis Detection Kit I, BD Biosciences,
Pharmingen, San Jose, CA, USA). A total of hDPCs (2 9 105 cells mL 1) were plated in 12-well
1 9 106 cells mL 1 were plated in 24-well plate, and plates. After 24 h of exposure to the cement extracts
the cells exposed to the cement extracts (1 : 8 dilu- (1 : 8 dilution) diluted in nonosteogenic and osteo-
tion) diluted in nonosteogenic medium serum-free for genic (50 lg mL 1 L-ascorbic acid and 5 mmol L 1
24 h. hDPCs treated with H2O2 (1 mmol L 1) were b-glycerophosphate), both serum-free, medium, the
used as positive control, and cells exposed to nonos- RNA was extracted from cells using Trizol (Invitro-
teogenic medium serum-free were the negative con- gen, Carlsbad, CA, USA), according to the instruc-
trol. Three independent experiments were performed tions provided by the supplier. The gene expression
in sextuplicate for each experimental group and out- was analysed by qPCR (StepOne, Applied Biosystems,
come. For this experiment, 100 000 events were Life Technologies, Grand Island, NY, USA) using
analysed for each sample. TaqMan chemistry and pre-designed primers and
probe sets (Gene Expression Assays, Applied Biosys-
tems). Primer/probe references were as follows: BMP2
Alkaline phosphatase activity
(Hs00154192_m1); OC (Hs01587814_g1); ALP
Alkaline phosphatase (ALP) activity was evaluated (Hs01029144_m1) and GAPDH (Hs02758991_g1)
using a commercial kit (Labtest; Lagoa Santa, MG, (Applied Biosystems, Life Technologies). Triplicates

782 International Endodontic Journal, 50, 780–789, 2017 © 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd
 Rodrigues et al. Pulp cells response to MTA and MTA plus

were prepared for each reaction, and the experiment

was repeated three times independently (n = 9/
group). The levels of target gene expression for each
sample group were calculated with the ΔΔCt method
(fold expression = 2 (ΔΔCt  stdev)) compared to con-
trol (serum-free medium). To assess differences in the
mRNA expression, we tested the same cement extract
in nonosteogenic and osteogenic medium.

Statistical analysis
Comparisons of cell viability, apoptosis, ALP activity
and qPCR were made by two-way analysis of variance
(ANOVA) with Bonferroni post-test. Comparisons of ARS
were made by one-way ANOVA followed by Tukey’s Figure 1 Human dental pulp cell (hDPC) viability evaluated
test. A level of significance of 5% was chosen to by the MTT assay after exposure for 24 h to different dilu-
denote difference between means. Data are presented tions (1 : 2, 1 : 4 and 1 : 8) of MTA and MTA Plus extracts
as mean  standard error of the mean. in nonosteogenic a-MEM medium serum-free. Bars with dif-
ferent letters represent significant differences between cement
extracts and control groups. MTA P, MTA Plus; NC, negative
Results control; PC, positive control.

Cell viability ALP activity

The hDPC viability tests revealed that MTA and MTA Even though viability was decreased after 1 day of
P had no cytotoxic effects as cell viability was higher cells exposed to the cement extracts prepared in both
(P < 0.05) or similar to the negative control nonosteogenic and osteogenic medium (Fig. 3(a,b)),
(P > 0.05) for 1 day of cells exposure to the extracts. ALP activities for all groups were increased. In gen-
Except for the 1 : 2 dilution, there was no difference eral, MTA had significantly lower ALP activity than
in cell viability of the cells exposed to MTA and MTA the control groups (P < 0.05), and MTA P had signifi-
P (P > 0.05). The positive control promoted cell death cantly higher ALP activity in relation to MTA and the
in hDPCs (25% of cell viability). At 1 : 8 dilution, cell control (P < 0.05) (Fig. 3(c,d)). Also, it was observed
proliferation was significantly higher, compared with that the osteogenic medium provided a significant
the dilution of the other extracts (P < 0.05), and for increase of ALP activity (P < 0.05) that reached its
this reason, all subsequent experiments were per- peak at 3 days (Fig. 3d) when compared to nonos-
formed using 1 : 8 dilution (Fig. 1). teogenic medium (Fig. 3c).

Apoptosis/necrosis Alizarin red staining

Exposure of hDPCs to MTA and MTA P cement After 14 days of cell exposure, MTA and MTA P had
extracts at 1 : 8 dilution for 24 h did not induce a significant stimulatory effect on the formation of
apoptosis, as the percentage of apoptotic cells was mineralized nodules (Fig. 3e) (P < 0.05). In the MTA
lower (P < 0.05) or similar (P > 0.05) to the negative group, there was a significantly higher percentage of
control group. Live cell population was higher mineralized area than those formed when the cells
(P < 0.05) or similar (P > 0.05) to the negative con- were exposed to MTA P extract (Fig. 3f) (P < 0.05).
trol in MTA and MTA P groups. There was, however,
a small albeit significant increase in the percentage of
qPCR analysis
necrotic cells (annexin V-negative, PI-positive cells)
associated with both MTA and MTA P materials On day one, hDPCs exposed to MTA in nonosteogenic
(P < 0.05). Apoptotic cell percentages were high in medium overexpressed osteogenic markers BMP2 by 8
the positive control (Fig. 2). times, and OC, 5 times that of the control (P < 0.05).

© 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd International Endodontic Journal, 50, 780–789, 2017 783
 Pulp cells response to MTA and MTA plus Rodrigues et al.

Figure 2 (a) Flow cytometry analysis of hDPCs viability, mortality by apoptosis or necrosis after 1 day exposure to MTA and
MTA P extracts in 1 : 8 dilution. (b) Representative two-dimensional flow cytometry dot plot of data derived from FITC-AnV
and Phycoerythrin (PE) (incorporated propidium iodide - PI) stained hDPCs of control, MTA and MTA P groups. Cement
extracts (1 : 8 dilution) and controls were prepared in nonosteogenic a-MEM medium serum-free. Bars with different letters
represent significant differences between groups in each population analysed. The population of living cells (LC) (annexin /
PI ), initial apoptosis (IA) (annexin+/PI ), late apoptosis or secondary necrosis (LA) (annexin+/PI+), and necrotic (N) (an-
nexin /PI+) cells is represented in the left inferior, right inferior, right superior and left superior quadrants, respectively. MTA
P, MTA Plus; NC, negative control; PC, positive control.

The same cells exposed to MTA P under these condi- (~1.5–2.0). Also, no OC and ALP mRNA expression
tions overexpressed BMP2 and OC mRNA by 4 and 2.5 was identified for either material prepared with nonos-
times (P < 0.05), respectively (Fig. 4a). There was no teogenic medium (Fig. 4c). In the osteogenic medium,
mRNA expression for ALP in both material groups the BMP2 transcript rate remained the same (~1.5) as
when compared to controls with serum-free medium. MTA, while the MTA P had no BMP2 mRNA expres-
The gene expression in cells exposed to cement extracts sion (Fig. 4d). Interestingly, expression of ALP was
prepared with osteogenic medium was lower when increased only in the MTA P (Fig. 4d) (P < 0.05).
compared with the mRNA expression level of cells cul-
tured in nonosteogenic medium (Fig. 4b).
Discussion
At day 3, the rate of all transcripts decreased signifi-
cantly (P < 0.05) (Fig. 4(c,d)). MTA and MTA P In the present study, the cytotoxicity and osteogenic
presented BMP2 gene expression at the same fold activity of MTA and MTA P were evaluated by

784 International Endodontic Journal, 50, 780–789, 2017 © 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd
 Rodrigues et al. Pulp cells response to MTA and MTA plus

Figure 3 (a and b) hDPC viability and (c and d) ALP activity evaluation after exposure to MTA and MTA P extracts (1 : 8
dilution). MTA and MTA P extracts were diluted in nonosteogenic a-MEM medium serum-free (a and c) and osteogenic a-MEM
medium (L-ascorbic and b-glycerophosphate) serum-free (b, d and e). (e) Alizarin red staining (ARS) statistical analysis, (f)
osteogenic cultures of hDPCs showed calcium deposition as demonstrated by positive ARS. *Significant difference between
cement extracts and control groups. Different letters represent significant difference between MTA and MTA P groups. MTA P,
MTA Plus; C, cells exposed to nonosteogenic medium; CO, cells exposed to osteogenic medium.

© 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd International Endodontic Journal, 50, 780–789, 2017 785
 Pulp cells response to MTA and MTA plus Rodrigues et al.

exposing hDPCs to cement extracts diluted in nonos- that MTA Angelus was not cytotoxic after 2 and
teogenic and osteogenic medium. 5 days of contact with human dermal fibroblasts
The standard assay used to evaluate the cytotoxic- (Hirschman et al. 2012). They are also consistent
ity of the materials was the MTT after 24 h of cells with a study included in the XTT assay that MTA
exposure, following the ISO 10993 parameters (ISO Plus with 1 : 5 and 1 : 10 dilutions had no toxic
2005). The MTT results showed that both MTA and effects on MDPC-23 cells (Eid et al. 2014).
MTA P were not cytotoxic for hDPCs at 1 : 2, 1 : 4 To double-check possible cytotoxic effects, apoptosis
and 1 : 8 dilutions. MTA has been reported to induce and necrosis was assessed by flow cytometry. The
proliferation of hDPCs by elution components such as results revealed that MTA and MTA P did not induce
calcium ions (Takita et al. 2006). High proliferation apoptosis, but there was a small increase in necrotic
of hDPCs cells was observed at 1 : 8 dilution of cells. Various mechanisms may be related with this
cement extracts. As none of the materials were cyto- minor increase in necrosis, but as it did not affect the
toxic, and a high proliferation was detected in 1 : 8 overall proportion of dead cells (combining apoptosis
dilution, the highest dilution of cement extract for all and necrosis), it does not affect the overall conclusion.
subsequent experiments assessing their biological Speculatively, this increase in necrosis observed with
activity was used. These results corroborate with pre- exposure to the materials may be related with the
vious studies which revealed using the MTT assay change in the pH of the medium, which could induce

Figure 4 mRNA expression levels of target genes in hDPCs exposed to MTA and MTA P extracts for 1 (a and b) and 3 days (c
and d) diluted in nonosteogenic a-MEM medium serum-free (a and c) and osteogenic a-MEM medium (L-ascorbic and b-glycer-
ophosphate) serum-free (b and d). Bars with different letters represent significant differences between groups. BMP2, bone mor-
phogenetic protein 2; OC, osteocalcin; ALP, alkaline phosphatase genes; C, cells exposed to nonosteogenic medium serum-free;
CO, cells exposed to osteogenic medium serum-free.

786 International Endodontic Journal, 50, 780–789, 2017 © 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd
 Rodrigues et al. Pulp cells response to MTA and MTA plus

biochemical stress and subsequent membrane disrup- was markedly reduced in all groups, which may be
tion. During the hydration process, MTA Angelus and indicative of negative feedback associated with the
MTA Plus calcium silicates leach OH ions producing early increase observed in day 1. Previous studies
an alkaline pH (Gandolfi et al. 2014, 2015a, Prati & have revealed that BMP2 is expressed along with its
Gandolfi 2015). The apoptosis results corroborated receptor in hDPCs and accelerates the differentiation
with a study which showed that exposure to fresh or of these cells into odontoblasts (Nakashima 1994, Gu
premixed ProRoot MTA did not cause an increase in et al. 1996, Saito et al. 2004). The efficacy of MTA to
detachment and rounding of mouse undifferentiated induce BMP2 expression and calcification in human
pulp cells (OD-21) from the culture surface indicating periodontal ligament cells (PDL) and human dental
apoptosis was not induced (Moghaddame-Jafari et al. pulp cells (hDPCs) is well established (Rashid et al.
2005). Furthermore, there was no significant increase 2003, Maeda et al. 2010, Paranjpe et al. 2011).
in the sub-G1 population of pulp cells exposed to Both materials stimulated an increase in mRNA
MTA, which demonstrated that these cells were not expression for OC during 1 day of exposure. In later
induced to undergo apoptosis (Moghaddame-Jafari stages of osteoblastic differentiation, osteocalcin is a
et al. 2005). Other studies have demonstrated that bone phenotypic marker highly expressed in hDPCs
MTA improves the proliferation of dental mesenchy- (Wang et al. 2013, Gandolfi et al. 2015b). The
mal cells (D’Anto et al. 2010), helps the odontoblastic expression of osteocalcin in human dental pulp stem
differentiation of pulp cells and causes no cell death cells (DPSCs) after 12 h by ProRoot MTA induction
(Moghaddame-Jafari et al. 2005, Mizuno & Banzai has been reported in the literature (Zhao et al.
2008, Paranjpe et al. 2011). 2012).
ALP is one of the major enzymes expressed during Only exposure to MTA P increased the expression of
the early maturation of osteoblasts and plays an ALP at day 3. A possible reason for the higher ALP
important role in mineral deposition (Lee et al. 2011). mRNA expression could be associated with the hydrox-
The ALP activity on the first day of exposure was yapatite nucleation process (Gandolfi et al. 2014,
low. On the seventh day, the ALP increased for 2015a), in which the ALP enzyme is induced by the
hDPCs exposed to cement extracts and for the control osteogenic medium plus a basic pH and an increase in
group. MTA P induced a greater increase in ALP calcium level promoted by MTA P. Moreover, L-ascorbic
activity when compared to the control and MTA acid present in osteogenic medium has been shown to
groups. This effect of MTA P on ALP activity may be enhance the expression of bone-type ALP mRNA in rat
caused by its higher calcium release and increase in marrow stromal cells (Peter et al. 1998).
local pH when compared to MTA (Gandolfi et al.
2014). The increase in ALP activity was greater in
Conclusion
hDPCs exposed to osteogenic medium for both groups,
and it can be explained by a possible cumulative effect Extracts from MTA and MTA Plus were not cytotoxic
of osteogenic factors from the medium. This factor and did not induce apoptosis in hDPCs. MTA P
can be involved in cell phenotype hDPCs odontoblas- induced greater ALP activity of hDPCs than MTA.
tic differentiation with an increased ALP activity Both materials induced mineralization nodule forma-
(Gronthos et al. 2000, Liu et al. 2012). MTA and tion by hDPCs with MTA being more pronounced
MTA P have stimulated mineralization nodules depo- than MTA P. Exposure of hDPCs to extracts of both
sition, and this effect was more pronounced for MTA. cements enhanced mRNA expression of osteogenic
The ability of MTA to induce calcium deposition has markers of these cells.
been reported in studies that evaluated its bioactivity
and potential to induce osteogenic differentiation
Conflict of interest
(Yasuda et al. 2008, Paranjpe et al. 2011).
The potential of MTA P to induce the gene expres- The authors have stated explicitly that there are no
sion of osteogenic markers, BMP2, OC and ALP in conflict of interests in connection with this article.
hDPCs was evaluated. The induction of BMP2 mRNA
was twofold smaller in hDPCs exposed to MTA P
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