doi:10.1111/iej.

12751

Cytotoxicity and bioactivity of various pulpotomy

materials on stem cells from human exfoliated
primary teeth

M. Collado-Gonza
lez1, D. Garc
ıa-Bernal1, R. E. On

~ ate-Sa

nchez2, P. S. Ortolani-Seltenerich3,


T. Alvarez-Muro 4
, A. Lozano , L. Forner , C. Llena , J. M. Moraleda1 &
3 3 3

F. J. Rodr
ıguez-Lozano1,2
1
Cellular Therapy and Hematopoietic Transplant Unit, Hematology Department, Virgen de la Arrixaca Clinical University
Hospital, IMIB, University of Murcia, Murcia, Spain; 2School of Dentistry, Faculty of Medicine, University of Murcia, Murcia,
Spain; 3Department of Stomatology, Universitat de Valencia, Valencia, Spain; and 4Dental Clinic Esdens, Alicante, Spain

Abstract Finally, the deposition of a calcified matrix in pres-

Collado-Gonza
lez M, Garc
ıa-Bernal D, On ~ ate-

staining. Statistical analysis was performed with anal-

nchez RE, Ortolani-Seltenerich PS, Alvarez-Muro
Sa
T, Lozano A, Forner L, Llena C, Moraleda JM,
Rodr
ıguez-Lozano FJ. Cytotoxicity and bioactivity of
various pulpotomy materials on stem cells from human
exfoliated primary teeth. International Endodontic Journal, 50,
e19–e30, 2017.
Aims To investigate the cytotoxicity and bioactivity
of several pulpotomy materials: Biodentine (Septodont,
Saint-Maur-des-Fosses, France) MTA (Angelus, Lon-
drina, PR, Brazil), Theracal LC (Bisco Inc., Scham-
burg, IL, USA) and IRM (Dentsply DeTrey GmbH,
Konstanz, Germany), after contact with stem cells iso-
lated from human exfoliated primary teeth (SHEDs).
Methodology SHEDs were cultured in the presence
of the eluates of various pulpotomy materials for 24,
48 and 72 h. Cell viability was determined by mito-
chondrial dehydrogenase enzymatic (MTT) assay.
Apoptosis and changes in cell phenotype were evalu-
ated by flow cytometry. Also, an in vitro scratch
wound-healing assay was used to determine their
effects on cell migration. To assess cell morphology
and attachment to the different pulpotomy materials,
SHEDs were directly seeded onto the material surfaces
and analysed by scanning electron microscopy (SEM).
ence of these materials was verified by Alizarin Red
ysis of variance and Bonferroni or Tukey post-test
(a = 0.05).
Results Cell viability in the presence of Biodentine
eluates was significantly higher to that obtained using
complete medium alone (control; P < 0.01) and was
also significantly higher than using MTA Angelus from
48 h of incubation (P < 0.01). However, Theracal LC
and IRM were associated with low rates of cell viability
(P < 0.001). Similar results were obtained in an apop-
tosis assay. In addition, SHEDs maintained their mes-
enchymal phenotype in all conditions although their
capacity to migrate was higher in the presence of Bio-
dentine. SEM studies revealed a suitable proliferation
rate, cell spreading and attachment, especially when
using Biodentine and MTA Angelus discs. Finally, Bio-
dentine eluates significantly induced calcified matrix
deposition from 7 days of culture (P < 0.01).
Conclusions Biodentine exhibited better cytocom-
patibility and bioactivity than MTA Angelus, Theracal
LC and IRM.
Keywords: cytotoxicity, mineral trioxide aggregate,
pulpotomy materials.
Received 31 October 2016; accepted 2 February 2017

Correspondence: Francisco Javier Rodr
ıguez Lozano, School of Introduction

Dentistry/ Cellular Therapy and Hematopoietic Transplant
Unit, IMIB-Arrixaca, Hospital Morales Meseguer 2pl. Av.
Marqu
es de los V
elez s/n 30008, University of Murcia, Murcia,
Spain (Tel.: +0034 868889518, e-mail: fcojavier@um.es).
One objective of Paediatric Dentistry is to maintain
pulpally involved primary teeth in a healthy state
until the time of normal exfoliation. In this respect,

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  lez et al.
Pulpotomy materials and stem cells Collado-Gonza
vital pulp therapy aims to treat reversible pulp inju-
ries, maintain vitality and function and preserve the
primary tooth until this time (Ritwik 2013, Parisay
et al. 2015). Conversely, nonvital pulp therapy
involves the removal of irreversibly inflamed or necro-
tic pulp tissue, cleaning of the root canals and filling
with a resorbable paste to restore and/or maintain
the health of the periradicular tissues until exfoliation
(Carrotte & Waterhouse 2009). Several methods have
been developed to preserve pulp vitality in primary
teeth with deep caries such as indirect or direct pulp
capping and vital pulpotomy (Kisby 2016).
Vital pulp methods require the use of biomaterials
to form a protective layer over the exposed vital pulp
in direct pulp capping and pulpotomy procedures
(Gandolfi et al. 2015, Prati & Gandolfi 2015). Such
materials should possess adequate biocompatibility
and bioactivity to promote dental pulp stem cell activ-
ity and pulp healing in primary teeth.
Stem cells from human exfoliated primary teeth
(SHED), also called immature dental pulp stem cells
(IDPSC), which are obtained from dental pulp
explants or by digestion of dental pulp tissue from
exfoliated deciduous teeth, have demonstrated
immunosuppressive properties (Rodr
ıguez-Lozano
et al. 2011). Although SHED are isolated from the
dental pulp, their differentiation potential is not only
limited to odontoblasts. In fact, SHED can differentiate
into several cell types including neurons, osteoblasts,
adipocytes and endothelial cells (Rosa et al. 2016).
Several studies have evaluated the cytotoxicity of
pulpotomy materials on human dental pulp stem cells
from permanent teeth, but few studies have used den-
tal pulp stem cells from primary teeth. Amongst these
materials, calcium hydroxide (Ca(OH)2) is one of the
most commonly used in pulp capping or pulpotomies
(Saghiri et al. 2016), with adequate biological
responses of SHEDs (Lutfi et al. 2010). On the other
hand, eugenol in combination with zinc oxide (ZOE)
is used in Paediatric Dentistry as a root canal sealer
in pulpectomies and as a pulp sedative in cases of
vital pulps. However, even very low concentrations of
eugenol produce high toxicity in human dental pulp
fibroblasts of primary teeth (Escobar-Garc
ıa et al.
2016, Lee et al. 2016). Another commonly used den-
tal material in pulp capping treatments or pulpo-
tomies is mineral trioxide aggregate (MTA), which
has been shown to have several advantages over Ca
(OH)2 and ZOE (Goyal et al. 2016, Rajasekharan et al.
2017). Recently, new biomaterials have been intro-
duced and studied. For example, Biodentine
e20 International Endodontic Journal, 50, e19–e30, 2017
(Septodont, Saint-Maur-des-Fosses, France) has been
demonstrated to have a suitable degree of biocompati-
bility with dental pulp stem cells (Luo et al. 2014,
Bortoluzzi et al. 2015), although its effects on SHED
have not been reported. In addition, Theracal LC
(Bisco Inc., Schamburg, IL, USA) is a new light-cured,
resin-modified, calcium silicate-filled base/liner mate-
rial designed for direct and indirect pulp capping,
although its cytotoxicity towards dental pulp cells has
not been extensively studied (Bortoluzzi et al. 2015,
Poggio et al. 2015).
Cell culture techniques are useful for evaluating the
biocompatibility of different materials (Peters 2013).
Indeed, in vitro assays with cell cultures are com-
monly used to elucidate the mechanisms involved in
different biological responses and to investigate cell
behaviour in specific situations. Even though the
results of these in vitro assays cannot be immediately
extrapolated to clinical conditions in humans, they
are clinically relevant because they represent an
appropriate model for the correct screening of differ-
ent dental materials properties, as well as for evaluat-
ing their potential health risks (Pires et al. 2016).
The aim of the present study was to compare and
assess the cytotoxicity and bioactivity of four pulpo-
tomy materials: Biodentine, MTA, Theracal and inter-
mediate restorative material (IRM) in contact with
human stem cells from deciduous teeth. The null
hypothesis was that there would be no significant
cytotoxic differences between the pulpotomy materi-
als.
Materials and methods
Preparation of test specimens
The materials tested were Biodentine (Septodont,
Saint-Maur-des-Fosses, France) MTA (Angelus, Lon-
drina, PR, Brazil), Theracal LC (Bisco Inc.) and IRM
(Dentsply DeTrey GmbH, Konstanz, Germany).
The materials were mixed according to the manufac-
turers’ instructions before sterilizing by ultraviolet irra-
diation for 15 min and then maintained in an
incubator at 37 °C for 48 h to achieve complete setting.
The materials were evaluated by incubating cultured
cells with extracts or eluates of the sealers, according to
the International Standard ISO 10993-5 (ISO 2005).
The extraction vehicle was DMEM medium serum-free
(Gibco Invitrogen, Paisley, UK) supplemented with
penicillin/streptomycin (PAA Laboratories, Pasching,
Austria).
© 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd

After extraction, the eluates were stored in culture
medium for 24 h at 37 °C in a humid atmosphere
containing 5% CO2. In accordance with the relevant
ISO standards, the ratio between the surface of the
sample and the volume of the medium was 6-
cm2 mL
1. The extraction media were then collected
at the end of this period and passed through a 0.22-
lm filter (Merck Millipore, Billerica, MA, USA). Subse-
quently, various dilutions (1/1, 1/2 and 1/4 vol/vol)
of these extraction media were prepared for analysis.
Isolation and characterization of SHEDs
This study was approved by the institutional review
board (IRB) of the University of Murcia. Samples were
obtained according to ethical guidelines of the Faculty
of Dentistry, University of Murcia, Murcia, Spain.
Signed informed consent was obtained FROM the par-
ents or guardians to use physiologically exfoliated pri-
mary teeth of the children (n = 8 teeth from 6- to 9-
year-old children).
Human dental pulp (hDP) was separated from
teeth, washed with Ca2+/Mg2+-free Hank’s balanced
salt solution (HBSS) (Gibco, Gaithersburg, MD, USA)
and digested using a solution containing 3 mg mL
1
collagenase-A (Sigma-Aldrich, St. Louis, MO, USA) for
1 h at 37 °C. Cells obtained after enzymatic digestion
were seeded at a density of 1.5 9 104 cells cm
2 in
25-cm2 plastic culture flasks (BD Biosciences,
Pharmingen, San Jose, CA, USA) in complete medium
and incubated at 37 °C in a humid atmosphere con-
taining 5% CO2 for 3 days. Then, red blood cells and
other nonadherent cells were removed, and fresh
medium was added to allow further growth. The
adherent cells were grown to 80% confluence and
were defined as passage zero (P0). For subsequent
passaging, cells were washed with Ca2+/Mg2+-free
phosphate-buffered saline (PBS) (Gibco Invitrogen)
and detached by incubating with 0.25% trypsin-EDTA
solution (Gibco Invitrogen) for 2–5 min at 37 °C. Cul-
ture medium was added to inactivate the trypsin
activity. Finally, SHEDs were centrifuged at 500 g for
5 min and plated in 75-cm2 flasks at a density of
5 9 103 cells cm
2.
Before the experiments, SHEDs phenotype were
analysed by immunofluorescence using specific anti-
bodies for CD90 (dilution 1 : 250; BD Biosciences,
Pharmingen), CD73 (1 : 200; Santa Cruz Biotechnol-
ogy Inc, Santa Cruz, CA, USA) and CD105 (1 : 100;
Abcam, Cambridge, UK). After three washes with
PBS, cells were incubated in the dark for 1 h with
© 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd
 lez et al. Pulpotomy materials and stem cells
Collado-Gonza
anti-mouse Alexa Fluorâ 488-conjugated secondary
antibody (1 : 500; Molecular Probes, Invitrogen,
Eugene, OR, USA). Microscope slides were mounted
with antifade solution (Vecta shield mounting med-
ium, Vector Laboratories, Hercules, CA, USA) contain-
ing 40 ,6-diamidino-2-phenylindole (DAPI; Molecular
Probes, 0.2 mg mL
1 in PBS) and examined in a fluo-
rescence microscope (Leica DMI 4000B, Wetzlar,
Germany).
MTT assay
The proliferation rate of SHEDs cultured in the pres-
ence of the different eluates was evaluated using the
MTT assay (MTT Cell Growth Kit, Chemicon, Rose-
mont, IL, USA) after 24, 48 and 72 h of culture,
using stem cells from human exfoliated deciduous
teeth cultured in complete medium as controls. After
incubation, MTT was added to each well, incubated
for 4 h, stopping the process by adding dimethyl
sulphoxide. Absorbance at 570 nm (Abs570) was
measured using an automatic microplate reader
(ELx800; Bio-Tek Instruments, Winooski, VT, USA)
using Abs690 as the reference wavelength. Each con-
dition was analysed in quintuplicate.
Analysis of expression of mesenchymal stem cell
surface markers on SHEDs exposed to pulpotomy
materials by means of flow cytometry
The expression of mesenchymal stem cell surface
molecules was analysed on cultures of SHEDs in pas-
sage 2–4 by flow cytometry. Briefly, cells were seeded
at a density of 3 9 104 cells cm
2 in 48-well plates
and treated with the different eluates for 72 days at
37 °C. Then, the cells were detached using a 0.25%
w/v trypsin-EDTA solution, rinsed twice with PBS
and incubated in the dark at 4 °C for 30 min with
fluorescence-conjugated specific monoclonal antibod-
ies for human CD73, CD90 and CD105 (Miltenyi Bio-
tec, Bergisch Gladbach, Germany), as recommended
by the International Society of Cellular Therapy
(ISCT) to confirm the mesenchymal phenotype of the
cells (Dominici et al. 2006). Lack of expression of the
haematopoietic markers CD14, CD20, CD34 and
CD45 was also analysed. Nonspecific fluorescence was
measured using specific isotype monoclonal antibod-
ies. Subsequently, cells were acquired using a BD
FACS Canto flow cytometer (BD Biosciences) and
analysed using Kaluza analysis software (Beckman
Coulter, Inc., Brea, CA, USA).
International Endodontic Journal, 50, e19–e30, 2017 e21
  lez et al.
Pulpotomy materials and stem cells Collado-Gonza
Detection of apoptosis and necrosis by flow
cytometry (Annexin-V/7-AAD staining)
SHEDs were cultured on the different eluates for 72 h, fol-
lowed by double staining with PE-conjugated Annexin-V
and 7-AAD (BD Biosciences, Pharmingen). Percentages
of live (Annexin-V
/7-AAD
), early apoptotic (Annexin-
V+/7-AAD
) or late apoptotic and necrotic cells
(Annexin-V+/7-AAD+ and Annexin-V
/7-AAD+) were
determined by flow cytometry. Subsequently, the per-
centages of each population were calculated.
Cell migration assay
The effects of the different pulpotomy agent eluates on
SHED migration were examined by the wound-healing
assay. Briefly, 5 9 104 SHEDs were seeded onto 60-mm
culture plates and allowed to reach a complete confluent
monolayer before incubating for 24 h in DMEM med-
ium without FBS. After this time, a ‘wound’ was
inflicted by making a scratch through the confluent
layer of cells using a 200-lL pipette tip. The cells were
then washed with PBS to remove cell debris and incu-
bated with various dilutions of eluates for up to 48 h to
allow cell migration back into the wound area. Images
of the scratch area were taken at 0, 24 h and 48 h
post-wounding using a phase-contrast microscope
(Nikon, Tokyo, Japan). The images obtained were anal-
ysed by ImageJ software (NIH, Bethesda, MD, USA).
Scanning electronic microscopy (SEM)
Different samples of Biodentine, MTA, IRM and Thera-
Cal LC were shaped into 1.6-mm thick discs of 5 mm
diameter using rubber moulds. Fifteen discs of each
material were prepared and subdivided into three
groups, each containing five parallel samples. SHEDs
were directly seeded onto each disc at a density of
5 9 104 cells mL
1. After 3 days of culture, the seeded
samples were removed from the culture wells, rinsed
with PBS, fixed with 3% glutaraldehyde and dehydrated
in a graded series of ethanol concentrations up to 100%.
The samples were then immersed in 100% hexamethyl-
disilazane, air-dried, mounted on aluminium stubs, and
sputter coated with gold/palladium. Finally, gold/palla-
dium-coated specimens were examined by SEM.
Analysis of matrix calcium deposition
The control and experimental groups were evaluated
for matrix calcium production at 7, 14 and 21 days
e22 International Endodontic Journal, 50, e19–e30, 2017
of treatment by staining with Alizarin Red, a dye that
binds to calcium salts and an anthraquinone deriva-
tive used to identify calcium containing osteocytes a
differentiated culture of both human and rodent
MSCs. For this, cells were fixed with 70% ethanol for
1 h at 4 °C, stained for 30 min with 2% Alizarin Red
solution (Sigma AB, Malm€ o, Sweden) and washed
three times with ultrapure water. To quantify the
level of staining, 1 mL of 10% cetylpyridinium chlo-
ride (CPC) (Sigma AB) was added to each well and
incubated for 20 min to elute the stain. The absor-
bance of the eluted stain was read at 550 nm using a
spectrophotometer, and a standard curve was pre-
pared using Alizarin Red stain and CPC.
Statistical analysis
Data from the MTT assay, cell migration and Alizarin
Red staining quantification were analysed using SPSS
version 15.0 statistical software (SPSS, Inc., Chicago,
IL, USA). Data were tested for normal distribution
using the Kolmogorov–Smirnov test. Comparisons
between groups in MTT, cell migration and Alizarin
Red staining quantification assays were analysed
using one-way ANOVA followed by Bonferroni post-
tests. A P value <0.05 was considered significant. All
values are presented as the mean  standard devia-
tion (SD).
Results
Isolation and characterization of SHED
The presence of stem cells derived from the remaining
dental pulp of exfoliated primary teeth was confirmed
and characterized using specific antibodies for CD73,
CD90 and CD105, towards which more than 95%
positive expression was recorded (Fig. 1).
MTT assay
To analyse the effects of the eluates of the different
pulpotomy materials on SHED proliferation rates, an
MTT assay was carried out (Fig. 2). The incubation of
SHEDs with various dilutions (1 : 1 or 1 : 2 < 1 : 4)
of IRM eluates resulted in a significant reduction
(P < 0.001) in cell proliferation rates compared with
that observed in the control group. In the presence of
Theracal eluate extracts, both undiluted and diluted
1 : 2, cell proliferation was almost completely inhib-
ited and was significantly lower than the rate
© 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd
  lez et al. Pulpotomy materials and stem cells
Collado-Gonza

(a) (b)

(c) (d)

Figure 1 Representative immunofluorescent images of stained SHEDs, using CD73 (a), CD90 (b), CD105 (c) and a negative
control (d). Cell nuclei (blue) accompanied by the respective cytoplasm (green) characterize stem cells (original magnification
9100).

Figure 2 Cell proliferation and viability were determined using the 3-(4,5-dimethyl-thiazol)-2,5-diphenyl-tetrazolium bromide
(MTT) assay; Theracal LC or IRM led to a significantly lower level of proliferation than the control (***P < 0.001). Also, cell
proliferation in the presence of Biodentine eluates was significantly higher than that obtained using complete medium (control;
**P < 0.01), and MTA Angelus from 48 h of incubation onwards (**P < 0.01). (*P < 0.05; **P < 0.01; ***P < 0.001) anal-
ysed by one- way ANOVA.

obtained in control specimens at all times during the

experiment (P < 0.001). However, MTA Angelus and
Biodentine led to significantly higher cell proliferation
rates than in the control from 48 h onwards
(P < 0.01 and P < 0.001, respectively). Biodentine,
particularly, induced SHED proliferation and led to a
significantly higher rate of proliferation than MTA
Angelus from 48 h of incubation onwards (P < 0.01).
Analysis of mesenchymal phenotype and
apoptosis/necrosis
To confirm the mesenchymal phenotype of SHEDs iso-
lated from primary dental pulp cultures and to deter-
mine possible phenotypic changes after culture with
the eluates of the different pulpotomy materials used,
flow cytometry studies were carried out. The MSC

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  lez et al.
Pulpotomy materials and stem cells Collado-Gonza

surface molecules, CD73, CD90 and CD105, were
expressed at levels >95%, whereas the expression of
the haematopoietic markers, CD14, CD20, CD34 and
CD45, was lower than 5% (Fig. 3). Importantly, after
incubation of SHEDs with the different dilutions of the
eluated extracts (undiluted, 1 : 1, 1 : 2, and 1 : 4),
the percentage of positive expression of these mes-
enchymal markers did not change significantly com-
pared to untreated SHEDs (control).
Representative two-dimensional dot plots of the dis-
tribution of live (Annexin-V
/7-AAD
), early apop-
totic (Annexin-V+/7-AAD
), or late apoptotic and
necrotic (Annexin-V+/7-AAD+ and Annexin-V
/7-
AAD+) cells in untreated SHEDs, or exposed to differ-
ent dilutions of the eluates of the studied pulpotomy
agents are shown in Fig. 4. Undiluted extracts of Bio-
dentine and MTA Angelus were associated with more
than 87% of viable cells after 72 h. By contrast,

Figure 3 Mesenchymal phenotype analysis of SHEDs after culture on pulpotomy material discs by flow cytometry. After 72 h
of culture, cells were detached and labelled with fluorescence-conjugated specific antibodies for the mesenchymal surface mark-
ers CD73, CD90 and CD105, and the haematopoietic markers CD14, CD20, CD34 and CD45. Insert numbers represent the
mean fluorescence intensity values from viable cells. Histograms show representative flow cytometry results obtained after
three independent experiments.

e24 International Endodontic Journal, 50, e19–e30, 2017 © 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd
  lez et al. Pulpotomy materials and stem cells
Collado-Gonza

Figure 4 SHEDs were cultured on Biodentine, MTA Angelus, Theracal LC, IRM and plastic (control) for 72 h, after which the cells
were labelled with Annexin-V and 7-AAD and analysed by flow cytometry. Numbers within the different quadrants represent the per-
centages of live (Annexin-V
/7-AAD
), early apoptotic (Annexin-V+/7-AAD
) or late apoptotic and necrotic cells (Annexin-V+/7-
AAD+ and Annexin-V
/7-AAD+). Dot plots display representative flow cytometry results obtained after three independent experiments.

© 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd International Endodontic Journal, 50, e19–e30, 2017 e25
  lez et al.
Pulpotomy materials and stem cells Collado-Gonza
TheraCal LC and IRM led to a decrease in cell viabil-
ity using the more concentrated eluates (undiluted
and 1 : 2), whilst this cytotoxic effect was lost when
using the most diluted extracts (1 : 4).
Cell migration
To study whether the various pulpotomy materials
could influence cell migration, cell monolayers were
‘wounded’ using a scraper and allowed to heal in the
presence or absence of these pulpotomy material
extracts. Treatment with the different dilutions of Bio-
dentine eluates for 24 h induced a slightly lower,
although still significant (P < 0.001) cell migration
rate than that observed using complete medium (con-
trol), The extracts of MTA Angelus were seen to have
significantly promoted wound closure after 48 h of
treatment, when levels were comparable to those
observed with the control extracts (P < 0.001). How-
ever, after 48 h of incubation IRM and TheraCal LC
(undiluted, 1 : 2 and 1 : 4), cell migration was signif-
icantly lower than the control (P < 0.01; Fig. 5).
SHEDs attachment on pulpotomy material discs
The morphology and adhesion of SHEDs on the sur-
face of Biodentine, MTA Angelus, TheraCal LC and
IRM discs after culture for 72 h are shown in Fig. 6.
SEM analyses revealed better cell growth and spread-
ing on Biodentine and MTA Angelus (Fig. 6a,b,
respectively) compared to that observed on TheraCal
LC or IRM (Fig. 6c,d). More cell-monolayer structures
Figure 5 Migration of SHEDs exposed to extracts of Biodentine, MTA Angelus, Theracal LC and IRM evaluated by the in vitro
scratch wound-healing assay. Confluent SHEDs were wounded and incubated with different dilutions of the pulpotomy material
extracts for up to 48 h. Cell migration is represented as the percentage of the open wound area for each condition compared
with the control. (*P < 0.05; **P < 0.01; ***P < 0.001) analysed by one- way ANOVA.
e26 International Endodontic Journal, 50, e19–e30, 2017
were formed on the surface of Biodentine and MTA
Angelus, suggesting that these pulpotomy agents
have a good porous microstructure that allows better
cell attachment. However, with the IRM and Theracal
LC discs, cell attachment was limited, with only a few
round cells appearing on the surface of the materials.
Alizarin Red Staining (ARS) for matrix calcium
deposition analysis
The deposition of a calcified matrix on the different
materials after culturing SHEDs was verified by Ali-
zarin Red staining. The cells cultured on Biodentine
exhibited a significantly higher level of Alizarin Red
staining than the control after only after 7 days of
cultures, unlike the IRM or Theracal cultures, where
staining was almost absent (P < 0.01; Fig. 7a,b).
Indeed, these differences became more pronounced
with the time of culture, and, after 21 days Alizarin
Red staining using Biodentine revealed a significant
increase in calcification compared to that obtained
with the control or Theracal LC/IRM (P < 0.01 and
P < 0.0001, respectively; Fig. 7b).
Discussion
Biocompatibility is an important property that should
be considered when selecting a material for vital pulp
therapy because of its direct contact with vital tissues
(Lee et al. 2014). In this study, biocompatibility-
related in vitro tests were performed to provide data
on the safe use of biomaterials for vital pulp therapy
© 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd

(a) (b)
(c) (d)
Figure 6 Scanning electron microscopic images of cells cultured on Biodentine (a), MTA Angelus (b), Theracal LC (c), and IRM (d).
The morphology of the cells seeded on Biodentine and MTA Angelus had similar characteristics, with substantial cell spreading
and the production of an extracellular matrix. However, using Theracal LC and IRM, cell attachment was limited. Scale bar: 1009.
in primary teeth. Biodentine, MTA Angelus, TheraCal
LC and IRM were tested to evaluate their cytocompat-
ibility towards SHEDs. Unlike previous reports who
used animal cell lines (de Menezes et al. 2009), the
present study used stem cells from exfoliated cells of
primary dental pulps. These stem cells were found to
be positive for the antibodies against human MSCs
markers CD73, CD90 and CD105 (Fig. 1). Similar
results have been reported previously, in which MSCs
isolated from other sources such as amniotic mem-
brane or adipose tissue were positive for CD54, CD29,
CD73, CD90, CD13, CD44, CD105 and CD166. The
results suggest that the immunophenotypical and
morphological profiles of SHEDs are the same as those
of MSCs isolated from bone marrow (Insausti et al.
2010, 2012).
A number of quantitative and qualitative in vitro
methods are available to evaluate biomaterial cytotox-
icity and potential adverse effects on cell behaviour
(Buttke et al.1993). For example, the colorimetric-
based MTT assay is a suitable technique to quantita-
tively assess cell viability or proliferation. Nonetheless,
this is only one aspect of biocompatibility and cannot
be used alone to characterize a material as being bio-
compatible or nonbiocompatible (Peters 2013).
Hence, cell apoptosis, cell migration, cell attachment
and mineralization in the presence of the aforesaid
© 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd
 lez et al. Pulpotomy materials and stem cells
Collado-Gonza
biomaterials was analysed to obtain more information
in terms of cytocompatibility. The MTT results
showed that neither Biodentine nor MTA was cyto-
toxic for SHEDs, at least in 1 : 1, 1 : 2 and 1 : 4 dilu-
tions. However, in the presence of IRM and Theracal
LC eluates, cell viability was very low or almost
absent. These results corroborate previous studies,
which revealed no significant differences in cell viabil-
ity between MTA Angelus and Biodentine extracts
and their corresponding controls up to 48 h of cul-
ture using dental pulp cells (DPCs) (Dalto
e et al.
2016). However, using the mouse odontoblast cell
line (MDPC-23), Poggio et al. (2015) reported that
Biodentine had better cell biocompatibility than MTA
Angelus and ProRoot MTA eluates after 72 h of
culture.
Although the stem cells derived from the dental
pulp of exfoliated primary teeth were initially charac-
terized by immunocytochemistry staining because of
the clear cytomorphological details the method
reveals, flow cytometry was also used because it per-
mits multiple characteristics to be correlated in single
cells and the identification of the presence of antigens
either on the surface of or within cells. Moreover, the
information obtained is both qualitative and quantita-
tive (Lutfi et al. 2010). With Biodentine and MTA
Angelus, the MSC surface molecules, CD73, CD90
International Endodontic Journal, 50, e19–e30, 2017 e27
  lez et al.
Pulpotomy materials and stem cells Collado-Gonza

Figure 7 Alizarin Red staining of SHEDs in the presence of the pulpotomy materials used. (a) Biodentine stimulated the deposi-
tion of mineralized nodules, an effect that was less pronounced in the case of MTA. (b) Quantification of Alizarin Red staining.
Results are representative of three independent experiments (n = 9). (*P < 0.05; **P < 0.01; ***P < 0.001) analysed by one-
way ANOVA.

and CD105, were expressed at levels >95%, whereas
the expression of the haematopoietic markers, CD34
and CD45, was <5%. However, only with a 1 : 4
dilution of IRM and Theracal LC was a similar expres-
sion obtained for CD 73, probably due to factors
related with cell death.
The method involving the double staining of cells
with Annexin-V and 7-AAD was chosen to evaluate
cell death. Annexin-V staining precedes the loss of
membrane integrity (which accompanies the last
stages of cell death) resulting from either apoptotic or
necrotic processes. Therefore, Annexin-V staining is
typically used in combination with a vital dye such as
7-AAD to identify apoptosis in its different stages. The
results revealed that MTA and Biodentine did not
induce apoptosis, whereas IRM and Theracal LC were
more cytotoxic than the other calcium silicate
cements used, leading to membrane permeability-
related apoptosis and necrosis. The apoptosis results
were consistent with a previous study which showed
that exposure to freshly mixed MTA Angelus, Bioden-
tine, freshly light-cured TheraCalLC or freshly-mixed
IRM caused a significant difference in the percentage
of healthy, nonapoptotic and non-necrotic cells, Ther-
acal LC and IRM being the most cytotoxic (Bortoluzzi
et al. 2015).
In very deep cavities with remaining dentine thick-
ness between 0.25 and 0.01 mm or in pulp exposure
cavities, dental pulp stem cells proliferate, migrate to
the site of injury and differentiate to form odontoblas-
toid cells (Lutfi et al. 2010). Cell migration is neces-
sary for homoeostatic tissue maintenance and the
regeneration of injured organs and tissues (Rennert
et al. 2012). So, dental materials should facilitate
migration of these cells to repair or protect the dental
pulp. In this respect, cell migration of SHEDs has been
tested in the presence of the eluates of various
medicaments. Biodentine and MTA exhibited very
good cell migration at 48 h, especially at the 1 : 4
dilution, whereas IRM and Theracal LC eluates did

e28 International Endodontic Journal, 50, e19–e30, 2017 © 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd

not stimulate cell migration of SHEDs. These cell
migration results agree with those of a study, which
showed that exposure to Biodentine, induced a signifi-
cant increase in cell migration and adhesion of discs
(Luo et al. 2014).
Scanning electronic microscopy (SEM) is a useful
tool for examining the morphology and cell attach-
ment of primary cultured cells seeded on dental mate-
rials (Ahmed et al. 2014). In the present study, SEM
revealed a limited level of cells adhering to IRM and
Theracal LC discs, whereas MTA Angelus and Bioden-
tine permitted extensive cellular colonization of the
discs, allowing the formation of a cell monolayer cov-
ering the entire surface. Differences in cellular attach-
ment and spreading level on different materials may
be attributed to various factors such as chemical com-
position, particle size, surface topography and the
bioactivity of samples (Butt et al. 2014, Utneja et al.
2015). Interestingly, Widbiller et al. (2016) obtained
similar results of cell attachment using hDPSCs and
Biodentine discs.
Extracellular matrix mineralization was also anal-
ysed in the presence of the four pulpotomy materials
tested, using the Alizarin Red staining method.
Amongst them, Biodentine stimulated mineralization
nodule deposition to a significant extent, an effect that
was less pronounced in the case of MTA Angelus.
However, the ability of MTA Angelus to induce cal-
cium deposition has been reported in studies that eval-
uated its bioactivity and potential to induce osteogenic
differentiation (Yasuda et al. 2008, Paranjpe et al.
2011), but the lack of studies evaluating the bioactiv-
ity of Biodentine on SHEDs precludes direct compar-
ison with the results of the present investigation.
Conclusions
Biodentine exhibited better cytocompatibility and
bioactivity than MTA Angelus, Theracal LC and IRM.
Acknowledgements
This work was supported by the Spanish Net of Cell
Therapy (TerCel) provided by the Carlos III Institute
of Health (ISCiii) to JMM (RD12/0019/0001).
Conflict of interest
The authors have stated explicitly that there are no
conflict of interests in connection with this article.
© 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd
 lez et al. Pulpotomy materials and stem cells
Collado-Gonza
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