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Effects of HEMA and TEDGMA on the in vitro odontogenic differentiation

potential of human pulp stem/progenitor cells derived from deciduous teeth

Article  in  Dental materials: official publication of the Academy of Dental Materials · January 2011

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journal homepage: www.intl.elsevierhealth.com/journals/dema

Effects of HEMA and TEDGMA on the in vitro odontogenic

differentiation potential of human pulp stem/progenitor
cells derived from deciduous teeth

Athina Bakopoulou a , Gabriele Leyhausen b , Joachim Volk b , Asterios Tsiftsoglou c ,

Pavlos Garefis a , Petros Koidis a , Werner Geurtsen b,∗,1
a Department of Fixed Prosthesis & Implant Prosthodontics, School of Dentistry, Aristotle University of Thessaloniki, Greece
b Department of Conservative Dentistry, Periodontology & Preventive Dentistry, Medical University of Hannover, Germany
c Department of Pharmacology, School of Pharmaceutical Sciences, Aristotle University of Thessaloniki, Greece

a r t i c l e i n f o a b s t r a c t

Article history: Objectives. The aim of this study was to investigate the effects of HEMA and TEGDMA on the
Received 1 September 2010 odontogenic differentiation potential of dental pulp stem/progenitor cells.
Received in revised form Methods. Dental stem/progenitor cell cultures were established from pulp biopsies of human
19 December 2010 deciduous teeth of 1–3 year-old children (Deciduous Teeth Stem Cells-DTSCs). Cultures
Accepted 10 March 2011 were characterized for stem cell markers, including STRO-1, CD146, CD34, CD45 using
flow cytometry. Cytotoxicity was evaluated with the MTT assay. DTSCs were then induced
for osteo/odontogenic differentiation by media containing dexamethasone, KH2 PO4 ,␤-
Keywords: glycerophosphate and l-ascorbic acid phosphate in the presence of nontoxic concentrations
Resinous monomers of HEMA (0.05–0.5 mM) and TEGDMA (0.05–0.25 mM) for 3 weeks. Additionally, the effects of
Biocompatibility a single exposure (72 h) to higher concentrations of HEMA (2 mM) and TEGDMA (1 mM) were
Stem/progenitor pulp cells also evaluated.
Odontogenic differentiation Results. DTSCs cultures were positive for STRO-1 (7.53 ± 2.5%), CD146 (91.79 ± 5.41%), CD34
Biomineralization (11.87 ± 3.02%) and negative for CD45. In the absence of monomers cell migration, differen-
Reparative dentinogenesis tiation and production of mineralized dentin-like structures could be observed. Cells also
progressively expressed differentiation markers, including dentin sialophosphoprotein-
DSPP, bone sialoprotein-BSP, osteocalcin-OCN and alkaline phosphatase-ALP. On the
contrary, long-term exposure to nontoxic concentrations of HEMA and TEGDMA signifi-
cantly delayed the differentiation and mineralization processes of DTSCs, whereas, one
time exposure to higher concentrations of these monomers almost completed inhibited
mineral nodule formation. BSP, OCN, ALP and DSPP expression were also significantly down-
regulated.
Significance. These findings suggest that HEMA and TEGDMA can severely disturb the odon-
togenic differentiation potential of pulp stem/progenitor cells, which might have significant
consequences for pulp tissue homeostasis and repair.
© 2011 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

∗
Corresponding author at: Tel.: +49 0511 532 4815; fax: +49 0511 532 4811.
E-mail address: Geurtsen.Werner@mh-hannover.de (W. Geurtsen).
1
Professor and Chairman, School of Dentistry, Medical University of Hannover, Carl-Neuberg- Str. 1, 30625, Hannover, Germany; Affiliate
Professor of Restorative Dentistry University of Washington, Seattle, USA.
0109-5641/$ – see front matter © 2011 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.dental.2011.03.002
 d e n t a l m a t e r i a l s 2 7 ( 2 0 1 1 ) 608–617
1. Introduction
Dental composite resin-based materials have been widely
studied for cytotoxicity and genotoxicity in various cell cul-
ture systems [1,2]. These effects have been attributed to the
release of residual monomers or other substances, derived
either from incomplete polymerization or resin degrada-
tion [3]. Among the compounds released from resin-based
materials, the comonomers TEGDMA (triethylene-glycol-
dimethacrylate) and HEMA (2-hydroxy-ethyl-methacrylate)
have been found to induce to a variable level genetic and cellu-
lar toxicologic effects on different mammalian cell types [4,5].
HEMA is one of the most common components of dentin-
adhesive systems, in a concentration ranging from 30 to 55%
and has a pivotal role during the dentin impregnation pro-
cess [6]. Because of its low molecular weight and its relative
hydrophilicity, HEMA can diffuse through the residual dentin
and affect the underlying odontoblast vitality and pulp phys-
iological activity [7]. TEGDMA, on the other hand, is released
in high amounts from polymerized dental resins into aqueous
media and accounts for most of their unreacted double bonds
[8]. Moreover, TEGDMA is a component of dentin adhesives in
contents varying from 25 to 50% [9]. Due to its lipophilic nature,
TEGDMA can easily penetrate the cytosol and membrane lipid
compartments of mammalian cells, causing several cytotoxic
effects [10,11].
There are already studies supporting that these monomers
are able to cause inflammatory responses and to disturb repar-
ative dentinogenesis when directly applied to the human pulp
tissue [12,13]. In addition, previous in vitro studies have shown
that these monomers can cause even at non toxic concen-
trations significant perturbation of the normal differentiation
process of pulp fibroblasts into odontoblasts [14]. They are
also able to affect the physiological mineralization proce-
dures of terminally differentiated cells, such as osteoblasts
[15]. However, there is to our knowledge no information con-
cerning the effects of nontoxic concentrations of these resin
monomers on the odontogenic differentiation potential of
putative dental mesenchymal stem cells (MSCs), which is
essential for the regeneration and repair of the dentin/pulp
complex.
A few years ago, Gronthos et al. identified a popula-
tion of post-natal stem cells in the human dental pulp of
both adult teeth (Dental Pulp Stem Cells, DPSCs) and exfo-
liated deciduous teeth (Stem cells from Human Exfoliated
Deciduous teeth, SHED) [16,17]. These cells represent a pop-
ulation of undifferentiated MSCs, which are characterized by
unlimited self-renewal, colony forming capacity and multipo-
tent differentiation potential into several cell lineages, such
as osteo/odontogenic, neurogenic, adipogenic, chondrogenic
and myogenic, when grown under defined culture conditions
[18]. They remain in a quiescent state in the dental pulp and
can perform continuous cell division during dental pulp tis-
sue injury/regeneration [19]. In addition, these authors have
found that stem cells from the pulp of deciduous teeth repre-
sent a more immature cell population compared those of adult
teeth, as they are characterized by a higher proliferation rate,
increased cell population doublings and higher osteoinductive
capacity in vivo [17].
609
Therefore, it was the objective of this study to investi-
gate the hypothesis that the resinous monomers HEMA and
TEGDMA may play a role in the physiological odontogenic
differentiation process of pulp stem/progenitor cells, which
is indispensible to the repair of the dentin/pulp complex as
a response to external stimuli [20]. Here this hypothesis is
tested in an in vitro system of cultured dental stem/progenitor
cells derived from the pulp of human deciduous teeth (Decid-
uous teeth Stem Cells-DTSCs). The data presented in this
study add significant information concerning the toxicologi-
cal effects of these monomers on matured (differentiated) cell
populations (odontoblasts, osteoblasts), by further clarifying
how pathways regulating cellular homeostasis, dentinogene-
sis and tissue repair may be modified by concentrations well
below those which cause acute toxicity.
2. Materials and methods
2.1. Chemicals and reagents
The monomers TEGDMA and HEMA were gifts from
VOCO (Cuxhaven, Germany). Dulbecco’s modified Eagle’s
medium (DMEM, containing l-glutamine and 2.0 g/l NaHCO3 ),
Trypsin/EDTA and penicillin/streptomycin/amphotericin
B were purchased from Biochrom AG (Berlin, Germany)
and Fetal Bovine Serum (FBS) from LONZA (Verviers,
Belgium). The chemicals MTT [3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide], dexamethasone disodium
phosphate, monopotassium phosphate, ␤-glycerophosphate,
l-ascorbic acid, Alizarin Red S, neutral buffered forma-
lin, cetylpyridinium chloride, Naphtol-AS-MX Phosphate,
N,N-dimethylformamide, Fast Blue BB Salt and Tris-
(hydroxymethyl)-aminomethane were purchased from
Sigma–Aldrich (Taufkirchen, Germany). The mouse anti-
human antibodies CD146-PE, CD34-APC and CD45-PE were
purchased from BD Biosciences (Heidelberg, Germany). The
mouse anti-human antibodies STRO-1-FITC and anti-DSP
(LFMb-21) and the broad spectrum immunoperoxidase ABC
kit were obtained from Santa Cruz Biotechnology, Inc (CA,
U.S.A.). The NucleoSpin RNA II isolation kit was purchased
from Macherey–Nagel (Düren, Germany) and the Robus T
I RT-PCR kit (F-580L) from Finnzymes (Espoo, Finland). The
primers used for the RT-PCR analysis were synthesized by
Biozym Scientific GmbH (Hess. Oldendorf, Germany).
2.2. Cell culture
The human DTSCs cultures used in this study were estab-
lished from the dental pulp of human extracted deciduous
teeth of children aged 1–3 years old. All teeth were healthy
and were extracted due to malposition in the dental arch.
The collection of the samples was performed according to
the guidelines of the Institutional Review Board and the
parents of all donors signed an informed consent form.
For the establishment of cell cultures teeth were disin-
fected and cut around the cementum–enamel junction to
expose the pulp chamber. The pulp tissue was minced into
small fragments, which were placed in 25 cm2 culture flasks
with DMEM, supplemented with 10% FBS, 100 Units/ml peni-
610 d e n t a l m a t e r i a l s 2 7 ( 2 0 1 1 ) 608–617
cillin, 100 mg/ml streptomycin, 0.25 mg/ml Amphotericin B
( = complete culture medium-CCM) and incubated at 37 ◦ C in
10% CO2 . After reaching confluency cells were collected by
treatment with 0.25% trypsin/0.25 mM EDTA and then con-
tinuously passed for further experiments. Cultured DTSCs
in passage numbers from 2–6 from at least three differ-
ent donors were used for all the experiments with similar
results.
2.3. Surface epitope characterization of DTSCs cultures
Before any experiment, DTSCs cultures used in this study were
characterized using surface epitope markers commonly used
for the characterization of MSCs of dental origin [18], includ-
ing STRO-1, CD146 (MUC18), CD34 and CD45. Briefly, cells
were first harvested by trypsinization and washed three times
with PBS. For each analysis 106 cells/tube were first Fc-blocked
with 1 ␮g of human IgG for 10 min at room temperature (RT)
and subsequently stained by incubation with the mouse anti-
human antibodies STRO-1-FITC, CD146-PE, CD34-APC and
CD45-PE for 20 min in the dark at RT. Then, cells were washed
with 2 ml FACS wash solution (dPBS + 1%BSA + 0.1%NaN3 ) and
centrifuged for 5 min at 230 × g. Supernatant was removed,
cells were re-suspended in 200 ␮l FACS solution and analyzed
with a BD LSR II Flow Cytometer (BD Biosciences). A total
of 100,000 events were acquired for each sample. Data were
analyzed using Summit 5.1 software (Beckman Coulter, Inc,
U.S.A.).
2.4. Exposure of DTSCs to HEMA and TEGDMA and
MTT cytotoxicity assay
TEGDMA and HEMA were dissolved in absolute ethanol and
sequentially diluted to get different concentrations of stock
solutions. The monomers were freshly diluted in the culture
medium prior to each experiment. The final concentration
of ethanol did not exceed 0.25% (v/v). Cells incubated with
medium containing 0.25% ethanol served as control. For the
assessment of cytotoxicity DTSCs were seeded in 96-well
plates (5.000 cells/well) and allowed to grow for 24 h. Sub-
sequently, DTSCs were treated with HEMA (0.1–8 mM) and
TEGDMA (0.05–5 mM), for 24, 48 or 72 h. Cell viability was
assessed using the MTT cell viability assay to determine the
mitochondrial dehydrogenase activity. Briefly, at the end of
each incubation period the culture medium was discarded
and 100 ␮l of 5 mg/ml MTT in PBS was added to each well.
The cells were incubated in the dark for 3 h at 37 ◦ C and 10%
CO2 . Then, the MTT solution was discarded and the insolu-
ble formazan was dissolved with DMSO for 20 min RT. The
absorbance was measured against blank (DMSO) at a wave-
length of 570 nm by a microplate reader (Spectra Max 250,
MWG Biotech).
2.5. Induction of odontogenic differentiation in the
presence of HEMA and TEGDMA
For the odontogenic differentiation experiments DTSCs were
exposed to concentrations of HEMA (0.05, 0.1 and 0.5 mM)
and TEGDMA (0.05, 0.1 and 0.25 mM), which were found-based
on the MTT analysis- to have minimal or no cytotoxicity to
the cells (cell viability ≥85% for both monomers after 72-h
exposure). Both control and monomer treated cultures were
induced for odontogenic differentiation by being exposed
to DMEM complete culture medium (CCM), supplemented
additionally with 0.01 ␮M dexamethasone disodium phos-
phate (Dexa), 1.8 mM monopotassium phosphate (KH2 PO4 )
and 5 mM ␤-glycerophosphate (␤-GP) and 100 ␮M l-ascorbic
acid phosphate (l-ascorbic). Cells were treated for a total
period of 3 weeks with the differentiation medium containing
the different concentrations of the monomers being changed
every 3-4 days (long-term exposure). Cultures exposed to nor-
mal CCM without the additional supplements for the same
3-week period were used as negative control (uninduced
control).
In a second series of experiments, DTSCs cultures were
exposed only once to higher concentrations of HEMA (2 mM)
and TEGDMA (1 mM), which were found in the MTT assay to
reduce cell viability by 20–30% after a 72 h exposure. Then, the
medium with the monomers was washed out with PBS and
replaced by differentiation medium (containing Dexa, KH2 PO4 ,
␤-GP and l-ascorbic) without monomers, that was changed
every 3-4 days for the same period of three weeks (short-term
exposure). Purpose of this second series of experiments was to
assess whether a single exposure to these monomers would
be able to irreversibly affect their normal differentiation pro-
cesses. At the end of each week, control and monomer-treated
cultures of both long-term and short-term experiments were
evaluated for mineralization by Alizarin Red S (AR-S) staining
and processed for immunocytochemical analysis of alkaline
phosphatase (ALP) activity.
2.6. Alizarin Red S mineralization assay
For the assessment of in vitro mineralization, cell cultures
were washed twice with PBS (−) (without Ca2+ and Mg2+ )
and fixed with 10% neutral buffered formalin (NBF) for 1 h
at RT. Then, cultures were stained with 1% AR-S (pH 4.2)
for 20 min at RT, followed by rinsing three times with deion-
ized water (dH2 O). Mineralized nodules were photographed
using an inverted microscope (Olympus Optical Co, Ltd, Japan)
equipped with a digital camera (Olympus E-410, Olympus
Optical Co, Ltd, Japan). Quantification of the total mineral-
ized tissue produced per well was performed by extracting
the AR-S from the stained sites by adding 2 ml of cetylpyri-
dinium chloride (CPC) buffer (10%, w/v) in 10 mM Na2 HPO4
(pH 7) for 2 h at 37 ◦ C. Subsequently, 200 ␮l aliquots were
transferred to a 96-well plate and the OD550nm was measured
using a microplate reader (Spectra Max 250, MWG Biotech).
Mineralized nodule formation was represented as OD per
␮g of total cellular protein, determined by Bradford Protein
assay.
2.7. Histhochemical detection of alkaline phosphatase
(ALP) activity
Cells in 6-well-plates were washed twice with PBS (−) and
fixed with 10% NBF, as described in the AR-S protocol.
ALP activity was visualized by incubating the cells for 2 h
at 37 ◦ C with 0.1 mg/ml Naphtol-AS-MX Phosphate in N,N-
dimethylformamide and 0.6 mg/ml Fast Blue BB Salt in 0.2 M
 d e n t a l m a t e r i a l s 2 7 ( 2 0 1 1 ) 608–617 611

Fig. 1 – Single-parameter histograms showing the expression of STRO-1, CD146, CD34 and CD45 in DTSCs cultures
established from the dental pulp of human extracted deciduous teeth of children aged 1-3 years old (Red line: isotype
control, Green line: marker of interest). DTSCs cells were positive for STRO-1, CD34 and CD146 and negative for CD45.
Results from one representative experiment are shown. (For interpretation of the references to color in this figure legend,
the reader is referred to the web version of the article.)

Tris- (hydroxymethyl)-aminomethane buffer (pH 8.9). The
cells were rinsed with dH2 O and evaluated for ALP activ-
ity under an inverted microscope (Olympus Optical Co, Ltd,
Japan).
2.8. Semi-quantitative reverse
transcription/polymerase chain reaction (RT)-PCR analysis
Total RNA was extracted from cells with NucleoSpin RNA II
kit at days 9 and 15 after induction of differentiation. For the
RT-PCR reactions 0.5 ␮g of total RNA was diluted in a 25 ␮l
PCR reaction of 1X PCR reaction buffer containing 1.5 mM
MgCl2 /200 mM each of dNTP/0.04 units/␮l of DyNAzyme EXT
DNA Polymerase/0.1 Units/␮l of AMV Reverse Transcriptase
(RT) and 10 pmol of each human-specific primer sets: bone
sialoprotein (BSP) (sense: 5 -ATGGAGAGGACGCCACGCCT-3 ,
antisense: 5 -GGTGCCCTTGCCCTGCCTTC-3 ), osteocalcin
(OCN) (sense: 5 -GACTGTGACGAGTTGGCTGA-3 , antisense:
5 -AAGAGGAAAGAAGGGTGCCT-3 ), dentin sialophospho-
protein (DSPP) sense: 5 -GGG ACACAGGAAAAGCAGAA-3 ,
antisense: 5 -TGCTCCATTCCCACTAGGAC-3 and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
(sense: 5 -GAAGGTGAAGGTCGGAGT-3 , antisense: 5 -
GAAGATGGTGATGGGATTTC-3 ).  The reactions were
performed in a PCR thermal cycler (Bio-Rad iCycler, Munich,
Germany) at 50 ◦ C for 30 min for cDNA synthesis, 94 ◦ C
for 2 min for one cycle and then 94 ◦ C/(45 s), 56 ◦ C/(60 s),
72 ◦ C/(60 s) for 30 cycles, with a final 10-min extension at
72 ◦ C. RT-PCR products were analyzed by 1.5%, w/v agarose gel
electrophoresis and visualized by ethidium bromide staining.
2.9. Immunocytochemical detection of dentin
sialophosphoprotein (DSPP) expression
DTSCs cultures exposed to HEMA and TEGDMA were pro-
cessed for immunocytochemical detection of DSPP expression
14 days after induction of differentiation. Cells were washed
with PBS (−) and fixed with 10% NBF for 30 min at RT. Cells
were incubated first with 1.5% blocking serum in PBS to
avoid non-specific staining and then with mouse anti human
DSP (LFMb-21) primary antibody (dilution 1:100) for 1 h at RT.
Then cells were incubated with goat anti-mouse secondary
antibody (dilution 1:200) for 1 h at RT and processed for enzy-
matic immunohistochemical staining using a broad spectrum
immunoperoxidase ABC kit according to the manufacturer’s
protocol. Finally, cells were counterstained with hematoxylin
and examined under an inverted microscope.
2.10. Statistical analysis
Each experiment was performed in triplicates and repeated at
least three times. Values were expressed as the mean ± SD.
Statistical analysis of the data was performed using one-
way analysis of variance (ANOVA). Follow-up comparisons
between groups were then carried out using the Tukey multi-
ple comparison test (p < 0.05).
612 d e n t a l m a t e r i a l s 2 7 ( 2 0 1 1 ) 608–617
Fig. 2 – Cytotoxic effects of (a) HEMA and (b) TEGDMA on
the mitochondrial dehydrogenase activity (cell viability) of
DTSCs cells. The cells were exposed to various
concentrations of the monomers for 24, 48 or 72 h and the
mitochondrial activity was determined by measuring the
tetrazolium reduction relative to the negative control (MTT
assay), which was set to 100%. Results are expressed
means ± SD of three independent experiments in triplicate
(n = 3). Asterisks indicate statistically significant differences
from the untreated control group (one-way ANOVA,
followed by Tukey post hoc test, p < 0.05).
3. Results
3.1. Immunophenotypic profiles of DTSCs
The DTSCs cultures used in this study (n = 4) were found to
express the MSCs markers STRO-1 (7.53 ± 2.5%) and CD34
(11.87 ± 3.02%), as well as the perivascular marker CD146,
which was positive in the majority of the cell population
(91.79 ± 5.41%). In contrast, DTSCs did not express the leuko-
cyte precursor marker CD45 (0.88 ± 0.2%), which indicates the
stromal origin of these cells and the absence of hematopoietic
precursor contamination (Fig. 1).
3.2. Cytotoxicity of HEMA and TEGDMA in DTSCs cells
HEMA and TEGDMA caused a time- and concentration-
dependent reduction of the mitochondrial dehydrogenase
activity in DTSCs cells (Fig. 2a and b). HEMA reduced
cell viability by 4–68% at concentrations of 0.1–8 mM and
TEGDMA by 7–72% at concentrations 0.05–5 mM, respec-
tively, after 72-h treatment. Statistically significant differences
compared to the control (p < 0.05) were observed for concen-
trations of HEMA > 0.5 mM and TEGDMA > 0.25 mM. However,
0.05–0.5 mM of HEMA and 0.05–0.25 mM of TEGDMA showed
very little or no effect on the viability of DTSCs cells and for
this reason these concentrations were used for the subsequent
long-term mineralization experiments.
3.3. In vitro mineralization
One week after induction of odontogenic differentiation with
the selected media containing Dexa, ␤-GP, KH2 PO4 and l-
ascorbic, cells of the DTSCs-control cultures started to migrate
inside the confluent monolayers in an oriented manner
(Fig. 3a) and to aggregate forming colony-like clusters or more
organized elongated 3-D structures (Fig. 3b). In this case, an
obvious cell body elongation and polarization of the migrat-
ing cells could be observed (Fig. 3b). Immunocytochemical
analysis also revealed that these cells were strongly posi-
tive for DSPP, which confirms their odontoblastic phenotype
(Fig. 3c and d). The mineralization process in the control cul-
tures initiated inside these cellular aggregates (Fig. 4a and
b) and gradually increased, covering 70–80% of the mono-
layer at the end of the 3-week observation period (Fig. 4c). On
the other hand, the mineralization remained very low in the
uninduced-control cultures, exposed to normal medium with-
out the additional supplements for the same 3-week period
and was only restricted to a few mineralized nodules formed
spontaneously (Fig. 4d–f).
On the contrary, both long-term and short-term exposure
to HEMA and TEGDMA significantly disturbed the normal
differentiation and mineralization processes of DTSCs. More
specifically, in cultures exposed continuously for 3 weeks to
nontoxic concentrations of HEMA (0.05–0.5 mM) and TEGDMA
(0.05–0.25 mM) the production of mineralized matrix was sig-
nificantly more delayed and less extensive compared to the
control cultures. In these cultures, a lower number of miner-
alized nodules, which were of smaller size could be observed
at all time points (7, 14, 21 days) compared to the induced-
control cultures (Fig. 4g–l). On the other hand, mineralization
was significantly disrupted in cultures exposed short-term
(72 h) to higher concentrations of HEMA (2 mM) and TEGDMA
(1 mM) (Fig. 4m–r). In this case, clear morphological alterations
could be observed, especially in TEGDMA-treated cultures,
where cells presented signs of cellular damage (e.g. retraction,
decrease in cellular density, rounding or blebbing), 1 week after
induction of differentiation (Fig. 4p). Despite the fact that these
morphological alterations diminished during the next 3 week
period (Fig. 4q and r), the production of mineralized matrix
remained at low levels, being restricted to a few mineralized
nodules.
These observations were further evaluated by spectropho-
tometric quantification of the mineralized tissue produced,
using the CPC extraction method (Fig. 4). The analysis showed
that the inhibition of mineralization in cultures treated
with the monomers for long-term periods (21 days) was
concentration-dependent and therefore, more pronounced in
cultures exposed to the higher concentrations HEMA (0.5 mM)
and TEGDMA (0.25 mM) tested. In addition, the effects on
 d e n t a l m a t e r i a l s 2 7 ( 2 0 1 1 ) 608–617 613

Fig. 3 – Representative phase contrast microscopy photographs of DTSCs cells 9 days after induction of differentiation. Cells
in adherent monolayers (a) started migrating and forming 3D rounded aggregates or more organized elongated
3D-structures (b). Immunocytochemical analysis revealed a pronounced expression of DSPP, especially inside the organized
structures and in migrating cells forming these structures, which confirms their odontoblastic phenotype (c and d). These
dentinogenic cells showed an obvious elongation and polarization of their cell bodies vertically to the structures and were
finally entrapped within the newly formed dentin matrix (Scale Bars 50 ␮m).

mineralization were significantly more severe during the first the fact that ALP activity progressively increased during the
2 weeks in cultures exposed long-term to HEMA compared to three-week observation period, it did not finally reach the lev-
TEGDMA (p < 0.05). Overall, at the end of the 3-week observa- els of the induced control cultures.
tion period all types of monomer-treated cultures presented a
statistically significant decrease in the amount of mineralized
3.5. Expression of the differentiation markers BSP,
matrix produced, compared to the control cultures (p < 0.05)
DSPP, OCN
(Fig. 4).

DTSCs cultures induced for differentiation progressively

3.4. Alkaline phosphatase (ALP) activity
ALP was strongly expressed in the majority of the cell popu-
lation (80–100%) in DTSCs induced-control cultures, as early
as one week after induction of differentiation (Fig. 5a–c), but
remained very low (<25%) in the uninduced control cultures
(Fig. 5d–f).
The ALP activity was, however, significantly reduced
(50–60% of the cell population) compared to control in DTSCs
cultures induced for differentiation in the presence of HEMA
(0.5 mM and 0.1 mM) (Fig. 5g–i) and TEGDMA (0.25 mM, 0.1 mM)
(Fig. 5j–l), while no significant effect on ALP activity could be
observed for the lower (0.05 mM) HEMA and TEGDMA con-
centrations tested. ALP activity was also strongly inhibited
in DTSCs cultures exposed short term (72 h) to 2 mM HEMA
(Fig. 5m–o) or 1 mM TEGDMA (Fig. 5p–r). In this latter case,
the ALP expression was restricted to less than 50% of the cell
population in HEMA- and less than 25% in TEGDMA-treated
cultures one week after induction of differentiation. Despite
expressed mineralization markers, normally present during
reparative dentinogenesis, including BSP, DSPP and OCN,
as shown by RT-PCR analysis (Fig. 6). The expression of
these markers was, however, obviously reduced or completely
inhibited in cultures exposed to HEMA and TEGDMA. More
specifically, the expression of BSP on day 9 was relatively
reduced compared to control in cultures exposed long-term
to nontoxic concentrations of HEMA (0.5 mM and 0.1 mM)
and TEGDMA (0.25 mM and 0.1 mM) and was significantly
diminished in those exposed short-term to 2 mM HEMA and
1 mM TEGDMA. On day 15, BSP expression recovered to lev-
els comparable to control in long-term exposed cultures, but
it remained significantly reduced in cultures with short-term
exposure. The expression of OCN followed similar patterns
with BSP, showing an obvious reduction on day 9 in all HEMA
and TEGDMA treated cultures, which persisted on day 15 for
the short-term exposed cultures. Finally, the most pronounced
effects were observed in the expression of DSPP, a marker typ-
ical of odontogenic differentiation. The expression of DSPP
614 d e n t a l m a t e r i a l s 2 7 ( 2 0 1 1 ) 608–617

Fig. 4 – Alizarin Red S staining of DTSCs cultures (Scale Bars 50 ␮m). In control cultures induced for differentiation with
Dexa, KH2 PO4 , ␤-GP and l-ascorbic the mineralization process initiated with single mineralized nodules at day 7 (a),
subsequently increased inside the cellular aggregates (day 14) (b) and finally the mineralized tissue covered almost 70–80%
of the monolayer 21 days after induction of differentiation (c). On the contrary, in uninduced control cultures (d–f), exposed to
normal culture medium (CCM) without the additional supplements, the mineralization was very limited. In cultures induced
for differentiation in the continuous presence of non-toxic concentrations of HEMA (g–i) and TEGDMA (j–l) for 21 days the
production of mineralized matrix was significantly more delayed and less extensive compared to the induced-control
cultures. In cultures exposed short-term (72 h) to 2 mM HEMA (m–o) and 1 mM TEGDMA (p–r) the mineralization process was
almost completely inhibited, being restricted to few, sparse mineralized nodules even after three weeks. These data were
also confirmed by spectrophotometric quantification of the AR-S staining, using the CPC extraction method. Data are shown
as mean OD/␮g of total protein ± SD of 3 independent experiments in 6 replicates (n = 3). Asterisks indicate statistically
significant differences in mineralized tissue deposition of HEMA and TEGDMA-treated cultures compared to the
induced-control cultures at each time-point (7, 14, 21 days) (one-way ANOVA, followed by Tukey post hoc test, p < 0.05).

was severely reduced in all types of HEMA- and TEGDMA-
treated cultures without showing any significant recovery on
day 15 (Fig. 6). Overall, the above data suggest that the expres-
sion of differentiation markers was significantly reduced in
monomer-treated cultures, especially to those exposed for
shorter periods (72 h) to higher concentrations of HEMA and
TEGDMA.
4. Discussion
Clinical data and experimental observations have repeatedly
demonstrated that mature dental pulp responds naturally to
external irritations by producing reparative dentin [19–21]. In
cases of a mild pulp injury -caused for example by non cav-
itated stages of enamel caries, slowly progressing dentinal
caries, mild abrasion, erosion, mechanic-chemical irritation
or fracture involving enamel–dentin- the underneath odon-
toblast layer may survive and is stimulated to form tertiary
dentin matrix beneath the injury (reactionary dentin) [22].
On the other hand, in more severe dentinal injuries, such as
those usually occurring during restorative procedures, includ-
ing cavity preparation, acid etching treatment and application
of restorative materials, such as composite resins, especially
in deep cavities with small remaining dentin thickness (RDT)
 d e n t a l m a t e r i a l s 2 7 ( 2 0 1 1 ) 608–617 615

Fig. 5 – Histochemical staining showing ALP activity in DTSCs cultures exposed to various concentrations of HEMA and
TEGDMA. In induced-control cultures ALP was strongly expressed (80–100% of the cell population) as early as 1week (a) after
induction of osteo/odontogenic differentiation and remained stable during the 2nd (b) and 3rd (c) week, whereas in
uninduced- control cultures (d–f) ALP activity was very low (<25%). In cultures exposed continuously for 3 weeks (long-term)
to non toxic concentrations of HEMA (0.5, 0.1 mM) (g–i) and TEGDMA (0.25, 0.1 mM) (j–l) ALP activity was restricted to 50–60%
of the cell population. In cultures exposed short-term (72 h) to 2 mM HEMA (m–o) and 1 mM TEGDMA (p–r) ALP activity was
strongly inhibited to less than 50% of the cell population in HEMA- and less than 25% in TEGDMA-treated cultures (Scale
Bars 50 ␮m).

the odontoblasts subjacent to the affected dentin are often
destroyed and can, therefore, no longer perform repair of the
lesion [20–22]. In this case, the cascade of inflammatory events
occurring in the area of degenerating odontoblasts triggers
a different procedure, in which stem/progenitor cells from
the pulp core recruit toward the dentin-pulp border. These
Fig. 6 – Representative agarose gels containing RT-PCR
products from DTSCs cultures exposed to various
concentrations of HEMA and TEGDMA, at 9 and 15 days
after induction of odontogenic differentiation. Lane 1:
induced control, Lane 2: uninduced control, Lane 3: HEMA
0.5 mM long-term exposure (LT), Lane 4: HEMA 0.1 mM (LT),
Lane 5: HEMA 0.05 mM (LT), Lane 6: TEGDMA 0.25 mM (LT),
Lane 7: TEGDMA 0.1 mM (LT), Lane 8: TEGDMA 0.05 mM
(LT), Lane 9: HEMA 2 mM short-term exposure (ST), Lane 10:
TEGDMA 1 mM (ST). [BSP, bone sialoprotein (product:
322 bp), OC, osteocalcin (product: 137 bp), DSPP, dentin
sialophosphoprotein (product: 422 bp), GAPDH,
glyceraldehyde-3-phosphate dehydrogenase (product:
226 bp)]. a.
cells are competent to differentiate into odontoblast-like or
osteoblast-like cells producing reparative dentin or – in a
non-appropriate pulp environment- a less specific atubular
bone-like matrix (osteo-dentin) [20,22].
Previous studies have shown that this reparative process
can be stimulated as a result of the chemical activity of
restorative materials [20]. It has been also demonstrated that
components frequently present in adhesive and restorative
resins, such as HEMA and TEGDMA respectively, have the
capacity to diffuse through the dentinal tubules and reach
the pulp tissue at significantly high concentrations in the mil-
limolar range [23]. Diffusion of these monomer is increased
when the remaining dentin thickness is decreased or after acid
etching treatment of the dentin [20,23]. HEMA may reach con-
centrations as high as 1.5–8 mmol/L in the pulp [24], whereas
TEGDMA concentrations could be in the range of 4 mmol/L [8].
These concentrations have been found to cause significant
cytotoxicity through mechanisms associated with oxidative
stress, ROS production, depletion of intracellular glutathione
and finally induction of cell death, mainly via apoptosis [25,26].
In this study, we attempted to shed more light on the effects
of these two monomers on the normal differentiation pro-
cess of pulp stem/progenitor cells into odontoblasts, which
is indispensible for the repair of the dentin/pulp complex as a
response to external stimuli [20]. Our study design simulates
very well the in vivo situation, as it has been shown that these
monomers leach from composites in high amounts during
the first days after initial polymerization, but may continue at
lower concentrations for a significant period of time [3,24]. For
this reason, we have evaluated, on the one hand, the impact
of long-term exposure to nontoxic concentrations of HEMA
and TEGDMA on the odontogenic differentiation potential of
616 d e n t a l m a t e r i a l s 2 7 ( 2 0 1 1 ) 608–617
pulp progenitors cells and on the other hand the possibility of
recovery of this normal differentiation procedure after expo-
sure only once to higher concentrations of these monomers.
In the latter case, it should be emphasized that the concen-
trations of HEMA (2 mM) and TEGDMA (1 mM) selected in our
short-term experimental design are well below those reported
to be released by resin-based materials during the first days
after initial polymerization [3,5,8,24].
For the evaluation of these effects we have used a bio-
logical model of cell cultures established from the pulp of
healthy deciduous teeth of children aged 1–3 years old. Previ-
ous studies have shown that the pulp of deciduous teeth hosts
a population of more premature stem/progenitor cells com-
pared to that of adult teeth [17]. In addition, the young age of
the teeth donors secures a very high dentinogenic potential, as
the proportion of competent cells seems to reduce with aging
[27]. To the best of our knowledge, this is the first study eval-
uating the effects of resinous monomers on the odontogenic
differentiation potential of premature stem/progenitor popu-
lations derived from deciduous teeth. The immunophenotypic
characterization of the DTSCs cultures revealed the exis-
tence of a significant percentage of progenitor cells expressing
the stem cell surface markers STRO-1 (7.53 ± 2.5%), CD146
(91.79 ± 5.41%) and CD34 (11.87 ± 3.02%) (Fig. 1), which in accor-
dance with previous data [17]. The absence of expression of the
leukocyte precursor marker CD45 is confirmatory of the stro-
mal origin of these cells and the absence of hematopoietic
precursor contamination.
The evaluation of cytotoxicity of HEMA and TEGDMA in
DTSCs cells showed a time- and concentration-dependent
reduction of the mitochondrial dehydrogenase activity (Fig. 2a
and b), which is in accordance with previous studies
[25,26,28,29]. However, in our study the cytotoxicity of both
monomers was detectable at relatively lower concentrations
(HEMA > 0.5 mM and TEGDMA > 0.25 mM), compared to previ-
ous studies. This can be attributed to the different cells lines
used in various studies, but also to the fact that in our study
cells were seeded for the MTT assay at a relatively low density
(5000 cells/well), which has most probably increased the sen-
sitivity of our culture system, making possible to detect minor
cytotoxic effects at relatively low concentrations.
In this study, we induced cell cultures to differentiate using
media containing Dexa, KH2 PO4, ␤-GP and l-ascorbic. All of
these supplements have been reported to play a significant
role in the enhancement of extracellular mineralized matrix
formation. Dexa enhances extracellular gene expression [30],
l-ascorbic is necessary for the formation of collagenous
matrix, whereas ␤-GP is required for subsequent mineral-
ization. The latter is mainly cell-mediated through the ALP
activity expressed by differentiated odonto/osteogenic cells
[31]. Moreover, KH2 PO4 and ␤-GP act as inorganic and organic
phosphate ion sources respectively, which are necessary for
biomineralization [30].
We have shown that 3-week exposure of DTSCs cultures
to nontoxic concentrations of HEMA and TEGDMA could
significantly delay the physiological migration, differentia-
tion and mineralization processes of these cells (Fig. 3) in a
concentration-dependent manner. The overall production of
mineralized matrix was significantly reduced in all concentra-
tions and time-points evaluated (p < 0.05) (Fig. 4). In addition,
these effects were more evident in cultures exposed long-
term to HEMA as compared to TEGDMA (p < 0.05). This finding
is in accordance with previous mineralization studies using
cultures of pulp fibroblasts [14] and osteoblasts [15], which
have demonstrated that HEMA can severely disrupt the nor-
mal differentiation and mineralization process of these cells
at very low concentrations. On the other hand, the effects on
cell differentiation were very pronounced in cultures exposed
short-term (72 h) to 2 mM HEMA and 1 mM TEGDMA, where
a significant inhibition of the mineralization process could be
observed (Fig. 4). This finding implies that even one time expo-
sure to relatively high concentrations of HEMA and TEGDMA,
such as those released immediately after polymerization, may
significantly disrupt the reparative process of the pulp tissue.
The delay on the mineralized matrix deposition was also
accompanied with significant down-regulation of the expres-
sion of several differentiation markers, including ALP, OCN,
BSP and DSPP (Figs. 5 and 6). Among the markers tested,
DSPP was the most strongly influenced in all types of HEMA-
and TEGDMA-treated cultures. DSPP is the initial transla-
tional product of DSPP mRNA that is then cleaved to DSP and
DPP [32]. Despite the fact that DSPP has been found in trace
amounts in bone [33], it is considered a representative marker
of odontoblastic differentiation, having an active role in the
mineralization of dentin matrix [32]. The very low expres-
sion of DSPP in cultures treated long-term with HEMA and
TEGDMA suggests that even non-toxic concentrations of these
monomers are able to significantly disturb the potential of
DTSCs cells to acquire odontoblastic competence and to dif-
ferentiate into functional odontoblasts producing reparative
dentin. It is also strongly possible that the mineralized matrix
produced by DTSCs cells in HEMA- and TEGDMA-treated cul-
tures was rather in the form of a non-specific bone-like tissue
(osteodentin), which is usually the case in a non-appropriate
pulp environment [22]. On the other hand, due to its pivotal
role in dentin matrix mineralization, the complete absence
of DSPP in cells exposed short-term to 2 mM HEMA and 1 mM
TEGDMA may explain the severe inhibition of mineral nodules
deposition in these cultures.
5. Conclusion
In conclusion, our experiments provide evidence that
long-term exposure to nontoxic concentrations of HEMA
(0.05–0.5 mM) and TEGDMA (0.05–0.25 mM) is able to signifi-
cantly delay the physiological migration, odontogenic differ-
entiation and mineralization process of pulp stem/progenitor
cells of human deciduous teeth in a concentration-dependent
manner. In addition, only one time exposure to higher con-
centrations of HEMA (2 mM) and TEGDMA (1 mM) can almost
completely inhibit reparative dentin formation. Therefore,
our hypothesis that HEMA and TEGDMA can significantly
affect physiological pulp tissue regeneration/repair processes
is clearly supported by our results. Consequently, the data
presented in this study raise significant questions about the
safety of clinical procedures, such as direct pulp capping with
dental adhesives or restoration with dental composites in
deep cavities without the use of cavity liners. In such cases, the
diffusion of resinous monomers into the pulp space may inter-
 d e n t a l m a t e r i a l s 2 7 ( 2 0 1 1 ) 608–617
fere with the critical step of stem\progenitor cells recruitment
and differentiation into functional odontoblasts producing a
reparative dentin barrier. The latter stresses the importance
of a meaningful risk assessment, which should take into
account several factors, such as the pulp condition before
performing a restoration, the properties and handling of the
restorative materials and most importantly the significant
role of the remaining dentin thickness in clinical decision-
making.
Acknowledgement
This study was supported by a grant of DAAD (German Aca-
demic Exchange Service).
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