608 617 Dental-Materials

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d e n t a l m a t e r i a l s 2 7 ( 2 0 1 1 ) 608617

a v a i l a bl e a t w w w . s c i en c e d i r e c t .c o m

j o u r n a l h o m e p a g e : w w w . i n t l . e l s e v i e r h e a l t h . c o m / j o u r n a l s / d e m a

Effects of HEMA and TEDGMA on the in vitro odontogenic

differentiation potential of human pulp stem/progenitor

cells derived from deciduous teeth

Athina Bakopouloua, 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, Germanyc Department of Pharmacology, School of Pharmaceutical Sciences, Aristotle University of Thessaloniki, Greece

a r t i c l e i n f o

Article history:

Received 1 September 2010

Received in revised form

19 December 2010

Accepted 10 March 2011

Keywords:Resinous monomers

Biocompatibility

Stem/progenitor pulp cells

Odontogenic differentiation

Biomineralization

Reparative dentinogenesis

a b s t r a c t

Objectives. The aim of this study was to investigate the effects of HEMA and TEGDMA on the

odontogenic differentiation potential of dental pulp stem/progenitor cells.

Methods. Dental stem/progenitor cell cultures wereestablished from pulp biopsies of human

deciduous teeth of 13 year-old children (Deciduous Teeth Stem Cells-DTSCs). Cultures

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, KH2PO4,-

glycerophosphateand l-ascorbic acid phosphate in the presence of nontoxic concentrationsof HEMA (0.050.5mM) and TEGDMA (0.050.25 mM) for 3 weeks. Additionally, the effects of

a single exposure (72 h) to higher concentrations of HEMA (2 mM) and TEGDMA (1mM) were

also evaluated.

Results. DTSCs cultures were positive for STRO-1 (7.532.5%), CD146 (91.795.41%), CD34

(11.87 3.02%) and negative for CD45. In the absence of monomers cell migration, differen-

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 expressionwere 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: [email protected] (W. Geurtsen).

1 Professor and Chairman, School of Dentistry, Medical University of Hannover, Carl-Neuberg- Str. 1, 30625, Hannover, Germany;AffiliateProfessor 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
http://dx.doi.org/10.1016/j.dental.2011.03.002mailto:[email protected]://dx.doi.org/10.1016/j.dental.2011.03.002http://dx.doi.org/10.1016/j.dental.2011.03.002mailto:[email protected]://dx.doi.org/10.1016/j.dental.2011.03.002


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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 aqueousmedia and accounts for most of their unreacted double bonds

[8]. Moreover, TEGDMA is a component of dentin adhesives in

contentsvaryingfrom 25 to 50%[9]. Dueto itslipophilic 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

areableto cause inflammatory responsesand to disturbrepar-

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 arealso 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 higherosteoinductive

capacity in vivo [17].

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). Dulbeccos modified Eagles

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 fromSigmaAldrich (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 MachereyNagel (Dren, 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 13 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 cementumenamel 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-
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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 dH2O 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 0.5g of total RNA was diluted in a 25l

PCR reaction of 1X PCR reaction buffer containing 1.5mM

MgCl2/200mM each of dNTP/0.04 units/l of DyNAzyme EXT

DNA Polymerase/0.1Units/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 30min for cDNA synthesis, 94C

for 2 min for one cycle and then 94C/(45s), 56 C/(60s),

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 dentinsialophosphoprotein (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 30min 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 1h at RT and processed for enzy-matic immunohistochemical staining using a broad spectrum

immunoperoxidase ABC kit according to the manufacturers

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 meanSD.

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).


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612 d e n t a l m a t e r i a l s 2 7 ( 2 0 1 1 ) 608617

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

meansSD 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 (7.532.5%) 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 468% at concentrations of 0.18 mM and

TEGDMA by 772% at concentrations 0.055mM, respec-

tively, after 72-h treatment. Statisticallysignificantdifferences

compared to the control (p < 0.05) were observed for concen-

trations of HEMA > 0.5mM and TEGDMA > 0.25 mM. However,

0.050.5mM of HEMA and 0.050.25mM of TEGDMA showed

very little or no effect on the viability of DTSCs cells and for

this reasonthese concentrations wereused forthe subsequent

long-term mineralization experiments.

3.3. In vitro mineralization

One week after induction of odontogenic differentiation with

the selected media containing Dexa, -GP, KH2PO4 and l-

ascorbic, cells of the DTSCs-controlcultures 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 7080% 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 normalmedium with-

out the additional supplements for the same 3-week period

and was only restricted to a few mineralized nodules formed

spontaneously (Fig. 4df).

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 (0.050.5mM) and TEGDMA

(0.050.25 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. 4gl). On the other hand, mineralization

was significantly disrupted in cultures exposed short-term

(72 h) to higher concentrations of HEMA (2 mM) and TEGDMA

(1mM)(Fig. 4mr). 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).Despitethefactthatthese

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 (21days) was

concentration-dependent and therefore, more pronounced in

cultures exposed to the higher concentrations HEMA (0.5 mM)

and TEGDMA (0.25mM) tested. In addition, the effects on


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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 50m).

mineralization were significantly more severe during the first2 weeks in cultures exposed long-term to HEMA compared to

TEGDMA (p < 0.05). Overall, at the end of the 3-week observa-

tion period all types of monomer-treated cultures presented a

statistically significant decrease in the amount of mineralized

matrix produced, compared to the control cultures (p


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Fig. 4 Alizarin Red S staining of DTSCs cultures (Scale Bars 50m). In control cultures induced for differentiation withDexa, KH2PO4, -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 7080%

of the monolayer 21 days after induction of differentiation (c). On the contrary, in uninduced control cultures (df), 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 (gi) and TEGDMA (jl) 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 (mo) and 1 mM TEGDMA (pr) 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 proteinSD 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 [1921]. 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 enameldentin- 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)


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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 (80100% 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 inuninduced- control cultures (df) ALP activity was very low (


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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 (1mM) selected in our

short-term experimental design arewell 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 13 years old. Previ-

ous studies have shown thatthe 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 donorssecures 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-

lationsderived fromdeciduous teeth.The immunophenotypic

characterization of the DTSCs cultures revealed the exis-tence of a significant percentage of progenitorcells expressing

the stem cell surface markers STRO-1 (7.532.5%), CD146

(91.79 5.41%)and CD34 (11.87 3.02%)(Fig.1), whichin accor-

dance with previousdata [17]. Theabsence 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 > 0.5mM 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, KH2PO4, -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, KH2PO4 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 wassignificantly reduced in all concentra-

tions and time-points evaluated (p


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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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