d e n t a l m a t e r i a l s 2 7 ( 2 0 1 1 ) 608–617
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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
Athina Bakopoulou a , Gabriele Leyhausen b , Joachim Volk b , Asterios Tsiftsoglou c , Pavlos Gareﬁs a , Petros Koidis a , Werner Geurtsen b,∗,1
a b c
Department of Fixed Prosthesis & Implant Prosthodontics, School of Dentistry, Aristotle University of Thessaloniki, Greece Department of Conservative Dentistry, Periodontology & Preventive Dentistry, Medical University of Hannover, Germany 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
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 were established from pulp biopsies of human deciduous teeth of 1–3 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, KH2 PO4 , -
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
glycerophosphate and l-ascorbic acid phosphate in the presence of nontoxic concentrations of HEMA (0.05–0.5 mM) and TEGDMA (0.05–0.25 mM) for 3 weeks. Additionally, the effects of a single exposure (72 h) to higher concentrations of HEMA (2 mM) and TEGDMA (1 mM) were also evaluated. Results. DTSCs cultures were positive for STRO-1 (7.53 ± 2.5%), CD146 (91.79 ± 5.41%), CD34 (11.87 ± 3.02%) and negative for CD45. In the absence of monomers cell migration, differentiation and production of mineralized dentin-like structures could be observed. Cells also progressively expressed differentiation markers, including dentin sialophosphoproteinDSPP, bone sialoprotein-BSP, osteocalcin-OCN and alkaline phosphatase-ALP. On the contrary, long-term exposure to nontoxic concentrations of HEMA and TEGDMA signiﬁcantly 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 downregulated. Signiﬁcance. These ﬁndings suggest that HEMA and TEGDMA can severely disturb the odontogenic 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
2. Here this hypothesis is tested in an in vitro system of cultured dental stem/progenitor cells derived from the pulp of human deciduous teeth (Deciduous teeth Stem Cells-DTSCs).5-diphenyltetrazolium bromide]. Due to its lipophilic nature. l-ascorbic acid. increased cell population doublings and higher osteoinductive capacity in vivo . these authors have found that stem cells from the pulp of deciduous teeth represent a more immature cell population compared those of adult teeth. In addition. HEMA can diffuse through the residual dentin and affect the underlying odontoblast vitality and pulp physiological activity . osteoblasts). U. The pulp tissue was minced into small fragments. neutral buffered formalin. derived either from incomplete polymerization or resin degradation .
Materials and methods
Chemicals and reagents
The monomers TEGDMA and HEMA were gifts from VOCO (Cuxhaven. Alizarin Red S. which were placed in 25 cm2 culture ﬂasks with DMEM.2]. 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.5-dimethylthiazol-2-yl)2. The mouse antihuman antibodies CD146-PE. chondrogenic and myogenic.
Dental composite resin-based materials have been widely studied for cytotoxicity and genotoxicity in various cell culture systems [1.S. They are also able to affect the physiological mineralization procedures of terminally differentiated cells. DPSCs) and exfoliated deciduous teeth (Stem cells from Human Exfoliated Deciduous teeth. Finland). the comonomers TEGDMA (triethylene-glycoldimethacrylate) and HEMA (2-hydroxy-ethyl-methacrylate) have been found to induce to a variable level genetic and cellular toxicologic effects on different mammalian cell types [4. by further clarifying how pathways regulating cellular homeostasis. Germany). These effects have been attributed to the release of residual monomers or other substances. However. in a concentration ranging from 30 to 55% and has a pivotal role during the dentin impregnation process . adipogenic. it was the objective of this study to investigate the hypothesis that the resinous monomers HEMA and TEGDMA may play a role in the physiological odontogenic differentiation process of pulp stem/progenitor cells. These cells represent a population of undifferentiated MSCs. previous in vitro studies have shown that these monomers can cause even at non toxic concentrations signiﬁcant perturbation of the normal differentiation process of pulp ﬁbroblasts into odontoblasts . HEMA is one of the most common components of dentinadhesive systems. causing several cytotoxic effects [10. The chemicals MTT [3-(4. is released in high amounts from polymerized dental resins into aqueous media and accounts for most of their unreacted double bonds . N. TEGDMA.1. identiﬁed a population of post-natal stem cells in the human dental pulp of both adult teeth (Dental Pulp Stem Cells. TEGDMA is a component of dentin adhesives in contents varying from 25 to 50% . as they are characterized by a higher proliferation rate. Fast Blue BB Salt and Tris(hydroxymethyl)-aminomethane were purchased from Sigma–Aldrich (Taufkirchen. Oldendorf. containing l-glutamine and 2. The primers used for the RT-PCR analysis were synthesized by Biozym Scientiﬁc GmbH (Hess. Naphtol-AS-MX Phosphate. In addition. monopotassium phosphate.13].
Therefore. which are characterized by unlimited self-renewal.d e n t a l m a t e r i a l s 2 7 ( 2 0 1 1 ) 608–617
2. Among the compounds released from resin-based materials.11]. Germany) and Fetal Bovine Serum (FBS) from LONZA (Verviers. such as osteoblasts .
The human DTSCs cultures used in this study were established from the dental pulp of human extracted deciduous teeth of children aged 1–3 years old. Dulbecco’s modiﬁed Eagle’s medium (DMEM.A. For the establishment of cell cultures teeth were disinfected and cut around the cementum–enamel junction to expose the pulp chamber. there is to our knowledge no information concerning the effects of nontoxic concentrations of these resin monomers on the odontogenic differentiation potential of putative dental mesenchymal stem cells (MSCs). Belgium). The NucleoSpin RNA II isolation kit was purchased from Macherey–Nagel (Düren. dentinogenesis and tissue repair may be modiﬁed by concentrations well below those which cause acute toxicity. TEGDMA can easily penetrate the cytosol and membrane lipid compartments of mammalian cells. 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. Because of its low molecular weight and its relative hydrophilicity. -glycerophosphate. The data presented in this study add signiﬁcant information concerning the toxicological effects of these monomers on matured (differentiated) cell populations (odontoblasts. which is indispensible to the repair of the dentin/pulp complex as a response to external stimuli . colony forming capacity and multipotent differentiation potential into several cell lineages. All teeth were healthy and were extracted due to malposition in the dental arch. CD34-APC and CD45-PE were purchased from BD Biosciences (Heidelberg. There are already studies supporting that these monomers are able to cause inﬂammatory responses and to disturb reparative dentinogenesis when directly applied to the human pulp tissue [12. on the other hand.0 g/l NaHCO3 ). Trypsin/EDTA and penicillin/streptomycin/amphotericin B were purchased from Biochrom AG (Berlin. supplemented with 10% FBS.5].2. 100 Units/ml peni-
.N-dimethylformamide. Gronthos et al.). which is essential for the regeneration and repair of the dentin/pulp complex. such as osteo/odontogenic. dexamethasone disodium phosphate. Germany) and the Robus T I RT-PCR kit (F-580L) from Finnzymes (Espoo. when grown under deﬁned culture conditions . Inc (CA. A few years ago. Moreover. neurogenic. SHED) [16. Germany). cetylpyridinium chloride. Germany).17]. They remain in a quiescent state in the dental pulp and can perform continuous cell division during dental pulp tissue injury/regeneration . Germany).
1 software (Beckman Coulter. Then. Both control and monomer treated cultures were induced for odontogenic differentiation by being exposed to DMEM complete culture medium (CCM).to have minimal or no cytotoxicity to
2.6 mg/ml Fast Blue BB Salt in 0. 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. CD146-PE. that was changed every 3-4 days for the same period of three weeks (short-term exposure). KH2 PO4 .5. DTSCs cultures were exposed only once to higher concentrations of HEMA (2 mM) and TEGDMA (1 mM). For the assessment of cytotoxicity DTSCs were seeded in 96-well plates (5. the MTT solution was discarded and the insoluble formazan was dissolved with DMSO for 20 min RT.01 M dexamethasone disodium phosphate (Dexa). which were found in the MTT assay to reduce cell viability by 20–30% after a 72 h exposure.25% (v/v). supplemented additionally with 0. for 24. 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 processes. Supernatant was removed. The absorbance was measured against blank (DMSO) at a wavelength of 570 nm by a microplate reader (Spectra Max 250. Then. as described in the AR-S protocol. DTSCs cultures used in this study were characterized using surface epitope markers commonly used for the characterization of MSCs of dental origin . Ltd.2 M
. Then. Brieﬂy. the medium with the monomers was washed out with PBS and replaced by differentiation medium (containing Dexa.05.1%NaN3 ) and centrifuged for 5 min at 230 × g. 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 antihuman antibodies STRO-1-FITC. At the end of each week.7.05–5 mM). cultures were stained with 1% AR-S (pH 4. w/v) in 10 mM Na2 HPO4 (pH 7) for 2 h at 37 ◦ C. which were found-based on the MTT analysis. CD34-APC and CD45-PE for 20 min in the dark at RT. 48 or 72 h.
Surface epitope characterization of DTSCs cultures
Before any experiment. 1.25% trypsin/0.6.
2.25% ethanol served as control. The ﬁnal concentration of ethanol did not exceed 0. 0. CD34 and CD45. The monomers were freshly diluted in the culture medium prior to each experiment. cell cultures were washed twice with PBS (−) (without Ca2+ and Mg2+ ) and ﬁxed with 10% neutral buffered formalin (NBF) for 1 h at RT. DTSCs were treated with HEMA (0. Subsequently. cells were washed with 2 ml FACS wash solution (dPBS + 1%BSA + 0. Japan) equipped with a digital camera (Olympus E-410. Cell viability was assessed using the MTT cell viability assay to determine the mitochondrial dehydrogenase activity. ALP activity was visualized by incubating the cells for 2 h at 37 ◦ C with 0.3. The cells were incubated in the dark for 3 h at 37 ◦ C and 10% CO2 . 100 mg/ml streptomycin. 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). Histhochemical detection of alkaline phosphatase (ALP) activity
Cells in 6-well-plates were washed twice with PBS (−) and ﬁxed with 10% NBF. 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. cells were ﬁrst harvested by trypsinization and washed three times with PBS. After reaching conﬂuency cells were collected by treatment with 0.Ndimethylformamide and 0.5 mM) and TEGDMA (0. Ltd. Induction of odontogenic differentiation in the presence of HEMA and TEGDMA
For the odontogenic differentiation experiments DTSCs were exposed to concentrations of HEMA (0.610
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cillin.000 events were acquired for each sample.
Alizarin Red S mineralization assay
For the assessment of in vitro mineralization. Cells incubated with medium containing 0.25 mM EDTA and then continuously passed for further experiments. Mineralized nodule formation was represented as OD per g of total cellular protein. Then.25 mM).000 cells/well) and allowed to grow for 24 h.05. A total of 100.25 mg/ml Amphotericin B ( = complete culture medium-CCM) and incubated at 37 ◦ C in 10% CO2 . including STRO-1. 0. Cultured DTSCs in passage numbers from 2–6 from at least three different donors were used for all the experiments with similar results.8 mM monopotassium phosphate (KH2 PO4 ) and 5 mM -glycerophosphate ( -GP) and 100 M l-ascorbic acid phosphate (l-ascorbic). Olympus Optical Co. 0.
2. MWG Biotech).1–8 mM) and TEGDMA (0. Quantiﬁcation of the total mineralized tissue produced per well was performed by extracting the AR-S from the stained sites by adding 2 ml of cetylpyridinium chloride (CPC) buffer (10%. 200 l aliquots were transferred to a 96-well plate and the OD550nm was measured using a microplate reader (Spectra Max 250.2) for 20 min at RT. CD146 (MUC18).). MWG Biotech).A. U. Data were analyzed using Summit 5. cells were re-suspended in 200 l FACS solution and analyzed with a BD LSR II Flow Cytometer (BD Biosciences).4. In a second series of experiments.1 and 0. Japan). Inc. Cultures exposed to normal CCM without the additional supplements for the same 3-week period were used as negative control (uninduced control). -GP and l-ascorbic) without monomers. Mineralized nodules were photographed using an inverted microscope (Olympus Optical Co.
the cells (cell viability ≥85% for both monomers after 72-h exposure). followed by rinsing three times with deionized water (dH2 O). 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.1 mg/ml Naphtol-AS-MX Phosphate in N.S.1 and 0. determined by Bradford Protein assay.
2. Subsequently. Brieﬂy.
osteocalcin (OCN) (sense: 5 -GACTGTGACGAGTTGGCTGA-3 . 1 – Single-parameter histograms showing the expression of STRO-1.)
Tris. Then cells were incubated with goat anti-mouse secondary antibody (dilution 1:200) for 1 h at RT and processed for enzymatic immunohistochemical staining using a broad spectrum immunoperoxidase ABC kit according to the manufacturer’s protocol. Ltd.9).5 mM MgCl2 /200 mM each of dNTP/0. 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. the reader is referred to the web version of the article. antisense: 5 -GGTGCCCTTGCCCTGCCTTC-3 ).04 units/ l of DyNAzyme EXT DNA Polymerase/0. CD146.
2. w/v agarose gel electrophoresis and visualized by ethidium bromide staining.(hydroxymethyl)-aminomethane buffer (pH 8.9.05).8.
Each experiment was performed in triplicates and repeated at least three times. with a ﬁnal 10-min extension at
2. 72 ◦ C/(60 s) for 30 cycles. Germany) at 50 ◦ C for 30 min for cDNA synthesis. 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. CD34 and CD146 and negative for CD45. Follow-up comparisons between groups were then carried out using the Tukey multiple comparison test (p < 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 -TGCTCCATTCCCACTAGGAC-3 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (sense: 5 -GAAGGTGAAGGTCGGAGT-3 . RT-PCR products were analyzed by 1. 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. 56 ◦ C/(60 s).
2. (For interpretation of the references to color in this ﬁgure legend. Munich. Japan). Results from one representative experiment are shown. antisense: 5The reactions were GAAGATGGTGATGGGATTTC-3 ).10. Finally. Immunocytochemical detection of dentin sialophosphoprotein (DSPP) expression
DTSCs cultures exposed to HEMA and TEGDMA were processed for immunocytochemical detection of DSPP expression 14 days after induction of differentiation. DTSCs cells were positive for STRO-1. Values were expressed as the mean ± SD.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. The cells were rinsed with dH2 O and evaluated for ALP activity under an inverted microscope (Olympus Optical Co.
72 ◦ C.d e n t a l m a t e r i a l s 2 7 ( 2 0 1 1 ) 608–617
Fig. 94 ◦ C for 2 min for one cycle and then 94 ◦ C/(45 s). dentin sialophosphoprotein (DSPP) sense: 5 -GGG ACACAGGAAAAGCAGAA-3 . Green line: marker of interest). Statistical analysis of the data was performed using oneway analysis of variance (ANOVA).5%. Cells were washed with PBS (−) and ﬁxed with 10% NBF for 30 min at RT. antisense: 5 -AAGAGGAAAGAAGGGTGCCT-3 ). cells were counterstained with hematoxylin and examined under an inverted microscope. Cells were incubated ﬁrst with 1. performed in a PCR thermal cycler (Bio-Rad iCycler.
3. DTSCs did not express the leukocyte precursor marker CD45 (0. In contrast. more pronounced in cultures exposed to the higher concentrations HEMA (0.
Cytotoxicity of HEMA and TEGDMA in DTSCs cells
HEMA and TEGDMA caused a time.1–8 mM and
One week after induction of odontogenic differentiation with the selected media containing Dexa. In addition. Immunocytochemical analysis also revealed that these cells were strongly positive for DSPP. 48 or 72 h and the mitochondrial activity was determined by measuring the tetrazolium reduction relative to the negative control (MTT assay). 3c and d). 4m–r).
In vitro mineralization
Fig. exposed to normal medium without the additional supplements for the same 3-week period and was only restricted to a few mineralized nodules formed spontaneously (Fig.05–5 mM. 3b). 4c).53 ± 2. 1 week after induction of differentiation (Fig. the effects on
. after 72-h treatment.41%). 4d–f). 3a) and to aggregate forming colony-like clusters or more organized elongated 3-D structures (Fig.87 ± 3. HEMA reduced cell viability by 4–68% at concentrations of 0. The cells were exposed to various concentrations of the monomers for 24.5 mM of HEMA and 0. 2 – Cytotoxic effects of (a) HEMA and (b) TEGDMA on the mitochondrial dehydrogenase activity (cell viability) of DTSCs cells. -GP. On the other hand. In this case.612
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TEGDMA by 7–72% at concentrations 0. On the other hand.05) were observed for concentrations of HEMA > 0. 1). KH2 PO4 and lascorbic.05–0.5%) and CD34 (11. The mineralization process in the control cultures initiated inside these cellular aggregates (Fig. the mineralization remained very low in the uninduced-control cultures. In these cultures. the production of mineralized matrix remained at low levels. On the contrary. Results are expressed means ± SD of three independent experiments in triplicate (n = 3).
3. 2a and b). Statistically signiﬁcant differences compared to the control (p < 0. retraction. 4p).
3.25 mM) the production of mineralized matrix was signiﬁcantly more delayed and less extensive compared to the control cultures. especially in TEGDMA-treated cultures.and concentrationdependent reduction of the mitochondrial dehydrogenase activity in DTSCs cells (Fig. 4g–l). covering 70–80% of the monolayer at the end of the 3-week observation period (Fig. which conﬁrms their odontoblastic phenotype (Fig. In this case. followed by Tukey post hoc test. However. 14.05–0. 3b). a lower number of mineralized nodules.05–0. rounding or blebbing). both long-term and short-term exposure to HEMA and TEGDMA signiﬁcantly disturbed the normal differentiation and mineralization processes of DTSCs. an obvious cell body elongation and polarization of the migrating cells could be observed (Fig. which indicates the stromal origin of these cells and the absence of hematopoietic precursor contamination (Fig. which were of smaller size could be observed at all time points (7. which was set to 100%.
3. clear morphological alterations could be observed. Despite the fact that these morphological alterations diminished during the next 3 week period (Fig. More speciﬁcally.5 mM) and TEGDMA (0. cells of the DTSCs-control cultures started to migrate inside the conﬂuent monolayers in an oriented manner (Fig.
Immunophenotypic proﬁles of DTSCs
The DTSCs cultures used in this study (n = 4) were found to express the MSCs markers STRO-1 (7. decrease in cellular density.5 mM) and TEGDMA (0. using the CPC extraction method (Fig.25 mM.79 ± 5. 21 days) compared to the inducedcontrol cultures (Fig.3.2. 0. 4a and b) and gradually increased. 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.88 ± 0. where cells presented signs of cellular damage (e. 4q and r). mineralization was signiﬁcantly disrupted in cultures exposed short-term (72 h) to higher concentrations of HEMA (2 mM) and TEGDMA (1 mM) (Fig. These observations were further evaluated by spectrophotometric quantiﬁcation of the mineralized tissue produced. which was positive in the majority of the cell population (91.25 mM) tested. in cultures exposed continuously for 3 weeks to nontoxic concentrations of HEMA (0. Asterisks indicate statistically signiﬁcant differences from the untreated control group (one-way ANOVA. p < 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.02%).5 mM and TEGDMA > 0.05–0. as well as the perivascular marker CD146.05).1.g. 4). being restricted to a few mineralized nodules. respectively.2%).
The expression of these markers was. 4). showing an obvious reduction on day 9 in all HEMA and TEGDMA treated cultures.1 mM) and TEGDMA (0. as early as one week after induction of differentiation (Fig.25 mM. which conﬁrms their odontoblastic phenotype (c and d). 5a–c). Immunocytochemical analysis revealed a pronounced expression of DSPP.4. Despite
. especially inside the organized structures and in migrating cells forming these structures. obviously reduced or completely inhibited in cultures exposed to HEMA and TEGDMA. DSPP and OCN. however.05 mM) HEMA and TEGDMA concentrations tested. 5j–l).05) (Fig.25 mM and 0. but remained very low (<25%) in the uninduced control cultures (Fig. but it remained signiﬁcantly reduced in cultures with short-term exposure.
mineralization were signiﬁcantly more severe during the ﬁrst 2 weeks in cultures exposed long-term to HEMA compared to TEGDMA (p < 0.1 mM) (Fig. the ALP expression was restricted to less than 50% of the cell population in HEMA. normally present during reparative dentinogenesis. Overall. Finally. which persisted on day 15 for the short-term exposed cultures. On day 15. as shown by RT-PCR analysis (Fig.
Alkaline phosphatase (ALP) activity
ALP was strongly expressed in the majority of the cell population (80–100%) in DTSCs induced-control cultures. it did not ﬁnally reach the levels of the induced control cultures. 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). 5d–f). 5p–r). while no signiﬁcant effect on ALP activity could be observed for the lower (0. In this latter case. 5m–o) or 1 mM TEGDMA (Fig.and less than 25% in TEGDMA-treated cultures one week after induction of differentiation. 3 – Representative phase contrast microscopy photographs of DTSCs cells 9 days after induction of differentiation.1 mM) and was signiﬁcantly diminished in those exposed short-term to 2 mM HEMA and 1 mM TEGDMA. 5g–i) and TEGDMA (0. the most pronounced effects were observed in the expression of DSPP. ALP activity was also strongly inhibited in DTSCs cultures exposed short term (72 h) to 2 mM HEMA (Fig. including BSP.5 mM and 0. Cells in adherent monolayers (a) started migrating and forming 3D rounded aggregates or more organized elongated 3D-structures (b). DSPP.05).5. a marker typical of odontogenic differentiation.
3.5 mM and 0. The ALP activity was. signiﬁcantly reduced (50–60% of the cell population) compared to control in DTSCs cultures induced for differentiation in the presence of HEMA (0. More speciﬁcally. The expression of OCN followed similar patterns with BSP. however. 6). at the end of the 3-week observation period all types of monomer-treated cultures presented a statistically signiﬁcant decrease in the amount of mineralized matrix produced.1 mM) (Fig. 0.
the fact that ALP activity progressively increased during the three-week observation period.d e n t a l m a t e r i a l s 2 7 ( 2 0 1 1 ) 608–617
Fig. the expression of BSP on day 9 was relatively reduced compared to control in cultures exposed long-term to nontoxic concentrations of HEMA (0. OCN
DTSCs cultures induced for differentiation progressively expressed mineralization markers. BSP expression recovered to levels comparable to control in long-term exposed cultures. The expression of DSPP
3. compared to the control cultures (p < 0. Expression of the differentiation markers BSP.
was severely reduced in all types of HEMA. On the other hand. Data are shown as mean OD/ g of total protein ± SD of 3 independent experiments in 6 replicates (n = 3).614
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Fig. especially to those exposed for shorter periods (72 h) to higher concentrations of HEMA and TEGDMA. 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).
4. 6). such as composite resins. the above data suggest that the expression of differentiation markers was signiﬁcantly reduced in monomer-treated cultures. 21 days) (one-way ANOVA. 4 – Alizarin Red S staining of DTSCs cultures (Scale Bars 50 m). On the contrary. erosion. In cases of a mild pulp injury -caused for example by non cavitated stages of enamel caries.
Clinical data and experimental observations have repeatedly demonstrated that mature dental pulp responds naturally to
external irritations by producing reparative dentin [19–21]. 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 more severe dentinal injuries. using the CPC extraction method. such as those usually occurring during restorative procedures. In control cultures induced for differentiation with Dexa. in uninduced control cultures (d–f). followed by Tukey post hoc test. Overall. 14. p < 0. -GP and l-ascorbic the mineralization process initiated with single mineralized nodules at day 7 (a). KH2 PO4 . mild abrasion. 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. especially in deep cavities with small remaining dentin thickness (RDT)
. the mineralization was very limited. These data were also conﬁrmed by spectrophotometric quantiﬁcation of the AR-S staining. exposed to normal culture medium (CCM) without the additional supplements. sparse mineralized nodules even after three weeks. acid etching treatment and application of restorative materials. slowly progressing dentinal caries. mechanic-chemical irritation or fracture involving enamel–dentin. 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 (7.05). being restricted to few.the underneath odontoblast layer may survive and is stimulated to form tertiary dentin matrix beneath the injury (reactionary dentin) .and TEGDMAtreated cultures without showing any signiﬁcant recovery on day 15 (Fig. including cavity preparation.
but may continue at lower concentrations for a signiﬁcant period of time [3. 0.1 mM) (j–l) ALP activity was restricted to 50–60% of the cell population. HEMA may reach concentrations as high as 1.5. These
Fig.24]. OC.1 mM (LT). In this case. mainly via apoptosis [25. at 9 and 15 days after induction of odontogenic differentiation. 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. whereas TEGDMA concentrations could be in the range of 4 mmol/L . DSPP. It has been also demonstrated that components frequently present in adhesive and restorative resins.05 mM (LT). Diffusion of these monomer is increased when the remaining dentin thickness is decreased or after acid etching treatment of the dentin [20.a less speciﬁc atubular bone-like matrix (osteo-dentin) [20. as it has been shown that these monomers leach from composites in high amounts during the ﬁrst days after initial polymerization.d e n t a l m a t e r i a l s 2 7 ( 2 0 1 1 ) 608–617
Fig.23]. Previous studies have shown that this reparative process can be stimulated as a result of the chemical activity of restorative materials . Lane 5: HEMA 0. therefore. no longer perform repair of the lesion [20–22]. In cultures exposed continuously for 3 weeks (long-term) to non toxic concentrations of HEMA (0. the impact of long-term exposure to nontoxic concentrations of HEMA and TEGDMA on the odontogenic differentiation potential of
. we have evaluated. Lane 1: induced control. whereas in uninduced. 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. the cascade of inﬂammatory events occurring in the area of degenerating odontoblasts triggers a different procedure. GAPDH. Lane 6: TEGDMA 0.
cells are competent to differentiate into odontoblast-like or osteoblast-like cells producing reparative dentin or – in a non-appropriate pulp environment. Lane 4: HEMA 0.1 mM) (g–i) and TEGDMA (0.5 mM long-term exposure (LT). 6 – Representative agarose gels containing RT-PCR products from DTSCs cultures exposed to various concentrations of HEMA and TEGDMA.25 mM (LT). Lane 2: uninduced control.25. [BSP. Lane 7: TEGDMA 0.control cultures (d–f) ALP activity was very low (<25%). such as HEMA and TEGDMA respectively. glyceraldehyde-3-phosphate dehydrogenase (product: 226 bp)]. a. These concentrations have been found to cause signiﬁcant cytotoxicity through mechanisms associated with oxidative stress. have the capacity to diffuse through the dentinal tubules and reach the pulp tissue at signiﬁcantly high concentrations in the millimolar range . on the one hand. Lane 9: HEMA 2 mM short-term exposure (ST). depletion of intracellular glutathione and ﬁnally induction of cell death.and less than 25% in TEGDMA-treated cultures (Scale Bars 50 m). in which stem/progenitor cells from the pulp core recruit toward the dentin-pulp border.
the odontoblasts subjacent to the affected dentin are often destroyed and can. For this reason.26]. Lane 10: TEGDMA 1 mM (ST). which is indispensible for the repair of the dentin/pulp complex as a response to external stimuli . Our study design simulates very well the in vivo situation. bone sialoprotein (product: 322 bp). 5 – Histochemical staining showing ALP activity in DTSCs cultures exposed to various concentrations of HEMA and TEGDMA.05 mM (LT).22]. Lane 8: TEGDMA 0. dentin sialophosphoprotein (product: 422 bp). 0. osteocalcin (product: 137 bp).1 mM (LT). we attempted to shed more light on the effects of these two monomers on the normal differentiation process of pulp stem/progenitor cells into odontoblasts. ROS production. In this study. Lane 3: HEMA 0.5–8 mmol/L in the pulp .
including ALP. The absence of expression of the leukocyte precursor marker CD45 is conﬁrmatory of the stromal origin of these cells and the absence of hematopoietic precursor contamination. the effects on cell differentiation were very pronounced in cultures exposed short-term (72 h) to 2 mM HEMA and 1 mM TEGDMA.87 ± 3.
5. where a signiﬁcant inhibition of the mineralization process could be observed (Fig. This can be attributed to the different cells lines used in various studies.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 exposure only once to higher concentrations of these monomers. which is in accordance with previous studies [25. 4). The latter is mainly cell-mediated through the ALP activity expressed by differentiated odonto/osteogenic cells .
these effects were more evident in cultures exposed longterm to HEMA as compared to TEGDMA (p < 0. having an active role in the mineralization of dentin matrix .41%) and CD34 (11. The delay on the mineralized matrix deposition was also accompanied with signiﬁcant down-regulation of the expression of several differentiation markers. The very low expression 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 differentiate into functional odontoblasts producing reparative dentin. CD146 (91. but also to the fact that in our study cells were seeded for the MTT assay at a relatively low density (5000 cells/well). In addition. which are necessary for biomineralization . our experiments provide evidence that long-term exposure to nontoxic concentrations of HEMA (0. compared to previous studies.29]. only one time exposure to higher concentrations of HEMA (2 mM) and TEGDMA (1 mM) can almost completely inhibit reparative dentin formation. l-ascorbic is necessary for the formation of collagenous matrix. In this study. In such cases. which have demonstrated that HEMA can severely disrupt the normal differentiation and mineralization process of these cells at very low concentrations.05) (Fig. Therefore. Previous studies have shown that the pulp of deciduous teeth hosts a population of more premature stem/progenitor cells compared to that of adult teeth . The overall production of mineralized matrix was signiﬁcantly reduced in all concentrations and time-points evaluated (p < 0. 3) in a concentration-dependent manner. due to its pivotal role in dentin matrix mineralization. In addition. Dexa enhances extracellular gene expression . making possible to detect minor cytotoxic effects at relatively low concentrations. Moreover.79 ± 5. may signiﬁcantly disrupt the reparative process of the pulp tissue. For the evaluation of these effects we have used a biological model of cell cultures established from the pulp of healthy deciduous teeth of children aged 1–3 years old.5 mM and TEGDMA > 0. OCN. the diffusion of resinous monomers into the pulp space may inter-
. DSPP is the initial translational product of DSPP mRNA that is then cleaved to DSP and DPP . It is also strongly possible that the mineralized matrix produced by DTSCs cells in HEMA.
In conclusion. We have shown that 3-week exposure of DTSCs cultures to nontoxic concentrations of HEMA and TEGDMA could signiﬁcantly delay the physiological migration. However. 5 and 6).26. which in accordance with previous data . we induced cell cultures to differentiate using media containing Dexa. which is usually the case in a non-appropriate pulp environment . All of these supplements have been reported to play a signiﬁcant role in the enhancement of extracellular mineralized matrix formation. our hypothesis that HEMA and TEGDMA can signiﬁcantly affect physiological pulp tissue regeneration/repair processes is clearly supported by our results.8. such as direct pulp capping with dental adhesives or restoration with dental composites in deep cavities without the use of cavity liners. which has most probably increased the sensitivity of our culture system.05–0. odontogenic differentiation and mineralization process of pulp stem/progenitor cells of human deciduous teeth in a concentration-dependent manner. This ﬁnding is in accordance with previous mineralization studies using cultures of pulp ﬁbroblasts  and osteoblasts . On the other hand. this is the ﬁrst study evaluating the effects of resinous monomers on the odontogenic differentiation potential of premature stem/progenitor populations derived from deciduous teeth. in our study the cytotoxicity of both monomers was detectable at relatively lower concentrations (HEMA > 0.24].and TEGDMA-treated cultures was rather in the form of a non-speciﬁc bone-like tissue (osteodentin). On the other hand. Despite the fact that DSPP has been found in trace amounts in bone .25 mM) is able to signiﬁcantly delay the physiological migration.5. it is considered a representative marker of odontoblastic differentiation.28.25 mM). whereas -GP is required for subsequent mineralization. -GP and l-ascorbic. 2a and b). 4).5%). Consequently.53 ± 2. In addition. the data presented in this study raise signiﬁcant questions about the safety of clinical procedures. such as those released immediately after polymerization. 1). BSP and DSPP (Figs. To the best of our knowledge.05). it should be emphasized that the concentrations 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. This ﬁnding implies that even one time exposure to relatively high concentrations of HEMA and TEGDMA. 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. In the latter case. DSPP was the most strongly inﬂuenced in all types of HEMAand TEGDMA-treated cultures. as the proportion of competent cells seems to reduce with aging .5 mM) and TEGDMA (0. KH2 PO4 and -GP act as inorganic and organic phosphate ion sources respectively. The immunophenotypic characterization of the DTSCs cultures revealed the existence of a signiﬁcant percentage of progenitor cells expressing the stem cell surface markers STRO-1 (7. the young age of the teeth donors secures a very high dentinogenic potential.05–0. differentiation and mineralization processes of these cells (Fig. The evaluation of cytotoxicity of HEMA and TEGDMA in DTSCs cells showed a time. Among the markers tested. KH2 PO4.and concentration-dependent reduction of the mitochondrial dehydrogenase activity (Fig.02%) (Fig.
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