Effects of resinous monomers on the odontogenic differentiation and mineralization potential of highly proliferative and clonogenic cultured apical papilla stem cells

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

Effects of resinous monomers on the odontogenic differentiation and mineralization potential of highly proliferative and clonogenic cultured apical papilla stem cells Athina Bakopoulou a,b , Gabriele Leyhausen b , Joachim Volk b , Petros Koidis a , Werner Geurtsen b,∗,1 a

Department of Fixed Prosthesis & Implant Prosthodontics, School of Dentistry, Aristotle University of Thessaloniki, Thessaloniki GR-54124, Greece b Department of Conservative Dentistry, Periodontology & Preventive Dentistry, School of Dentistry, Hannover Medical School, Hannover D-30625, Germany

a r t i c l e

i n f o

a b s t r a c t

Article history:

Objective. The aim of this study was to investigate the effects of resinous monomers on the

Received 29 September 2011

odontogenic differentiation and mineralization potential of apical papilla stem cells (SCAP).

Received in revised form

Methods. Cultures were established from developing third molars of healthy donors aged

1 December 2011

14–18 years-old and were extensively characterized for proliferation rate, colony form-

Accepted 4 January 2012

ing unit efficiency and expression of stem cell markers (STRO-1, CD146, CD34, CD45, CD105, CD117-c-Kit, CD24, CD90, Nanog, Oct3/4), in order to select those with enhanced stem cell and odontogenic differentiation properties. SCAP enriched cultures were then

Keywords:

induced for odontogenic differentiation in the continuous presence of low concentrations

Resinous monomers

(0.05–0.5 mM) of the monomers 2-hydroxy-ethyl-methacrylate-HEMA and triethylene-

Biomineralization

glycol-dimethacrylate-TEGDMA for 3 weeks (long-term exposure). Additionally, the effects

Dental pulp repair

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

Odontogenic differentiation

were evaluated.

Apical papilla stem cells

Results. The results showed that both types of monomer-exposure significantly delayed the odontogenic differentiation and mineralization processes of SCAP cells. A downregulation followed by recovery in the expression of differentiation markers, including dentin sialophosphoprotein-DSPP, bone sialoprotein-BSP, osteocalcin-OCN and alkaline phosphatase-ALP was recorded. This was accompanied by reduction of the mineralized matrix produced by monomer-treated-compared to non-treated contol cultures. Furthermore, a concentration-dependence was observed for both monomers during long-term exposure, whereas the effects of HEMA were evident at much lower concentrations compared to TEGDMA.

∗

Corresponding author at: Department of Conservative Dentistry, Periodontology and Preventive Dentistry, School of Dentistry, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany. Tel.: +49 0511 532 4815, fax: +49 0511 532 4811. E-mail address: [email protected] (W. Geurtsen). 1 Address: Affiliate Professor of Restorative Dentistry, University of Washington, Seattle, USA. 0109-5641/$ – see front matter © 2012 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.dental.2012.01.002

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Significance. These findings suggest that resinous monomers can delay the odontogenic differentiation of SCAP cells, potentially disturbing the physiological repair and/or developmental processes of human permanent teeth. © 2012 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

1.

Introduction

Residual substances released from resin-based dental restorative materials have been well-documented with respect to cytotoxicity and genotoxicity in various cell culture systems [1–3]. These effects have been attributed to the release of residual monomers or other substances derived from incomplete polymerization or resin degradation over time. Some of these compounds, such as the monomers 2-hydroxy-ethylmethacrylate (HEMA) and triethylene-glycol-dimethacrylate (TEGDMA) have been also found able to diffuse through the dentinal tubules and reach the pulp tissue at significantly high concentrations in the low millimolar range, even in the presence of an intact dentin barrier [4–6]. These concentrations are able to cause significant cytotoxicity in oral fibroblasts through mechanisms associated with oxidative stress, expressed via production of Reactive Oxygen Species (ROS), depletion of intracellular glutathione (GSH), cell cycle delays and finally induction of cell death, mainly via apoptosis [7–10]. However, there is until now only fragmentary information available on the biological effects of resinous substances released at low concentrations for extended periods of time during the long-term clinical service of these materials on primary recipient tissues, such as the dental pulp. Two previous in vitro studies have shown that long-term exposure to non-toxic concentrations of resinous monomers is able to cause significant disturbance of the differentiation processes of dental pulp cells [11,12]. In addition, in vivo studies on pulp responses have shown that direct capping of exposed pulps with resin adhesives causes a persistent chronic inflammatory reaction associated with lack of mineralized barrier formation [13–15], whereas application of resinous materials into deep cavities can be also associated with slight to moderate inflammation depending on the materials used and the remaining dentin thickness [16,17]. These clinical data suggest that resinous materials may be a continuous and critical source of xenobiotics leaching into the pulp cavity. However, only scarce data exist on the possible underlying mechanisms leading to a compromised pulp repair response, due to continuous exposure of pulp cells to resinous substances. Most recently, we have shown that two frequently and in high amounts eluted resin comonomers (HEMA and TEGDMA) at very low (non-toxic) concentrations were able to significantly disturb or completely inhibit the physiologic migration, odontogenic differentiation and mineralization processes of human pulp stem/progenitor cells derived from deciduous teeth [18]. Taking a step forward, we further evaluated in this study the long-term effects of non-toxic concentrations of these monomers on a highly proliferative and clonogenic stem cell population derived from the apical papilla of human permanent developing teeth. These cells were

extensively characterized with respect to stem cell properties and presented significant expression of mesenchymal and embryonic stem cell markers, high colony forming efficiency and most importantly pronounced odontogenic differentiation and mineralization potential. The rationale of this study was to give more insight into the effects of very low concentrations of resinous monomers on dental pulp stem cell populations possessing a very high regenerative potential and that could play a significant role on the repair/regeneration of the dentin/pulp complex as a response to external stimuli [19,20]. Our research hypothesis was that non-toxic concentrations of resinous monomers commonly diffusing into the pulp space are able to disturb the physiological differentiation processes of highly potent stem/progenitor cells, therefore jeopardizing the physiological repair and/or developmental processes of human permanent teeth.

2.

Materials and methods

2.1.

Chemicals and reagents

The monomers TEGDMA and HEMA were gifts from VOCO (Cuxhaven, Germany). The culture medium (␣-Modification of Eagle’s Medium-␣-MEM with nucleosides) and the enzyme collagenase type I were purchased from Gibco/Invitrogen (Karlsruhe, Germany), whereas the enzyme dispase from Roche Diagnostics GmbH (Mannheim, Germany). 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 formalin, 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 phycoerythrin (PE)conjugated CD146, allophycocyanin (APC)-conjugated CD34, PE-conjugated CD45, Peridinin-Chlorophyll-Protein-cyanin 5.5 dye (PerCP-Cy5.5)-conjugated CD117, Fluorescein isothiocyanate (FITC)-conjugated CD105, PE-conjugated Nanog and Alexa Fluor 647-conjugated Oct3/4 were purchased from BD Biosciences (Heidelberg, Germany). The fixation, permeabilization and staining buffers for flow cytometry were also purchased from BD Biosciences. The mouse anti-human antibody FITC-conjugated STRO-1 was obtained from Santa Cruz Biotechnology, Inc. (California, U.S.A.) and the mouse antihuman APC-conjugated CD24 and FITC-conjugated CD90 from BioLegend (San Diego, 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

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synthesized by Biozym Scientific GmbH (Hess. Oldendorf, Germany). The Bicinchoninic Acid (BCA) Protein assay was obtained from Thermo Fisher Scientific (Schwerte, Germany).

2.2.

Cell culture

The human SCAP cultures used in this study were established from the apical papilla of normal impacted third molars at the stage of root development (two thirds of the root completed). The teeth were extracted from young healthy donors aged 14–18 years for orthodontic reasons or due to lack of adequate space for eruption. The collection of the samples was performed according to the guidelines of the Institutional Review Board and all donors or their parents signed an informed consent form. Cell cultures were established using the enzymatic dissociation method, as described in previous publications by our group [20,21]. Briefly, teeth were washed, disinfected and the apical papilla was retrieved through the apical part of the incomplete roots. The tissue was minced into small segments and digested in a solution of 3 mg/ml collagenase type I and 4 mg/ml dispase for 1 h at 37 ◦ C. Single-cell suspensions were obtained by passing the cells through a 70 ␮m cell strainer. Cells were seeded at a density of 104 /cm2 using ␣-MEM culture medium, supplemented with 15% FBS, 100 ␮M l-ascorbic acid phosphate, 2 mM l-glutamine, 100 units/ml penicillin, 100 mg/ml streptomycin and 0,25 mg/ml Amphotericin B (=complete culture medium – CCM) and incubated at 37 ◦ C in 5% CO2 . After reaching 80–90% confluency, cells were collected by treatment with 0.25% trypsin/0.25 mM EDTA and then continuously passed for further experiments. Cultured SCAP cells in passage numbers from 2 to 6 were used for all the experiments with similar results.

2.3.

Characterization of SCAP cultures

Before any experiment, SCAP cultures used in this study were extensively characterized with respect to growth characteristics, colony forming unit fibroblasts (CFU-F) efficiency and expression of mesenchymal, as well as embryonic stem cell markers using flow cytometry. A total number of 10 SCAP cultures (n = 10) derived from different donors were analyzed with respect to the above mentioned characteristics in order to select the cultures with the most enhanced stem cell properties, i.e. highly proliferative and clonogenic potential and increased expression of stem cell markers to be used for further experiments.

2.5. assay

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Colony forming unit fibroblasts (CFU-F) efficiency

The assay was carried out by plating early passage (p.2) SCAP cells in 6-well plates at densities ranging from 100 to 200 cells/well in three replicates (n = 3). After plating, the dishes were placed in an incubator (37 ◦ C, 5% CO2 ) and left there for 10 days before being fixed with 10% neutral buffered formalin (NBF) for 1 h at room temperature (RT). Crystal violetstained colonies were macroscopically counted. A CFU-F was defined as a group of at least 50 cells. For each sample, colony forming unit efficiency (%) was calculated as follows: (mean number of colonies/total number of seeded cells) × 100. The CFU-F assay was repeated in three independent experiments. When more than 200 cells/well were initially seeded then too many individual colonies were overlapping and no counting could be performed.

2.6.

Flow cytometric analysis of stem cell markers

For flow cytometric analysis cells were first harvested by trypsinization and washed two times with ice-cold PBS. For surface epitope analysis, 106 cells/tube were first Fc-blocked with 1 ␮g of human IgG for 10 min at RT and subsequently stained by incubation with the fluorochromes conjugated mouse anti-human antibodies STRO-1-FITC, CD146-PE, CD34APC, CD45-PE, CD117-PerCP-Cy5.5, CD105-FITC, CD24-APC and CD90-FITC in various combinations for 20 min in the dark at RT. In addition, for intracellular staining for the embryonic markers Nanog and Oct3/4, cells were first Fcblocked and subsequently fixed with a 4% paraformaldehyde buffer, permeabilized with a saponin 0.1% (w/v) buffer and then stained with each of the fluorochrome conjugated mouse anti-human antibodies Oct3/4-Alexa Fluor 647 and Nanog-PE, as described above. After staining, cells were washed twice with 1 ml flow cytometry staining 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 staining 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.). After characterization of SCAP cultures derived from 10 different cell donors, SCAP cells with enhanced stem cell properties (proliferation rate, CFU-F efficiency and high expression of stem cell markers) were selected for the subsequent cytotoxicity and mineralization assays in the presence of the resinous monomers.

2.7. Cytotoxicity of HEMA and TEGDMA on highly proliferative and clonogenic human SCAP cells 2.4.

Cell growth analysis

For cell growth analysis, cells were seeded at 5 × 104 cells/well in 6-well plates. The cell number was assessed every 24 h with a hemocytometer, after harvesting the cells of the corresponding wells (6 replicates for each time point of 24, 48, 72, 96, 120 and 144 h respectively) with trypsinization and the corresponding growth curves were calculated.

TEGDMA and HEMA were dissolved in absolute ethanol and sequentially diluted to obtain different concentrations of stock solutions. The monomers were freshly diluted in 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, SCAP cells were seeded in 96well plates (5000 cells/well) and allowed to grow for 24 h.

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Subsequently, SCAP 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 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 5% CO2 . Then, the MTT solution was discarded and the insoluble formazan was dissolved with DMSO for 30 min at RT. The absorbance was measured against blanks (DMSO) at a wavelength of 570 nm by a microplate reader (Spectra Max 250, MWG Biotech).

2.8. Induction of odontogenic differentiation of SCAP cells in the presence of HEMA and TEGDMA Before proceeding to the odontogenic differentiation experiments in the presence of the monomers, well-characterized (as described above) SCAP cultures derived from at least 3 different donors were also additionally screened with respect to their odontogenic differentiation and mineralization potential using the Alizarin Red S (AR-S) Method (see below), in order to select the most potent cells with respect to these properties for further analysis of monomer long-term cytotoxicity. For the odontogenic differentiation experiments, SCAP cells were exposed to concentrations of 0.05, 0.1 and 0.5 mM of HEMA and TEGDMA, which were found in 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 a-MEM complete culture medium (CCM), supplemented additionally with 0.01 ␮M dexamethasone disodium phosphate (Dexa), 1.8 mM monopotassium phosphate (KH2 PO4 ) and 5 mM ␤-glycerophosphate (␤-GP) (=differentiation medium). 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 normal CCM without the additional supplements for the same 3-week period served as negative control (uninduced-control cultures), whereas cultures exposed for 3 weeks to differentiation medium without the presence of the monomers served as positive control (inducedcontrol cultures). In a second series of experiments, SCAP 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 almost 35% after a 72 h-exposure. Then, the medium with the monomers was washed out with PBS and replaced by differentiation medium without monomers that was changed every 3–4 days for the same period of three weeks (short-term exposure). This second series of experiments was performed in order to assess whether a single exposure to these monomers would be able to irreversibly affect their normal differentiation processes. At the end of each week, control and monomer-treated cultures of both long-term and shortterm experiments were evaluated for mineralization by AR-S staining.

2.9.

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% 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 de-ionized water. 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 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%, w/v) in 10 mM Na2 HPO4 (pH = 7) for 12 h at 37 ◦ C. Subsequently, 200 ␮l aliquots were transferred to a 96-well plate and the OD550 nm was measured using a microplate reader (Spectra Max 250, MWG Biotech). Mineralized nodule formation was represented as nmol AR-S per ␮g of total cellular protein, determined by Bicinchoninic Acid (BCA) Protein assay.

2.10. Histochemical detection of alkaline phosphatase (ALP) activity One week after induction of odontogenic differentiation, 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 Tris-(hydroxymethyl)-aminomethane buffer (pH 8.9). The cells were rinsed with dH2 O and evaluated for ALP activity under an inverted microscope (Olympus Optical Co., Ltd., Japan).

2.11. Semi-quantitative reverse transcription/polymerase chain reaction (RT)-PCR analysis Total RNA was extracted from cells with NucleoSpin RNA II kit at days 7 and 14 after induction of differentiation. For the RT-PCR reactions 0.5 ␮g of total RNA was diluted in a 25 ␮l PCR reaction of 1× 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 sialophosphoprotein (DSPP) (sense: 5 -GGG ACACAGGAAAAGCAGAA-3 , 5 -TGCTCCATTCCCACTAGGAC-3 antisense: and dehydrogenase (GAPDH) glyceraldehyde-3-phosphate 5 -GAAGGTGAAGGTCGGAGT-3 , antisense: 5 (sense: 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.

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2.12.

Statistical analysis

Each experiment was performed with 3–6 replicates and repeated at least three times. Values were expressed as means ± 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 multiple comparison test (p < 0.05).

3.

Results

3.1.

Characterization of SCAP cells

Significant variability in growth patterns, CFU-F efficiency and stem cell immunophenotypic profiles could be recorded in SCAP cultures established from different healthy donors of similar age and stage of third molar root development (two thirds of the root completed). Fig. 1 shows representative growth curves (Fig. 1a) and CFU-F efficiency assay results (Fig. 1b–d) of SCAP cultures derived from different donors. The CFU-F efficiency usually ranged between 20 and 45% in most SCAP cultures (Fig. 1d). SCAP cells with pronounced expression of stem cell properties, including high growth potential, shown by the inclination of their respective growth curves at the logarithmic phase (Fig. 1a), high clonogenicity (CFU-F efficiency > 35%) (Fig. 1b–d), as well as enhanced odontogenic differentiation/mineralization potential, as shown by AR-S staining (Fig. 1e) were selected for further experiments with the resin compounds (Fig. 1, donor 1). These cells were also characterized by a high expression of mesenchymal and embryonic stem cell markers, including STRO-1 (19.5 ± 1.5%), CD146 (64.3 ± 3.3%), CD34 (5.2 ± 1.1%), CD105 (85.3 ± 5.3%), CD24 (4.6 ± 0.6), CD90 (98.1 ± 0.7), Nanog (55 ± 4.3%), Oct3/4 (6.1 ± 1.2%), as shown in the respective flow cytometric histograms (Fig. 1f). CD45 and CD117 (c-Kit), were negative in these cells, which indicates their stromal origin and the absence of hematopoietic precursor contamination (Fig. 1f).

3.2.

Cytotoxicity of HEMA and TEGDMA in SCAP cells

HEMA and TEGDMA caused a time- and concentrationdependent reduction of the mitochondrial dehydrogenase activity in SCAP cells (Fig. 2A and B). HEMA reduced cell viability by 13–83.2% at concentrations of 0.1–8 mM and TEGDMA by 7.1–88.5% at concentrations between 0.05 and 5 mM respectively, after 72-h treatment. Statistically significant differences in cell survival compared to the control (p < 0.05) were observed for all concentrations of HEMA > 1 mM and TEGDMA > 1 mM after a 72-h treatment. Therefore, 0.05–0.5 mM of HEMA and TEGDMA showed very little or no effect on SCAP cell viability and for this reason these ‘sub-toxic’ concentrations were used for the subsequent long-term mineralization experiments. In addition, 2 mM of HEMA and 1 mM of TEGDMA both reduced cell viability by almost 35% and for this reason they were chosen for the short term-exposure experiments, taking into account that they are relevant to the concentrations of these monomers released immediately after the initial polymerization of the resinous materials. In addition, no severe signs of

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cytotoxicity or cell morphological alterations (e.g. cell retraction, decrease in cellular density, vacuolization, rounding or blebbing) could be observed with these concentrations (data not shown).

3.3. In vitro differentiation/mineralization by SCAP cells after exposure to the monomers Overall, the results showed that both types of monomerexposure (long-term and short-term) significantly delayed the differentiation and mineralization processes of SCAP cells. In addition, a concentration-dependence could be recorded in the long-term treated cultures. More specifically, at week 1, the mineralization was very limited and only a few sparse mineralized nodules could be observed in all types of cultures, including induced-control (Fig. 3a–c), uninducedcontrol (Fig. 3d–f) and cultures induced for differentiation in the presence of the monomers, both for the long-term (Fig. 3g–l) and short-term (Fig. 3m–r) exposure patterns. No statistically significant differences were measured in the total amount of mineralized matrix produced by monomer-treated cells compared to the control cells (both induced and uninduced), as shown by the AR-S quantification, using the CPC extraction method (Fig. 4). However, at week 2 after induction of differentiation a pronounced delay in mineralization process compared to the induced-control cultures could be recorded in cultures continuously treated (long-term exposure) with all non-toxic concentrations of HEMA tested (0.05, 0.1 and 0.5 mM), but also in cultures treated only once for 72 h with 2 mM HEMA (shortterm exposure) and subsequently exposed for 3 weeks to differentiation medium without any monomers (Figs. 3 and 4). On the other hand, concerning the monomer TEGDMA, these pronounced delay could be observed at week 2 only in cultures continuously treated with its highest non-toxic concentration tested (0.5 mM) (Figs. 3 and 4). In addition, significant effects could be recorded in cultures treated short-term (72 h) with 1 mM of TEGDMA (Figs. 3 and 4). Finally, at week 3 after induction of differentiation, all types of TEGDMA and HEMA-treated cultures (both short-term and long-term) showed a statistically significant reduction (p < 0.05) in the total amount of mineralized matrix produced compared to the induced-control cultures (Figs. 3 and 4). This was the case for all concentrations of monomer-treated cultures, except those exposed to the lowest concentration of TEGDMA (0.05 mM) (Figs. 3 and 4). Overall, these effects were much more pronounced in HEMA-treated cultures, whereas long-term exposure to only the highest concentration of TEGDMA (0.5 mM) was able to cause a very severe delay of the differentiation/mineralization processes of SCAP cells (Figs. 3j–l and 4). It must be noted, however, that despite the statistically significant delays observed in the differentiation/mineralization processes of monomer-treated SCAP cultures after 3 weeks, a complete inhibition could not be recorded. On the contrary, there was an overall tendency for a gradual recovery of these processes with time in almost all cases, so that at the end of the 3-week observation period a considerable amount of mineralized matrix – although statistically lower compared to control – could be observed in most types of monomer-treated

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Fig. 1 – SCAP culture characterization. (a) Representative growth curves of SCAP cultures (passage 2), established from the apical papilla of third molars at the stage of root development, extracted from three different healthy donors, aged 14–18 years old. (b) Macroscopic - and (c) microscopic pictures of CFU-Fs formed by SCAP cells derived from the same donors and stained with crystal violet. (d) CFU-F efficiency (%) of SCAP cultures established from these donors, after initial seeding of 100 or 200 cells per well and counting of the colonies (>50 cells) after 10 days. (e) Odontogenic differentiation potential of SCAP cultures derived from these donors, as shown by AR-S staining. (f) Representative single-parameter flow cytometry diagrams showing the expression of mesenchymal and embryonic stem cell markers. SCAP were positive for STRO-1, CD146, CD34, CD24, CD90, CD105, Nanog, Oct3/4, but were negative for CD45 and CD117. Due to the pronounced expression of stem cell properties (high proliferation rate, clonogenicity, mineralization potential and expression of stem cell markers) well-characterized SCAP cultures established from donor 1 were used for further experiments.

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of the cell population) and 0.1 mM (50–75%) HEMA, as well as in the presence of 0.5 mM (25–50%) and 0.1 mM TEGDMA (50–75%), while no significant effect on ALP activity could be observed for the lowest HEMA and TEGDMA concentrations tested (0.05 mM). ALP activity was also strongly inhibited (25–50% of the cell population) in SCAP cultures exposed short term (72 h) to 2 mM HEMA and 1 mM TEGDMA, at levels comparable to those of the uninduced-control cultures. These data indicate that ALP is one of the early differentiation markers that are affected after exposure to the resinous monomers.

3.5. Expression of the differentiation markers BSP, DSPP, OCN after monomer exposure

Fig. 2 – Cytotoxic effects of (a) HEMA and (b) TEGDMA on the mitochondrial dehydrogenase activity (cell viability) of SCAP 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 six replicates (n = 6). Asterisks indicate statistically significant differences from the ethanol control group (one-way ANOVA, followed by Tukey post hoc test, p < 0.05).

SCAP cultures induced for odontogenic differentiation progressively expressed mineralization markers, usually expressed by mature odontoblastic cells, including BSP, DSPP and OCN, as shown by RT-PCR analysis (Fig. 6). The expression of these markers was, however, reduced in cultures exposed to HEMA and TEGDMA in a concentration-dependent manner. Both BSP and OCN were significantly reduced in cultures continuously exposed to 0.5 and 0.1 mM HEMA, as well as 0.5 mM TEGDMA (mainly OCN) at day 7 after induction of differentiation. In addition, a significant reduction on the expression of these two markers could be also recorded at day 7 in cultures exposed short-term to the higher concentrations of HEMA (2 mM) and TEGDMA (1 mM). On the other hand, there was a very low DSPP expression both in control and monomer-treated cultures at the 7-day time-point. On day 14, there was a gradual recovery of BSP and OCN expression in monomer-treated cultures to levels comparable to the induced-control, whereas an obvious expression of DSPP could be now recorded in all types of cultures, being however lower in short-term treated cultures and in cultures continuously treated with 0.5 mM TEGDMA (Fig. 6). Overall, the above data suggest that the expression of differentiation markers was delayed in monomer-treated cultures in a concentration-dependent manner and this delay was initially more pronounced in those exposed short-term (72 h) to higher concentrations of these monomers.

4. cultures (Figs. 3 and 4). Exception to this, were cultures continuously exposed to 0.5 mM TEGDMA, where a very limited amount of mineralized matrix could be observed (Fig. 3l).

3.4.

Alkaline phosphatase (ALP) activity

Fig. 5 shows the ALP activity 7 days after induction of differentiation in induced-control (Fig. 5a), uninduced-control (Fig. 5b) and monomer-treated cultures, both long-term (Fig. 5c–h) and and short-term (Fig. 5i and j). According to the results, ALP was strongly expressed in 75–100% of the cell population in induced-control cultures, as early as one week after induction of differentiation, but was significantly lower (25–50%) in the uninduced-control cultures treated with CCM without the additional supplements. On the contrary, ALP activity was significantly reduced compared to the induced-control cultures in SCAP cultures continuously exposed to 0.5 mM (25–50%

Discussion

Pulp tissue response to dental restorative materials is a complicated process that is influenced by many factors, including the composition of the material, the chemistry and concentration of any eluted components or degradation products and the ability of the tissue to respond to these agents [22]. During the past few years, our understanding on the regenerative/repair potential of the dental pulp and how materials may impact on this capacity has significantly advanced [23]. It is already established that dental pulp responds to mild injury, as for example slowly progressing dentinal caries through reactionary dentinogenesis. During this physiological repair process, reactivated odontoblasts and/or cells located in the Hoehl’s layer produce a mineralized dentin barrier, in the form of tubular orthodentin or as an osteodentin-like layer [23]. However, in more severe dentinal injuries, such as pulp exposure or application of restorative materials in deep cavities,

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Fig. 3 – AR-S staining of SCAP cultures. In control cultures induced for differentiation with media containing Dexa, KH2 PO4 , ␤-GP the mineralization process initiated with single mineralized nodules at day 7 (a) and subsequently increased during the following two weeks (b, 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 delayed compared to the induced-control cultures, in all concentrations of HEMA(0.05, 0.1 and 0.5 mM), but only in the highest concentrations (0.1 and 0.5 mM) of TEGDMA-treated cultures. Finally, in cultures exposed short-term (72 h) to 2 mM HEMA (m–o) or 1 mM TEGDMA (p–r), despite the statistically significant delays (see Fig. 4) observed after 2 weeks (only for HEMA) and 3 weeks (for both HEMA and TEGDMA), there was an overall tendancy for a gradual recovery of the mineralization processes over time.

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Fig. 4 – Spectrophotometric quantification of the AR-S staining, using the CPC extraction method. Data are shown as mean nmol AR-S/␮g of total protein ± SD of 3 independent experiments in 4 replicates. 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).

the subjacent odontoblastic layer may be severely affected or destroyed [16,24]. In this case, stem/progenitor cells from the pulp core are triggered by the inflammatory signals to recruit toward the dentin–pulp border. These cells then differentiate into secreting odontoblast- or osteoblast-like cells, producing a reparative ortho- or osteo-dentin barrier, in order to repair the damage [23]. Several groups during the past few years have attempted to isolate and characterize these putative stem/progenitor cell populations residing in dental pulp stem cell niches and to analyze their functional role in pulp tissue repair [25,26]. Most studies clearly indicate that stem/progenitor cells isolated from the dental pulp and

expanded in vitro are characterized by significant heterogeneity, expressed through multiple phenotypic differences which most probably reflect distinct functional properties [27]. There is already evidence that there are significant variations in the odontogenic potential of single colony-derived populations isolated from the dental pulp, reflecting differences in their genotypic and protein expression patterns [28]. In addition, in immature teeth, a distinct highly potent stem cell population has been isolated and characterized in the apical papilla (SCAP) with potential role in odontoblast differentiation but also in pulp healing, as evidenced by a number of clinical cases showing that apexogenesis can occur in infected immature

Fig. 5 – Histochemical staining showing ALP activity 7 days after induction of differentiation in SCAP cultures exposed to various concentrations of HEMA and TEGDMA. In induced-control cultures (a) ALP was strongly expressed in the majority (75–100%) of the cell population, whereas in uninduced-control cultures (b) ALP activity was much lower (25–50%). In cultures exposed continuously for 3 weeks (long-term) to non-toxic concentrations of HEMA (c–e) and TEGDMA (f–h) there was a concentration-dependent reduction in ALP activity observed mainly for the higher concentrations (0.5 and 0.1 mM) of these monomers tested. In cultures exposed short-term (72 h) to 2 mM HEMA (i) and 1 mM TEGDMA (j) ALP activity was significantly inhibited to 25–50% of the cell population, which was comparable to the expression levels of the uninduced-control cultures.

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Fig. 6 – Representative agarose gels containing RT-PCR products from SCAP cultures exposed to various concentrations of HEMA and TEGDMA, at 7 and 14 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 0.5 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).].

permanent teeth with periradicular periodontitis or abscess [29,30]. The latter favors the possibility that SCAP in the apical papilla are important in this healing process. It is expected that this heterogeneity can be the cause of interpretational difficulties of the results received from different research groups when these systems are used as biocompatibility models and illuminates the need for a comprehensive characterization of the culture systems used in order to come to reliable results. It is already established that immortalized cells lines, such as transformed mouse fibroblasts (e.g. 3T3 or L929 clones) provide a means of good reproducibility, but certainly lack the metabolic potential of the primary recipient cells (e.g. pulp cells) [31]. On the other hand, more sophisticated in vitro biocompatibility models based on primary pulp cells and using reparative dentinogenesis as a biological endpoint can be a much better predictor of the clinical situation, but in this case significant effort must be given on their extensive characterization in order to ensure reliable interpretation of the results. This complexity is also compounded by the capabilities of cells derived from various cell donors differing in their genetic predisposition to activate recovery pathways to extend cell survival after exposure to toxic stimuli, leading to unpredictable results, especially in long-term experiments [32]. For all the above reasons, we introduce in this study the use of well-characterized SCAP cultures as a study model of the effects of resinous monomers on the physiological repair processes of the dental pulp, creating the basis for more sophisticated in vitro systems for studying the long-term biocompatibility of dental materials on the pulp tissue. These cells were selected among others due to their enhanced stem cell properties during expansion culture, such as high proliferation rate, colony forming unit efficiency, expression of stem cell markers, but most importantly their increased odontogenic differentiation/mineralization potential (Fig. 1a–f). These data are further supported by recently published work by our group showing that SCAP cells isolated from the apical

part of the pulp of human developing teeth present a much higher mineralization potential compared to dental pulp stem cells (DPSCs) isolated from the coronal part of pulp of the same teeth [20]. Furthermore, the anatomical proximity of the apical papilla to the coronal pulp and its potential implication in pulp healing processes [30] qualifies the use of SCAP as an appropriate cell population for studying the long-term effects of restorative materials on pulp tissue repair. The combined expression of both mesenchymal (STRO1, CD146, CD34, CD105, CD24, CD90) and embryonic (Nanog, Oct3/4) stem cell markers (Fig. 1f) is indicative of the enriched stem/progenitor cell content of the SCAP cultures used. STRO1 is a widely accepted marker for stromal stem cells that recognizes a trypsin insensitive epitope on perivascular cells of the bone marrow and dental pulp [33]. Analysis was also performed for CD146 (MUC18), a marker expressed in perivascular MSCs [19], CD34, expressed in primitive pluripotent stem cells (both stromal and hematopoietic) [33], CD105 (endoglin), a membrane glycoprotein functioning as part of TGF-␤ receptor complex and known to participate in several developmental processes [34], CD24, which is known to be expressed on the surface of B cells, but has been also found selectively in SCAP populations [19] and CD90, a surrogate marker of various kinds of stem cells, including MSC [35]. Significant expression was found for the premature embryonic markers Nanog and to a less extent Oct3/4, which are transcription factors associated with the maintenance of the pluripotency of embryonic stem cells [35]. Negative expression was found for CD117, a transmembrane tyrosine-kinase receptor (encoded by the Kit gene) and for CD45, a marker of leukocyte precursors. Both markers are mainly expressed in hematopoietic stem cells [36], and their absence precludes the possibility of hematopoietic precursor contamination in our system. When comparing these data with few published studies on the characterization of SCAP cells, an enhanced expression of most of these markers could be recorded in cultures used in this study [19].

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Our study design of both short-term exposure to relatively higher concentrations of HEMA and TEGDMA, as well as long term-exposure to sub-toxic concentrations of these monomers closely simulates the in vivo situation, as it is well-established that resin compounds 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 [37]. Our results have shown that long-term exposure to sub-toxic concentrations of HEMA and TEGDMA (0.05–0.5 mM), but also only one time exposure to relatively higher (1–2 mM) concentrations of these monomers, relevant to those immediately released after the polymerization of the composite materials [37], were able to significantly delay the physiological odontogenic differentiation and mineralization processes of highly potent stem/progenitor cell populations. It must be, however, noted that these two monomers showed quite different long-term cytotoxicity patterns. HEMA caused significant perturbation of SCAP differentiation/mineralization processes even at very low concentrations, such as 0.05 and 0.1 mM, whereas these concentrations of TEGDMA did not cause any of these pronounced effects. However, after a certain limit (in this case 0.5 mM), TEGDMA caused a very strong inhibition of these processes, whereas these effects were not so pronounced for the same (0.5 mM) concentration of HEMA. When comparing these results with data recently published by our group on the effects of resinous monomers on dental pulp stem cell cultures established from deciduous teeth (DTSCs), we conclude that the effects on SCAP cells were not as detrimental as those observed in DTSCs cells [18]. In the latter case, a much more significant disturbance of the mineralization processes could be recorded in cultures treated long-term with non-toxic concentrations of HEMA and TEGDMA, whereas an almost total inhibition of mineralization without any recovery was recorded in cultures treated only once (short-term) with the same relatively low (1–2 mM) concentrations of these monomers. These differences can be explained by the fact that different cells were used, emphasizing that cells with an enhanced odontogenic differentiation/mineralization potential were selected in the present study. Another potential reason is also that different methods for their isolation were applied (enzymatic dissociation vs. outgrowth method), which have been shown in previous studies [21,38] to provide cell populations differing significantly in their odontogenic differentiation potential, with an obvious advantage of the enzymatic dissociation method. These data further emphasize the need for comprehensive characterization of the cell culture systems used for biocompatibility testing. They also imply that significant variability in pulp tissue response can be expected when resinous materials are applied in different patients, based on their pulp tissue repair capacity. In the present study, although there were statistically significant delays in the mineralization rate and the expression of differentiation-specific markers (BSP, ALP, OCN, DSPP) (Figs. 5 and 6) in monomer-treated SCAP cultures, a tendency for a gradual recovery of these processes with time was eventually observed in almost all cases after 3 weeks, with the exception of cultures continuously exposed to the highest concentrations – 0.5 mM – of TEGDMA, where a more severe

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inhibition was recorded (Figs. 3l and 4). Furthermore, the effects of HEMA were obvious in much lower concentrations compared to TEGDMA (Figs. 3 and 4), which is in accordance with already published data [11,18]. More specifically, in a study by About et al. [11] it was shown that concentrations as low as 10 ␮M of HEMA and TEGDMA were able to reduce the protein levels of collagen I, osteonectin and dentin sialoprotein in human pulp cells after 4 weeks, with the effects of HEMA being significantly more prominent compared to TEGDMA. This was also the case in a previous study by our group in deciduous teeth stem cell cultures, where HEMA caused a more severe inhibition of the mineralization processes, as well as reduction in the expression of mineralization markers (BSP, DSPP, OCN and ALP) compared to TEGDMA [18]. The data of the present study are also in accordance with those recently published by Galler et al. [12], which showed that 0.3 mM of TEGDMA caused a very early reduction of the expression levels of mineralization-related genes (collagen I, ALP, BSP, OCN, Runx2 and DSPP) by 5–20% after 4 h and by 50% after 12 h in primary human pulp cultures, whereas the calcium deposition and ALP activity were also significantly reduced. The results of the present study further support that the reduction in the expression of mineralization-related genes due to monomer exposure can be still recorded 7 days after induction of differentiation, showing a gradual recovery up to day 14. Significant scientific information is now available from a number of studies on the possible underlying cellular mechanisms altering basic cellular functions after exposure to resinous monomers, which have been already reviewed [1–3]. A number of studies support that oxidative damage occurring after the production of ROS and simultaneous reduction of the intracellular glutathione pool can be considered as one of the predominant mechanisms of monomer-induced cytotoxicity [7–10]. Oxidative stress can lead to DNA damage, cell cycle delays and eventually to cell death, mainly in the form of apoptosis, which is the case after exposure to relatively higher concentrations of these monomers [39]. In addition, monomer-induced apoptosis has been mainly associated with differential activation of mitogen activated protein kinases (MAPK), proteins that regulate a variety of basic cellular functions, such as cell viability, proliferation, expression of surface antigens and programmed cell death (apoptosis) [40,41]. It is also reported that even at non-toxic concentrations the generation of oxidative stress can induce down-regulation of genes controlling differentiation and mineralization processes, such as collagen I [42]. In a recent study, TEGDMA and HEMA were found to cause significant suppression of osteogenic differentiation in a human osteosarcoma (MG63) cell line [43]. These effects were however significantly diminished by N-acetylcysteine (NAC), a scavenger of ROS, which clearly suggests the involvement of oxidative stress in these effects. In similar studies, NAC also protected rat pulp cells [44], as well as oral mucosa fibroblasts [45] from poly-methylmethacrylate (PMMA)-induced inhibition of mineralization-related genes, such as ALP, collagen I and III and dentin sialoprotein. Overall, these data indicate that oxidative stress can be considered as a main mechanism leading to down-regulation of differentiation-related genes, reduced metabolic activity and reduced production of

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mineralization-related protein products, leading eventually to a compromised pulp repair response. It also remains an open question whether these alterations may accumulate and become apparent after an extended period of time, making cells more susceptible to subsequent injuries from other toxic stimuli, such as additional compounds leaching from new resin restorations. Although there is a significant clinical evidence that application of resinous materials into deep cavities or pulp exposure sites can be associated with slight to persistent inflammation and lack of mineralized barrier formation [13–17], accurate information on the amount of components that finally diffuse into the coronal and/or root pulp space and are able to affect the reparative and/or developmental processes of immature teeth are completely lacking. The results of this study provide a potential mechanism to substantiate the clinical observations of a compromised pulp response after resinous materials’ application that is due to the disturbance of the physiological differentiation processes of stem/progenitor cells. In addition, there are, as already mentioned, several reports showing the important role of SCAP in apexogenesis occurring in infected immature permanent teeth with periradicular lesions [29,30]. In such cases, a clinical protocol is established during which the infected teeth are irrigated and disinfected with intra-canal medication of a triple antibiotic and then sealed at the access cavity with a restorative material allowing for apexification to be conducted by the remaining SCAP. The results of this study suggest that composite resins and resin modified glass ionomer cements should probably be avoided as filling materials of the root canal tooth access because the direct release of their components into the pulp space could potentially disturb the differentiation of SCAP cells, and possibly lead to incomplete apexification. Further translational research is needed toward this direction.

5.

Conclusions

In conclusion, our experiments provide evidence that longterm exposure to low concentrations (0.05–0.5 mM) of HEMA and TEGDMA, but also even one time exposure to higher concentrations (1–2 mM) of these monomers are able to significantly delay the physiological odontogenic differentiation and mineralization process of highly proliferative and clonogenic apical papilla stem/progenitor cells in a concentration-dependent manner. The clinical significance of these data is that persistent exposure to low concentrations of eluted resin substances may have chronic negative effects on pulp cells. Even if not causing acute cytotoxicity (such as pulp inflammation and/or necrosis) the continuous release of such substances may significantly compromise dental pulp homeostasis and repair, therefore jeopardizing the pulps’ vitality and prognosis of the restored teeth. This study also proposes for the first time the use of well-characterized stem/progenitor cell culture systems, as a reliable in vitro model for the evaluation of the long-term cytotoxic effects of restorative materials on pulp physiological repair processes, allowing for a more relevant extrapolation of the results to the clinical situation compared to already existing systems. Finally, the results

of this study provide evidence for the replacement of these compounds from present materials and the development of new biocompatible alternatives, which can be modified to preferentially stimulate pulp tissue repair/regenerative processes. Further clinical translational research is, however, imperative in order to incorporate these findings into clinical application.

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