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.
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, KH 2 PO 4 ,β-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.
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 sialophosphoprotein-DSPP, 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 down-regulated.
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.
Dental composite resin-based materials have been widely studied for cytotoxicity and genotoxicity in various cell culture systems . These effects have been attributed to the release of residual monomers or other substances, derived either from incomplete polymerization or resin degradation . Among the compounds released from resin-based materials, the comonomers TEGDMA (triethylene-glycol-dimethacrylate) and HEMA (2-hydroxy-ethyl-methacrylate) have been found to induce to a variable level genetic and cellular toxicologic effects on different mammalian cell types . HEMA is one of the most common components of dentin-adhesive systems, in a concentration ranging from 30 to 55% and has a pivotal role during the dentin impregnation process . Because of its low molecular weight and its relative hydrophilicity, HEMA can diffuse through the residual dentin and affect the underlying odontoblast vitality and pulp physiological activity . TEGDMA, on the other hand, is released in high amounts from polymerized dental resins into aqueous media and accounts for most of their unreacted double bonds . Moreover, TEGDMA is a component of dentin adhesives in contents varying from 25 to 50% . Due to its lipophilic nature, TEGDMA can easily penetrate the cytosol and membrane lipid compartments of mammalian cells, causing several cytotoxic effects .
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 . In addition, 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 . They are also able to affect the physiological mineralization procedures of terminally differentiated cells, such as osteoblasts . However, 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), which is essential for the regeneration and repair of the dentin/pulp complex.
A few years ago, Gronthos et al. identified a population of post-natal stem cells in the human dental pulp of both adult teeth (Dental Pulp Stem Cells, DPSCs) and exfoliated deciduous teeth (Stem cells from Human Exfoliated Deciduous teeth, SHED) . These cells represent a population of undifferentiated MSCs, which are characterized by unlimited self-renewal, colony forming capacity and multipotent differentiation potential into several cell lineages, such as osteo/odontogenic, neurogenic, adipogenic, chondrogenic and myogenic, when grown under defined culture conditions . They remain in a quiescent state in the dental pulp and can perform continuous cell division during dental pulp tissue injury/regeneration . In addition, these authors have found that stem cells from the pulp of deciduous teeth represent a more immature cell population compared those of adult teeth, as they are characterized by a higher proliferation rate, increased cell population doublings and higher osteoinductive capacity in vivo .
Therefore, 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, which is indispensible to the repair of the dentin/pulp complex as a response to external stimuli . 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). The data presented in this study add significant information concerning the toxicological effects of these monomers on matured (differentiated) cell populations (odontoblasts, osteoblasts), by further clarifying how pathways regulating cellular homeostasis, dentinogenesis and tissue repair may be modified by concentrations well below those which cause acute toxicity.
Materials and methods
Chemicals and reagents
The monomers TEGDMA and HEMA were gifts from VOCO (Cuxhaven, Germany). Dulbecco’s modified Eagle’s medium (DMEM, containing l -glutamine and 2.0 g/l NaHCO 3 ), 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, β-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 CD146-PE, CD34-APC and CD45-PE were purchased from BD Biosciences (Heidelberg, Germany). The mouse anti-human antibodies STRO-1-FITC and anti-DSP (LFMb-21) and the broad spectrum immunoperoxidase ABC kit were obtained from Santa Cruz Biotechnology, Inc (CA, U.S.A.). The NucleoSpin RNA II isolation kit was purchased from Macherey–Nagel (Düren, Germany) and the Robus T I RT-PCR kit (F-580L) from Finnzymes (Espoo, Finland). The primers used for the RT-PCR analysis were synthesized by Biozym Scientific GmbH (Hess. Oldendorf, Germany).
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. All teeth were healthy and were extracted due to malposition in the dental arch. The collection of the samples was performed according to the guidelines of the Institutional Review Board and the parents of all donors signed an informed consent form. For the establishment of cell cultures teeth were disinfected and cut around the cementum–enamel junction to expose the pulp chamber. The pulp tissue was minced into small fragments, which were placed in 25 cm 2 culture flasks with DMEM, supplemented with 10% FBS, 100 Units/ml penicillin, 100 mg/ml streptomycin, 0.25 mg/ml Amphotericin B ( = complete culture medium-CCM) and incubated at 37 °C in 10% CO 2 . After reaching confluency cells were collected by treatment with 0.25% trypsin/0.25 mM EDTA and then continuously passed for further experiments. Cultured DTSCs in passage numbers from 2–6 from at least three different donors were used for all the experiments with similar results.
Surface epitope characterization of DTSCs cultures
Before any experiment, DTSCs cultures used in this study were characterized using surface epitope markers commonly used for the characterization of MSCs of dental origin , including STRO-1, CD146 (MUC18), CD34 and CD45. Briefly, cells were first harvested by trypsinization and washed three times with PBS. For each analysis 10 6 cells/tube were first Fc-blocked with 1 μg of human IgG for 10 min at room temperature (RT) and subsequently stained by incubation with the mouse anti-human antibodies STRO-1-FITC, CD146-PE, CD34-APC and CD45-PE for 20 min in the dark at RT. Then, cells were washed with 2 ml FACS wash solution (dPBS + 1%BSA + 0.1%NaN 3 ) and centrifuged for 5 min at 230 × g . Supernatant was removed, cells were re-suspended in 200 μl FACS solution and analyzed with a BD LSR II Flow Cytometer (BD Biosciences). A total of 100,000 events were acquired for each sample. Data were analyzed using Summit 5.1 software (Beckman Coulter, Inc, U.S.A.).
Exposure of DTSCs to HEMA and TEGDMA and MTT cytotoxicity assay
TEGDMA and HEMA were dissolved in absolute ethanol and sequentially diluted to get different concentrations of stock solutions. The monomers were freshly diluted in the culture medium prior to each experiment. The final concentration of ethanol did not exceed 0.25% (v/v). Cells incubated with medium containing 0.25% ethanol served as control. For the assessment of cytotoxicity DTSCs were seeded in 96-well plates (5.000 cells/well) and allowed to grow for 24 h. Subsequently, DTSCs were treated with HEMA (0.1–8 mM) and TEGDMA (0.05–5 mM), for 24, 48 or 72 h. Cell viability was assessed using the MTT cell viability assay to determine the mitochondrial dehydrogenase activity. Briefly, at the end of each incubation period the culture medium was discarded and 100 μl of 5 mg/ml MTT in PBS was added to each well. The cells were incubated in the dark for 3 h at 37 °C and 10% CO 2 . Then, the MTT solution was discarded and the insoluble formazan was dissolved with DMSO for 20 min RT. The absorbance was measured against blank (DMSO) at a wavelength of 570 nm by a microplate reader (Spectra Max 250, MWG Biotech).
Induction of odontogenic differentiation in the presence of HEMA and TEGDMA
For the odontogenic differentiation experiments DTSCs were exposed to concentrations of HEMA (0.05, 0.1 and 0.5 mM) and TEGDMA (0.05, 0.1 and 0.25 mM), which were found-based on the MTT analysis- to have minimal or no cytotoxicity to the cells (cell viability ≥85% for both monomers after 72-h exposure). Both control and monomer treated cultures were induced for odontogenic differentiation by being exposed to DMEM complete culture medium (CCM), supplemented additionally with 0.01 μM dexamethasone disodium phosphate (Dexa), 1.8 mM monopotassium phosphate (KH 2 PO 4 ) and 5 mM β-glycerophosphate (β-GP) and 100 μM l -ascorbic acid phosphate ( l -ascorbic). Cells were treated for a total period of 3 weeks with the differentiation medium containing the different concentrations of the monomers being changed every 3-4 days (long-term exposure). Cultures exposed to normal CCM without the additional supplements for the same 3-week period were used as negative control (uninduced control).
In a second series of experiments, DTSCs cultures were exposed only once to higher concentrations of HEMA (2 mM) and TEGDMA (1 mM), which were found in the MTT assay to reduce cell viability by 20–30% after a 72 h exposure. Then, the medium with the monomers was washed out with PBS and replaced by differentiation medium (containing Dexa, KH 2 PO 4 , β-GP and l -ascorbic) without monomers, that was changed every 3-4 days for the same period of three weeks (short-term exposure). Purpose of this second series of experiments was to assess whether a single exposure to these monomers would be able to irreversibly affect their normal differentiation processes. At the end of each week, control and monomer-treated cultures of both long-term and short-term experiments were evaluated for mineralization by Alizarin Red S (AR-S) staining and processed for immunocytochemical analysis of alkaline phosphatase (ALP) activity.
Alizarin Red S mineralization assay
For the assessment of in vitro mineralization, cell cultures were washed twice with PBS (−) (without Ca 2+ and Mg 2+ ) and fixed with 10% neutral buffered formalin (NBF) for 1 h at RT. Then, cultures were stained with 1% AR-S (pH 4.2) for 20 min at RT, followed by rinsing three times with deionized water (dH 2 O). Mineralized nodules were photographed using an inverted microscope (Olympus Optical Co, Ltd, Japan) equipped with a digital camera (Olympus E-410, Olympus Optical Co, Ltd, Japan). Quantification of the total 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 Na 2 HPO 4 (pH 7) for 2 h at 37 °C. Subsequently, 200 μl aliquots were transferred to a 96-well plate and the OD 550nm was measured using a microplate reader (Spectra Max 250, MWG Biotech). Mineralized nodule formation was represented as OD per μg of total cellular protein, determined by Bradford Protein assay.
Histhochemical detection of alkaline phosphatase (ALP) activity
Cells in 6-well-plates were washed twice with PBS (−) and fixed with 10% NBF, as described in the AR-S protocol. ALP activity was visualized by incubating the cells for 2 h at 37 °C with 0.1 mg/ml Naphtol-AS-MX Phosphate in N,N-dimethylformamide and 0.6 mg/ml Fast Blue BB Salt in 0.2 M Tris- (hydroxymethyl)-aminomethane buffer (pH 8.9). The cells were rinsed with dH 2 O and evaluated for ALP activity under an inverted microscope (Olympus Optical Co, Ltd, Japan).
Semi-quantitative reverse transcription/polymerase chain reaction (RT)-PCR analysis
Total RNA was extracted from cells with NucleoSpin RNA II kit at days 9 and 15 after induction of differentiation. For the RT-PCR reactions 0.5 μg of total RNA was diluted in a 25 μl PCR reaction of 1X PCR reaction buffer containing 1.5 mM MgCl 2 /200 mM each of dNTP/0.04 units/μl of DyNAzyme EXT DNA Polymerase/0.1 Units/μl 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′, antisense: 5′-TGCTCCATTCCCACTAGGAC-3′ and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (sense: 5′-GAAGGTGAAGGTCGGAGT-3′, antisense: 5′-GAAGATGGTGATGGGATTTC-3′). The reactions were performed in a PCR thermal cycler (Bio-Rad iCycler, Munich, Germany) at 50 °C for 30 min for cDNA synthesis, 94 °C for 2 min for one cycle and then 94 °C/(45 s), 56 °C/(60 s), 72 °C/(60 s) for 30 cycles, with a final 10-min extension at 72 °C. RT-PCR products were analyzed by 1.5%, w/v agarose gel electrophoresis and visualized by ethidium bromide staining.
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. Cells were washed with PBS (−) and fixed with 10% NBF for 30 min at RT. Cells were incubated first with 1.5% blocking serum in PBS to avoid non-specific staining and then with mouse anti human DSP (LFMb-21) primary antibody (dilution 1:100) for 1 h at RT. Then cells were incubated with goat anti-mouse secondary antibody (dilution 1:200) for 1 h at RT and processed for enzymatic immunohistochemical staining using a broad spectrum immunoperoxidase ABC kit according to the manufacturer’s protocol. Finally, cells were counterstained with hematoxylin and examined under an inverted microscope.
Each experiment was performed in triplicates and repeated at least three times. Values were expressed as the mean ± SD. Statistical analysis of the data was performed using one-way analysis of variance (ANOVA). Follow-up comparisons between groups were then carried out using the Tukey multiple comparison test ( p < 0.05 ).