2.1

Chemicals and reagents

The monomers TEGDMA and HEMA were gifts from VOCO (Cuxhaven, Germany). Dulbecco’s modified Eagle’s medium (DMEM, containing l -glutamine and 2.0 g/l 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).

2.2

Cell culture

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.

2.3

Surface epitope characterization of DTSCs cultures

Before any experiment, DTSCs cultures used in this study were characterized using surface epitope markers commonly used for the characterization of MSCs of dental origin , 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.).

2.4

Exposure of DTSCs to HEMA and TEGDMA and MTT cytotoxicity assay

TEGDMA and HEMA were dissolved in absolute ethanol and sequentially diluted to get different concentrations of stock solutions. The monomers were freshly diluted in the culture medium prior to each experiment. The final concentration of ethanol did not exceed 0.25% (v/v). Cells incubated with medium containing 0.25% ethanol served as control. For the assessment of cytotoxicity DTSCs were seeded in 96-well plates (5.000 cells/well) and allowed to grow for 24 h. 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).

2.5

Induction of odontogenic differentiation in the presence of HEMA and TEGDMA

For the odontogenic differentiation experiments DTSCs were exposed to concentrations of HEMA (0.05, 0.1 and 0.5 mM) and TEGDMA (0.05, 0.1 and 0.25 mM), which were found-based on the MTT analysis- to have minimal or no cytotoxicity to the cells (cell viability ≥85% for both monomers after 72-h exposure). Both control and monomer treated cultures were induced for odontogenic differentiation by being exposed to DMEM complete culture medium (CCM), supplemented additionally with 0.01 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.

2.6

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.

2.7

Histhochemical detection of alkaline phosphatase (ALP) activity

Cells in 6-well-plates were washed twice with PBS (−) and fixed with 10% NBF, as described in the AR-S protocol. ALP activity was visualized by incubating the cells for 2 h at 37 °C with 0.1 mg/ml Naphtol-AS-MX Phosphate in N,N-dimethylformamide and 0.6 mg/ml Fast Blue BB Salt in 0.2 M 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).

2.8

Semi-quantitative reverse transcription/polymerase chain reaction (RT)-PCR analysis

Total RNA was extracted from cells with NucleoSpin RNA II kit at days 9 and 15 after induction of differentiation. For the RT-PCR reactions 0.5 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.

2.9

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.

2.10

Statistical analysis

Each experiment was performed in triplicates and repeated at least three times. Values were expressed as the mean ± SD. Statistical analysis of the data was performed using one-way analysis of variance (ANOVA). Follow-up comparisons between groups were then carried out using the Tukey multiple comparison test ( p < 0.05 ).