2.1

Establishment of DPSC cultures

DPSC cultures were established from third molars of healthy donors aged 18–24 years. The collection of the samples was performed according to the guidelines of the Institutional Review Board and all donors signed an informed consent form. The enzymatic dissociation method was used to establish cell cultures. Briefly, the teeth were disinfected and cut around the cementum-enamel junction to expose the pulp chamber. The tissue was minced into small fragments and digested in a solution of 3 mg/ml collagenase type I and 4 mg/ml dispase II (Invitrogen, Karlsruhe, Germany) for 1 h at 37 °C. Single cell suspensions were obtained by passing the cells through a 70 μm cell strainer (BD Biosciences, Heidelberg, Germany). Cells were expanded with a-MEM (Minimum Essential Media) culture medium (Invitrogen), supplemented with 15% FBS (EU-tested, Invitrogen), 100 mM l -ascorbic acid phosphate (Sigma–Aldrich, Taufkirchen, Germany), 100 units/ml penicillin, 100 mg/ml streptomycin and 0.25 mg/ml Amphotericin B (all from Invitrogen) (=Complete Culture Medium – CCM) and incubated at 37 °C in 5% CO ₂ . Cultured DPSCs in passage numbers from 2 to 6 from at least three donors were used for all experiments with similar results.

2.2

Characterization of DPSC cultures with flow cytometry

DPSC established cultures were characterized for mesenchymal (STRO-1, CD146/MUC18, CD105/endoglin, CD24, CD90/Thy-1, CD81-TAPA, CD34, CD49f/a6-integrin), neural (CD271/NGFR, nestin), hematopoietic (CD117/c-kit, CD45) and embryonic stem cell (SC) markers (Nanog, Oct3/4, SSEA-3, TRA-1-60) using flow cytometry. For surface epitope expression analysis, 10 ⁶ cells/probe were first Fc-blocked with 1 μg of human IgG for 20 min at room temperature (RT) and then stained with the following fluorochrome-conjugated mouse anti-human antibodies: STRO-1-FITC (Santa Cruz Biotechnology, Inc., CA, United States), CD146-PE (phycoerythrin), CD34-APC (allophycocyanin), CD117-PerCP-Cy5.5 (Peridinin-Chlorophyll-Protein-cyanin 5.5), CD45-PE, CD105-FITC, TRA-1-60-PE (all from BD Biosciences), CD24-APC, CD90-FITC, CD271-PE, CD49f-APC, CD81-FITC, SSEA-3-PE (all from BioLegend, Fell, Germany). Cells were incubated in the dark at RT with various combinations of antibodies. Additionally, for intracellular staining for Nanog, Oct3/4 and nestin, cells were first Fc-blocked and then additionally fixed with a 4% paraformaldehyde buffer, permeabilized with a saponin 0.1% (w/v) buffer (both from BD Biosciences) and then stained with the mouse anti-human antibodies Oct3/4-Alexa Fluor 647, Nanog-PE (both from BioLegend) and nestin-APC (R&D, Minneapolis, USA). Stained cells were washed twice with staining buffer (PBS + 1% BSA + 0.1% NaN3) and analyzed with a BD LSR II Flow Cytometer. A total of 100,000 events were acquired for each sample. Data were analyzed using Summit software, version 5.1 for Windows (Beckman Coulter, Inc. Krefeld, Germany).

2.3

Evaluation of cell viability/proliferation by MTT assay

Cell viability was determined using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, as previously described . DPSCs were exposed to the resinous monomer TEGDMA (provided free by VOCO, Cuxhaven, Germany) at concentrations 1 and 2 mM, which our previous study found to cause a low to medium cytotoxicity in these cells . TEGDMA was dissolved in absolute ethanol and sequentially diluted to obtain different concentrations of stock solutions. The monomer was 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 a negative control.

In parallel experiments, DPSCs were exposed to various concentrations (1–10 mM) of Lithium, in the form of LiCl (Sigma–Aldrich), which is known as a specific and noncompetitive GSK3β inhibitor . This second series of experiments was designed to evaluate the influence of various concentrations of LiCl on the viability of DPSCs. In further experiments, DPSCs were exposed to various concentrations (25, 50 and 100 ng/ml) of human recombinant Wnt-1 (hrWnt-1, Biochrom AG, Berlin, Germany), which is known to signal through β-catenin . This set of experiments intended to evaluate the influence of various concentrations of Wnt-1 on the viability of DPSCs. Finally, experiments combining the simultaneous exposure of DPSC to TEGDMA and LiCl (T/L) or TEGDMA and Wnt-1 (T/W) were also performed to evaluate potential synergistic or antagonistic effects on DPSC viability.

For the MTT assay, cells were seeded at 10 ⁴ cells/well in 96-well plates and left for 24 h to attach. Afterwards, the medium was replaced with medium containing the above mentioned concentrations of TEGDMA, LiCl, Wnt-1 or their combinations (and respective negative controls) and incubated for 24, 48 or 72 h. At the end of each time-point a 0.5 mg/ml MTT solution was added in each well and cells were incubated for 3 h at 37 °C and 5% CO ₂ . After this period the medium containing the MTT solution was discarded and the insoluble formazan was dissolved with DMSO for 1 h at RT. The absorbance was measured against blank (DMSO) at a wavelength of 545 nm and a reference filter of 630 nm by a microplate reader (Spectra Max 250, Molecular Devices, Biberach an der Riss, Germany). Experiments were performed in six replicates and repeated at least three times.

2.4

Cell-cycle analysis with flow cytometry

Cell cycle analysis was performed with flow cytometry. DPSCs were seeded in six well-plates at 2 × 10 ⁵ cells/well and allowed to attach for 24 h. Then, culture medium was replaced with CCM containing TEGDMA (1–2 mM), LiCl (1–10 mM), Wnt-1 (25–100 ng/ml) or their combinations. After 24, 48 or 72 h cells trypsinized, washed twice with ice-cold PBS and fixed in ice-cold ethanol. After washing with ice-cold PBS, cells were re-suspended in PBS containing 0.1% Triton-X, 20 μg/ml Propidium Iodide (PI) and 0.2 mg/ml RNase A (both from Sigma-Aldrich) and incubated in the dark for 30 min at RT. Samples were analyzed for DNA content with a BD FACSCalibur flow cytometer equipped with a 15 mW 488 nm argon ion laser (BD Biosciences). A total of 20,000 events were acquired for each sample using the CellQuest software. PI signals for analysis of DNA content were subjected to pulse processing for gating out cell debris and doublets. The FL2 fluorescence was detected in a linear/log histogram, and G0/G1, S, and G2/M phases were determined using multicycle cell analysis Software (Phoenix Flow Systems, San Diego, CA, USA). Three independent experiments were performed.

2.5

Quantitative real-time reverse-transcription polymerase chain reaction analysis

Total mRNA was isolated using the RNeasy kit (Qiagen, Hilden, Germany). cDNA synthesis using 1 μg template RNA was performed with Quanti Tect Reverse transcription kit (Qiagen), according to manufacturer’s instructions. Quanti Tect SYBR Green RT-PCR Kit (Qiagen) was used to perform all reactions. Rotor-Gene Q PCR device (Qiagen) was used to read the amplifications, which started with an initial step at 95 °C for 5 min to activate the Hotstart Taq DNA polymerase, followed by 40 cycles of PCR, comprising denaturation for 5 s at 95 °C and combined annealing/extension for 10 s at 60 °C. Beta-catenin/CTNB1 (NM_001098209, NM_001098210, NM_001904), LEF-1 (NM_001130713, NM_001166119, NM_016269), GSK3β (NM_001146156, NM_002093), Cyclin D1/CCND1 (NM_053056) and Cyclin B1/CCNB1 (NM_031966) mRNA expressions were quantified using respective QuantiTect primer assays. A standard melting curve was used to check quality of amplification and to ensure specificity. The results were adjusted by amplification efficiency (LinRegPCR) and genes were normalized to the two most stable housekeeping genes evaluated by geNorm (succinate dehydrogenase complex, subunit A, flavoprotein/SDHA and β-actin/ACTB, QuintiTect primer assays). All experiments were run in duplicates and repeated three times.

2.6

Western blot analysis

DPSCs were seeded in 60 mm dishes at 5 × 10 ⁵ cells/dish in CCM and allowed to attach for 24 h before being exposed for 24–72 h to various concentrations of TEGDMA, LiCl, Wnt-1 or their combinations. Cytosolic and nuclear extracts were separated from cultured cells using different lysis buffers. Briefly, cells were lysed on ice in buffer consisting of 25 mM Tris, 2 mM MgCl ₂ , 1 mM Na ₂ VO ₄ , 0.5% NP40 and a protease inhibitor mixture (mini-EDTA-free, Roche Applied Science, Mannheim, Germany). Cells were centrifuged at 5800 rpm for 5 min and the supernatant (cytosolic fraction) was stored at −20 °C The cell pellet (nuclei) was further lysed in buffer consisting of 10 mM Tris, 0.4 M LiCl, 20% Glycerol, 1 mM Na ₂ VO ₄ and the same protease inhibitor mixture. Cells were centrifuged at 14,000 rpm for 10 min and the supernatant (nuclear fraction) was stored at −20 °C. Protein concentrations of the extracted cytosolic and nuclear lysates were quantified using a BCA Protein Assay Kit (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions and measured spectrophotometrically at 562 nm by a microplate reader (Spectra Max 250, Molecular Devices). An equal amount of protein form each sample (2 μg for GAPDH and 20 μg for each β-catenin and Lamin B) was separated on a 10–15% sodium dodecyl sulfate polyacrylamide gel by electrophresis (SDS-PAGE) and then transferred onto a polyvinylidene fluoride (PVDF, Millipore, Bilerica, MA, USA) membrane at 240 mA for 1.5 h. After blocking in 3% BSA in PBS, the membranes were incubated overnight at 4 °C with the following primary antibodies (mouse- or rabbit-anti human diluted in 0.1% BSA in PBS): monoclonal mouse anti-β-catenin (1:500, BioLegend), monoclonal rabbit anti-GAPDH (1:10,000, Sigma), monoclonal mouse anti-Lamin B (1:500, Santa Cruz). GAPDH and Lamin B were used as the internal controls for the cytosolic and nuclear proteins respectively. The membranes were then washed with PBS containing Tween 20 (TPBS) and incubated with secondary goat anti-mouse or anti-rabbit conjugated horseradish peroxidase antibodies (1:10,000, DakoCytomation, Hamburg, Germany) for 1 h at RT and developed with the enhanced chemiluminescence (ECL) substrate (SuperSignal West Pico or Femto, Pierce, Rockford, IL, USA), using an ECL imaging system (Fusion SL-4-400WL, Vilber Lourmat, Eberhardzell, Germany).

2.7

Statistical analysis

At least three independent experiments, with two to six replicates, were performed. Results were analysed by one-way ANOVA Bonferroni’s post-test and two-tailed t -test using IBM SPSS Statistics 22 Software for Windows (International Business Machines Corporation, U.S.A) (* p < 0.05). Data were expressed as means ± standard deviation (SD).

2.1

Establishment of DPSC cultures

DPSC cultures were established from third molars of healthy donors aged 18–24 years. The collection of the samples was performed according to the guidelines of the Institutional Review Board and all donors signed an informed consent form. The enzymatic dissociation method was used to establish cell cultures. Briefly, the teeth were disinfected and cut around the cementum-enamel junction to expose the pulp chamber. The tissue was minced into small fragments and digested in a solution of 3 mg/ml collagenase type I and 4 mg/ml dispase II (Invitrogen, Karlsruhe, Germany) for 1 h at 37 °C. Single cell suspensions were obtained by passing the cells through a 70 μm cell strainer (BD Biosciences, Heidelberg, Germany). Cells were expanded with a-MEM (Minimum Essential Media) culture medium (Invitrogen), supplemented with 15% FBS (EU-tested, Invitrogen), 100 mM l -ascorbic acid phosphate (Sigma–Aldrich, Taufkirchen, Germany), 100 units/ml penicillin, 100 mg/ml streptomycin and 0.25 mg/ml Amphotericin B (all from Invitrogen) (=Complete Culture Medium – CCM) and incubated at 37 °C in 5% CO ₂ . Cultured DPSCs in passage numbers from 2 to 6 from at least three donors were used for all experiments with similar results.

2.2

Characterization of DPSC cultures with flow cytometry

DPSC established cultures were characterized for mesenchymal (STRO-1, CD146/MUC18, CD105/endoglin, CD24, CD90/Thy-1, CD81-TAPA, CD34, CD49f/a6-integrin), neural (CD271/NGFR, nestin), hematopoietic (CD117/c-kit, CD45) and embryonic stem cell (SC) markers (Nanog, Oct3/4, SSEA-3, TRA-1-60) using flow cytometry. For surface epitope expression analysis, 10 ⁶ cells/probe were first Fc-blocked with 1 μg of human IgG for 20 min at room temperature (RT) and then stained with the following fluorochrome-conjugated mouse anti-human antibodies: STRO-1-FITC (Santa Cruz Biotechnology, Inc., CA, United States), CD146-PE (phycoerythrin), CD34-APC (allophycocyanin), CD117-PerCP-Cy5.5 (Peridinin-Chlorophyll-Protein-cyanin 5.5), CD45-PE, CD105-FITC, TRA-1-60-PE (all from BD Biosciences), CD24-APC, CD90-FITC, CD271-PE, CD49f-APC, CD81-FITC, SSEA-3-PE (all from BioLegend, Fell, Germany). Cells were incubated in the dark at RT with various combinations of antibodies. Additionally, for intracellular staining for Nanog, Oct3/4 and nestin, cells were first Fc-blocked and then additionally fixed with a 4% paraformaldehyde buffer, permeabilized with a saponin 0.1% (w/v) buffer (both from BD Biosciences) and then stained with the mouse anti-human antibodies Oct3/4-Alexa Fluor 647, Nanog-PE (both from BioLegend) and nestin-APC (R&D, Minneapolis, USA). Stained cells were washed twice with staining buffer (PBS + 1% BSA + 0.1% NaN3) and analyzed with a BD LSR II Flow Cytometer. A total of 100,000 events were acquired for each sample. Data were analyzed using Summit software, version 5.1 for Windows (Beckman Coulter, Inc. Krefeld, Germany).

2.3

Evaluation of cell viability/proliferation by MTT assay

Cell viability was determined using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, as previously described . DPSCs were exposed to the resinous monomer TEGDMA (provided free by VOCO, Cuxhaven, Germany) at concentrations 1 and 2 mM, which our previous study found to cause a low to medium cytotoxicity in these cells . TEGDMA was dissolved in absolute ethanol and sequentially diluted to obtain different concentrations of stock solutions. The monomer was 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 a negative control.

In parallel experiments, DPSCs were exposed to various concentrations (1–10 mM) of Lithium, in the form of LiCl (Sigma–Aldrich), which is known as a specific and noncompetitive GSK3β inhibitor . This second series of experiments was designed to evaluate the influence of various concentrations of LiCl on the viability of DPSCs. In further experiments, DPSCs were exposed to various concentrations (25, 50 and 100 ng/ml) of human recombinant Wnt-1 (hrWnt-1, Biochrom AG, Berlin, Germany), which is known to signal through β-catenin . This set of experiments intended to evaluate the influence of various concentrations of Wnt-1 on the viability of DPSCs. Finally, experiments combining the simultaneous exposure of DPSC to TEGDMA and LiCl (T/L) or TEGDMA and Wnt-1 (T/W) were also performed to evaluate potential synergistic or antagonistic effects on DPSC viability.

For the MTT assay, cells were seeded at 10 ⁴ cells/well in 96-well plates and left for 24 h to attach. Afterwards, the medium was replaced with medium containing the above mentioned concentrations of TEGDMA, LiCl, Wnt-1 or their combinations (and respective negative controls) and incubated for 24, 48 or 72 h. At the end of each time-point a 0.5 mg/ml MTT solution was added in each well and cells were incubated for 3 h at 37 °C and 5% CO ₂ . After this period the medium containing the MTT solution was discarded and the insoluble formazan was dissolved with DMSO for 1 h at RT. The absorbance was measured against blank (DMSO) at a wavelength of 545 nm and a reference filter of 630 nm by a microplate reader (Spectra Max 250, Molecular Devices, Biberach an der Riss, Germany). Experiments were performed in six replicates and repeated at least three times.

2.4

Cell-cycle analysis with flow cytometry

Cell cycle analysis was performed with flow cytometry. DPSCs were seeded in six well-plates at 2 × 10 ⁵ cells/well and allowed to attach for 24 h. Then, culture medium was replaced with CCM containing TEGDMA (1–2 mM), LiCl (1–10 mM), Wnt-1 (25–100 ng/ml) or their combinations. After 24, 48 or 72 h cells trypsinized, washed twice with ice-cold PBS and fixed in ice-cold ethanol. After washing with ice-cold PBS, cells were re-suspended in PBS containing 0.1% Triton-X, 20 μg/ml Propidium Iodide (PI) and 0.2 mg/ml RNase A (both from Sigma-Aldrich) and incubated in the dark for 30 min at RT. Samples were analyzed for DNA content with a BD FACSCalibur flow cytometer equipped with a 15 mW 488 nm argon ion laser (BD Biosciences). A total of 20,000 events were acquired for each sample using the CellQuest software. PI signals for analysis of DNA content were subjected to pulse processing for gating out cell debris and doublets. The FL2 fluorescence was detected in a linear/log histogram, and G0/G1, S, and G2/M phases were determined using multicycle cell analysis Software (Phoenix Flow Systems, San Diego, CA, USA). Three independent experiments were performed.

2.5

Quantitative real-time reverse-transcription polymerase chain reaction analysis

Total mRNA was isolated using the RNeasy kit (Qiagen, Hilden, Germany). cDNA synthesis using 1 μg template RNA was performed with Quanti Tect Reverse transcription kit (Qiagen), according to manufacturer’s instructions. Quanti Tect SYBR Green RT-PCR Kit (Qiagen) was used to perform all reactions. Rotor-Gene Q PCR device (Qiagen) was used to read the amplifications, which started with an initial step at 95 °C for 5 min to activate the Hotstart Taq DNA polymerase, followed by 40 cycles of PCR, comprising denaturation for 5 s at 95 °C and combined annealing/extension for 10 s at 60 °C. Beta-catenin/CTNB1 (NM_001098209, NM_001098210, NM_001904), LEF-1 (NM_001130713, NM_001166119, NM_016269), GSK3β (NM_001146156, NM_002093), Cyclin D1/CCND1 (NM_053056) and Cyclin B1/CCNB1 (NM_031966) mRNA expressions were quantified using respective QuantiTect primer assays. A standard melting curve was used to check quality of amplification and to ensure specificity. The results were adjusted by amplification efficiency (LinRegPCR) and genes were normalized to the two most stable housekeeping genes evaluated by geNorm (succinate dehydrogenase complex, subunit A, flavoprotein/SDHA and β-actin/ACTB, QuintiTect primer assays). All experiments were run in duplicates and repeated three times.

2.6

Western blot analysis

DPSCs were seeded in 60 mm dishes at 5 × 10 ⁵ cells/dish in CCM and allowed to attach for 24 h before being exposed for 24–72 h to various concentrations of TEGDMA, LiCl, Wnt-1 or their combinations. Cytosolic and nuclear extracts were separated from cultured cells using different lysis buffers. Briefly, cells were lysed on ice in buffer consisting of 25 mM Tris, 2 mM MgCl ₂ , 1 mM Na ₂ VO ₄ , 0.5% NP40 and a protease inhibitor mixture (mini-EDTA-free, Roche Applied Science, Mannheim, Germany). Cells were centrifuged at 5800 rpm for 5 min and the supernatant (cytosolic fraction) was stored at −20 °C The cell pellet (nuclei) was further lysed in buffer consisting of 10 mM Tris, 0.4 M LiCl, 20% Glycerol, 1 mM Na ₂ VO ₄ and the same protease inhibitor mixture. Cells were centrifuged at 14,000 rpm for 10 min and the supernatant (nuclear fraction) was stored at −20 °C. Protein concentrations of the extracted cytosolic and nuclear lysates were quantified using a BCA Protein Assay Kit (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions and measured spectrophotometrically at 562 nm by a microplate reader (Spectra Max 250, Molecular Devices). An equal amount of protein form each sample (2 μg for GAPDH and 20 μg for each β-catenin and Lamin B) was separated on a 10–15% sodium dodecyl sulfate polyacrylamide gel by electrophresis (SDS-PAGE) and then transferred onto a polyvinylidene fluoride (PVDF, Millipore, Bilerica, MA, USA) membrane at 240 mA for 1.5 h. After blocking in 3% BSA in PBS, the membranes were incubated overnight at 4 °C with the following primary antibodies (mouse- or rabbit-anti human diluted in 0.1% BSA in PBS): monoclonal mouse anti-β-catenin (1:500, BioLegend), monoclonal rabbit anti-GAPDH (1:10,000, Sigma), monoclonal mouse anti-Lamin B (1:500, Santa Cruz). GAPDH and Lamin B were used as the internal controls for the cytosolic and nuclear proteins respectively. The membranes were then washed with PBS containing Tween 20 (TPBS) and incubated with secondary goat anti-mouse or anti-rabbit conjugated horseradish peroxidase antibodies (1:10,000, DakoCytomation, Hamburg, Germany) for 1 h at RT and developed with the enhanced chemiluminescence (ECL) substrate (SuperSignal West Pico or Femto, Pierce, Rockford, IL, USA), using an ECL imaging system (Fusion SL-4-400WL, Vilber Lourmat, Eberhardzell, Germany).

2.7

Statistical analysis

At least three independent experiments, with two to six replicates, were performed. Results were analysed by one-way ANOVA Bonferroni’s post-test and two-tailed t -test using IBM SPSS Statistics 22 Software for Windows (International Business Machines Corporation, U.S.A) (* p < 0.05). Data were expressed as means ± standard deviation (SD).