2.1

Chemicals

The monomer TEGDMA, the chemicals MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], L-ascorbic acid phosphate and DMSO (dimethyl sulfoxide), as well as all other chemicals used for fixation and staining of the cells were purchased from Sigma–Aldrich (Taufkirchen, Germany). The culture medium a-Modification of Eagle’s Medium (aMEM), trypsin/EDTA, penicillin, streptomycin, amphotericin B and fetal bovine serum (FBS-EU tested), Collagenase type I and Dispase II were purchased from Invitrogen (Karlsruhe, Germany). The mouse anti-human antibody STRO-1-FITC (fluorescein isothiocyanate) was purchased from Santa Cruz Biotechnology, Inc. (California, United States). The mouse anti-human antibodies CD146-PE (phycoerythrin), CD34-APC (allophycocyanin), CD117-PerCP-Cy5.5 (Peridinin-Chlorophyll- Protein-cyanin 5.5), CD45-PE, CD105-FITC, TRA-1-60-PE, Oct3/4-Alexa Fluor 647 and Nanog-PE were purchased from BD Biosciences (Heidelberg, Germany), whereas the mouse anti-human antibodies CD24-APC, CD90-FITC, CD271-PE, CD49f-APC, CD81-FITC, SSEA-3-PE were purchased from BioLegend (San Diego, United States). Finally, the mouse anti-human antibody for nestin-APC was purchased from R&D (Minneapolis, USA).

2.2

Cell culture

DPSC cultures were established from third molars derived from healthy donors aged 16–18 years old. 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 the enzymatic dissociation method was used . Briefly, teeth were disinfected and cut around the cementum–enamel junction to expose the pulp chamber. 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 μm cell strainer. Cells were expanded with a-MEM culture medium, supplemented with 15% FBS, 100 mM l -ascorbic acid phosphate, 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% CO ₂ . Cultured DPSCs in passage numbers from 4 to 8 from at least three different donors were used for all the experiments with similar results.

2.3

Characterization of DPSC cultures with flow cytometry

DPSC established cultures were characterized for embryonic stem cell (SC) markers (Nanog, Oct3/4, SSEA-3, TRA-1-60), mesenchymal SC markers (STRO-1, CD146/MUC18, CD105/endoglin, CD24, CD90/Thy-1, CD81-TAPA, CD34, CD49f/a6-integrin), neural SC markers (CD271/NGFR, nestin) and hemopoietic SC markers (CD117/c-kit, CD45).

For surface epitope expression analysis, 10 ⁶ cells/probe were first Fc-blocked with 1 mg of human IgG for 20 min at room temperature (RT) and subsequently stained with the following fluorochrome-conjugated mouse anti-human antibodies: STRO-1-FITC, CD146-PE, CD34-APC, CD117-PerCP-Cy5.5, CD45-PE, CD105-FITC, TRA-1-60-PE, CD24-APC, CD90-FITC, CD271-PE, CD49f-APC, CD81-FITC, SSEA-3-PE. Cells were incubated with various combinations of antibodies for 30 min in the dark at RT. Moreover, 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 and then stained with the mouse anti-human antibodies Oct3/4-Alexa Fluor 647, Nanog-PE and nestin-APC. Stained cells were washed twice with staining buffer (dPBS + 1%BSA + 0.1%NaN ₃ ) 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 5.1 software.

2.4

Collection of CM

CM was collected from DPSC cultures under two different conditions, i.e. from cultures grown under normal serum conditions (NSC) (15% FBS) and from cultures grown under serum deprivation conditions (SDC) (0.5% FBS). The cells were first cultured with normal CCM in 75 cm ² flasks, until reaching confluency of about 70–80%. Subsequently, in a first set of cultures, medium was changed with fresh CCM (FBS 15%) and the CM (CM1) of these NSC cultures was collected every 2 d for a total of 12 d. In second set of cultures the medium was changed with fresh CCM containing only 0.5% FBS (=serum deprived medium; SDM) and the CM (CM2) in these SDC cultures was collected every 4 d for a total of 24 d. Before medium change all cultures were washed twice with PBS. After collection at each time-point, CM1 and CM2 (containing the secretome of the cultured DPSCs under NSC and SDC respectively) was centrifuged at 200 × g for 5 min to remove any cell debris, the supernatant was filtered using a 0.2 μm filter and the samples were stored at −80 °C until used for further experiments. As negative controls, CCM (15% FBS) and SDM (0.5% FBS) were processed equally (non-conditioned medium; NCM).

2.5

Patterns of exposure of DPSCs to CM and evaluation of cell viability by MTT assay

In a first series of experiments, DPSCs were exposed to CM1 and CM2 in order to examine how CM may influence their viability. First, DPSCs were seeded in 96-well plates (10 ⁴ cells/well) and left for 24 h to attach. After this period, the supernatant was replaced with different types and concentrations of CM1 and CM2 in order to screen the optimum conditions favoring cell viability, as follows:

• 1.

  Medium was replaced with CM1 (15% FBS) from each of the six collection periods (2, 4, 6, 8, 10, 12 d) at two different concentrations (50% and 100%). Dilution to obtain the 50% concentration was performed using normal CCM (15% FBS), so that the final FBS concentration remained 15%.

• 2.

  Medium was replaced with CM2 (0.5% FBS) from each of the six collection periods (4, 8, 12, 16, 20, 24 d) at two different concentrations (50% and 100%). Dilution to obtain the 50% concentration was performed using SDM (0.5% FBS), so that the final FBS concentration remained 0.5% (=CM2a).

• 3.

  Medium was replaced with CM2 that was previously enriched at 5% FBS, from each of the six collection periods (4, 8, 12, 16, 20, 24 d) at two different concentrations (50% and 100%). Dilution to obtain the 50% concentration was performed using SDM (5% FBS), so that the final FBS concentration was 5% (=CM2b). Actually, CM2b was collected under SDC (0.5% FBS), but was enriched at 5% FBS before being exposed again to the cells.

• 4.

  Medium was replaced with CM2 that was previously enriched at 15% FBS, from each of the six collection periods (4, 8, 12, 16, 20, 24 d) at two different concentrations (50% and 100%). Dilution to obtain the 50% concentration was performed using CCM (15% FBS), so that the final FBS concentration was 15% (=CM2c). Actually, CM2c was collected under SDC (0.5% FBS), but was enriched at 15% FBS before being exposed again to the cells.

Cells were exposed to all the above mentioned different conditions (screening experiment) and after incubation period of 24, 48 or 72 h, MTT assay was performed. At the end of each time-point 20 μl of MTT (5 mg/ml in PBS) 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 room temperature (RT). The absorbance was measured against blank (DMSO) at a wave-length of 545 nm and a reference filter of 630 nm by a microplate reader (Epock, Biotek, Biotek Instruments, Inc, Vermont, USA). Experiments were performed in six replicates and repeated three times.

2.6

Exposure of DPSCs to CM2 in the presence of TEGDMA and evaluation of cell viability by MTT assay

In the second series of experiments, DPSCs were exposed to various concentrations of TEGDMA (0.25–2 mM) in the presence or absence of different concentrations (25, 50 and 75%) of CM2, which proved to be the most effective in enhancing cell viability in the previous set of experiments. The aim of this second series of experiments was to assess whether CM2 could counteract the toxic influence of TEGDMA on DPSCs viability. DPSCs were seeded in 96-well plates (10 ⁴ cells/well) in CCM and they were incubated for 24 h to allow attachment. Afterwards, the medium was replaced with CCM enriched with three (25, 50 and 75%) concentrations CM2c (=medium collected under serum deprivation conditions and enriched with 15% FBS) and containing 4 concentrations (0.25, 0.5, 1 and 2 mM) of TEGDMA. In this experiment we used CM2 collected after the first 4 d of serum deprivation (CM2-4d), which was found to produce the best effect in the previous set of experiments. In control cultures CCM was replaced by fresh CCM containing the same concentrations of TEGDMA, so as the effects of CM on cell viability could be compared between these two types of cultures (+CM or −CM). TEGDMA was dissolved in DMSO in order to obtain different concentrations of stock solutions. TEGDMA stock solutions were added at a dilution of 1/400 into the respective media (+CM and −CM) in order to have 4 final concentrations of TEGDMA (0.25, 0.5, 1.0 and 2.0 mM). The final concentration of DMSO did not exceed 0.25% (v/v), which was found not to be toxic in cells in pilot experiments. Cells incubated with medium (+CM or −CM) containing 0.25% DMSO served as negative controls with respect to TEGDMA. MTT assay was performed at 24, 48 and 72 h, as already described. Experiments were performed in six replicates and repeated three times.

2.7

Exposure of DPSCs to CM2 in the presence of TEGDMA and evaluation of cell migration by the in vitro scratch assay

DPSCs were exposed to various concentrations of TEGDMA (0.25–2 mM) in the presence or absence (+CM or −CM) of 50% of CM2c (enriched with 15% FBS), collected at day 4 after induction of SD, which was found to be the most effective in enhancing cell viability, as assessed by the MTT assay. For qualitative assessment of DPSCs migratory potential, the in vitro scratch assay was used, as previously described . Briefly, DPSCs were seeded in 6-well plates (5 × 10 ⁵ cells/well) in CCM and after reaching 90% confluency, DPSCs were exposed to TEGDMA (0.25, 0.5, 1.0, 2.0 mM) in the presence or absence of 50% CM2c. After a 24 h exposure to the above factors (TEGDMA ± CM2c), an even scratch was performed on the monolayer by using a p200 pipette tip. Every 12 h and for a total period of 72 h the scratch was observed under an inverted microscope and photographs were taken in order to determine under which conditions cells migrated more rapidly in order to perform “wound closure”. Cultures exposed to the same concentrations of TEGDMA under the presence or absence of CM2 (+CM or −CM) were comparatively evaluated. All samples were run in triplicate and the experiments were repeated three times.

2.8

Exposure of DPSCs to CM2 in the presence of TEGDMA and evaluation of cell migration by transwell inserts assay

For the quantitative assessment of the migratory potential of DPSCs under the influence of the same concentrations of TEGDMA and CM2c used for the in vitro scratch assay, a more sophisticated test, based on the potential of cells to migrate through a porous membrane as a response to a chemical attractant was used. DPSCs were first cultured in 75 cm ² flasks with CCM (15% FBS) and when the confluency was about 70–80% the culture medium was changed with SDM (0.5% FBS) for 24 h. Afterwards, cells were transferred at 2 × 10 ⁵ /100 μl density onto the polyester transwell (PET) inserts of 24-well plates with 8 μm membrane pore size (Corning, Life Sciences, Ltd). Transwell inserts were previously coated with a collagen I solution (10 μg/cm ² ). Cells were seeded at the top of the membranes, whereas at the lower chamber of the 24 well plates either CCM (−CM) or CCM enriched with 50% CM2c (+CM), both containing different TEGDMA concentrations (0.5, 1.0, 2.0 mM) were added as chemo-attractants. After 24 h the medium was aspirated and the upper surface of each membrane of the transwell insert was gently swabbed in order to remove the cells that were not migrated through the membrane. Membranes containing the migrated cells were fixed with 10% neutral buffered formalin (NBF) for 30 min at RT and stained with crystal violet solution (0.5%) in order to be counted. Photographs of the stained cells were taken from at least 5 random fields of view (20×) for each membrane under an inverted microscope equipped with a digital camera (Zeiss Axiovert 40, Carl Zeiss micro imaging, GmbH, Göttingen, Germany). All samples were run in triplicate and the experiments were repeated three times.

2.9

Exposure of DPSCs to CM in the presence of low (‘subtoxic’) concentrations of TEGDMA and evaluation of the mineralization potential by Alizarin Red S assay

In this set of experiments DPSCs were induced for osteo/odontogenic differentiation in the presence of low (0.25 and 0.5 mM) concentrations of TEGDMA and CM2c (50%, collected at day 4 after SD), as described for the viability and migration experiments. Osteo/odontogenic differentiation was induced by CCM (−CM) or CCM enriched with 50% CM2c (+CM), 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) (=differentiation medium ± CM2c). Cells were treated for a total period of 30 d with the differentiation medium (±fresh CM2c) containing the different concentrations of TEGDMA (0.25, 0.5 mM) or DMSO-control (0.25%) being changed every 2 d (=induced -CM or +CM cultures). Cultures exposed to normal CCM or CCM enriched with 50% CM2c but without the osteogenic supplements for the same 4-week period served as negative control (=uninduced -CM or +CM cultures). Mineralization was assessed at each time-point (12, 18, 22 and 30 d after induction of differentiation) using the Alizarin Red S (AR-S) mineralization assay, as previously described . Briefly, cell cultures were washed twice with PBS (−) (without Ca ²⁺ and Mg ²⁺ ) 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 equipped with a digital camera (Zeiss Axiovert 40, Carl Zeiss microimaging, GmbH, Göttingen, Germany). 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 12 h at 37 °C and the OD _550nm was measured using a microplate reader (Epock, Biotek, Biotek instruments, Inc, Vermont, USA). Mineralized nodule formation was represented as nmol AR-S per μg of total cellular protein, determined by Bicinchoninic Acid (BCA) Protein assay. All samples were run in duplicate and the experiments were repeated three times.

2.10

ELISA for the presence of TGF-β1 and FGF-2 in CM

The concentrations of FGF-2 and TGF-β1, two of the most important factors implicated in cell proliferation, migration and differentiation were measured in CM2, collected under SDC (0.5% FBS) conditions, as well as in cultures grown under NSC (15% FBS) conditions and exposed to various concentrations of TEGDMA (0.25–2 mM), the latter in order to assess whether TEGDMA could be an additional factor to stimulate DPSCs autocrine/paracrine effects. For this purpose, sandwich ELISA kits were obtained from R&D Systems (Minneapolis, MN, USA) and assays were performed following the manufacturer’s instructions. Baseline values for TGF-β1 and FGF-2 contained in SDM (0.5% FBS) and CCM (15% FBS) were subtracted from the values measured in conditioned media at different time-points of CM2 (SDC) collection or under different TEGDMA concentrations respectively. ELISAs were performed in triplicate with two replicates each.

2.11

Statistics

Statistical analysis of the data was performed using one-way and two-way analysis of variance (ANOVA). The comparisons with the control were performed with Dunnett test (2-sided) and follow up comparisons between groups that differed from control were carried out using Least Significance Difference (LSD) control. Analysis was performed with IBM Statistics 19 and the level of statistical difference was 0.05 ( p < 0.05).

2.1

Chemicals

The monomer TEGDMA, the chemicals MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], L-ascorbic acid phosphate and DMSO (dimethyl sulfoxide), as well as all other chemicals used for fixation and staining of the cells were purchased from Sigma–Aldrich (Taufkirchen, Germany). The culture medium a-Modification of Eagle’s Medium (aMEM), trypsin/EDTA, penicillin, streptomycin, amphotericin B and fetal bovine serum (FBS-EU tested), Collagenase type I and Dispase II were purchased from Invitrogen (Karlsruhe, Germany). The mouse anti-human antibody STRO-1-FITC (fluorescein isothiocyanate) was purchased from Santa Cruz Biotechnology, Inc. (California, United States). The mouse anti-human antibodies CD146-PE (phycoerythrin), CD34-APC (allophycocyanin), CD117-PerCP-Cy5.5 (Peridinin-Chlorophyll- Protein-cyanin 5.5), CD45-PE, CD105-FITC, TRA-1-60-PE, Oct3/4-Alexa Fluor 647 and Nanog-PE were purchased from BD Biosciences (Heidelberg, Germany), whereas the mouse anti-human antibodies CD24-APC, CD90-FITC, CD271-PE, CD49f-APC, CD81-FITC, SSEA-3-PE were purchased from BioLegend (San Diego, United States). Finally, the mouse anti-human antibody for nestin-APC was purchased from R&D (Minneapolis, USA).

2.2

Cell culture

DPSC cultures were established from third molars derived from healthy donors aged 16–18 years old. 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 the enzymatic dissociation method was used . Briefly, teeth were disinfected and cut around the cementum–enamel junction to expose the pulp chamber. 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 μm cell strainer. Cells were expanded with a-MEM culture medium, supplemented with 15% FBS, 100 mM l -ascorbic acid phosphate, 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% CO ₂ . Cultured DPSCs in passage numbers from 4 to 8 from at least three different donors were used for all the experiments with similar results.

2.3

Characterization of DPSC cultures with flow cytometry

DPSC established cultures were characterized for embryonic stem cell (SC) markers (Nanog, Oct3/4, SSEA-3, TRA-1-60), mesenchymal SC markers (STRO-1, CD146/MUC18, CD105/endoglin, CD24, CD90/Thy-1, CD81-TAPA, CD34, CD49f/a6-integrin), neural SC markers (CD271/NGFR, nestin) and hemopoietic SC markers (CD117/c-kit, CD45).

For surface epitope expression analysis, 10 ⁶ cells/probe were first Fc-blocked with 1 mg of human IgG for 20 min at room temperature (RT) and subsequently stained with the following fluorochrome-conjugated mouse anti-human antibodies: STRO-1-FITC, CD146-PE, CD34-APC, CD117-PerCP-Cy5.5, CD45-PE, CD105-FITC, TRA-1-60-PE, CD24-APC, CD90-FITC, CD271-PE, CD49f-APC, CD81-FITC, SSEA-3-PE. Cells were incubated with various combinations of antibodies for 30 min in the dark at RT. Moreover, 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 and then stained with the mouse anti-human antibodies Oct3/4-Alexa Fluor 647, Nanog-PE and nestin-APC. Stained cells were washed twice with staining buffer (dPBS + 1%BSA + 0.1%NaN ₃ ) 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 5.1 software.

2.4

Collection of CM

CM was collected from DPSC cultures under two different conditions, i.e. from cultures grown under normal serum conditions (NSC) (15% FBS) and from cultures grown under serum deprivation conditions (SDC) (0.5% FBS). The cells were first cultured with normal CCM in 75 cm ² flasks, until reaching confluency of about 70–80%. Subsequently, in a first set of cultures, medium was changed with fresh CCM (FBS 15%) and the CM (CM1) of these NSC cultures was collected every 2 d for a total of 12 d. In second set of cultures the medium was changed with fresh CCM containing only 0.5% FBS (=serum deprived medium; SDM) and the CM (CM2) in these SDC cultures was collected every 4 d for a total of 24 d. Before medium change all cultures were washed twice with PBS. After collection at each time-point, CM1 and CM2 (containing the secretome of the cultured DPSCs under NSC and SDC respectively) was centrifuged at 200 × g for 5 min to remove any cell debris, the supernatant was filtered using a 0.2 μm filter and the samples were stored at −80 °C until used for further experiments. As negative controls, CCM (15% FBS) and SDM (0.5% FBS) were processed equally (non-conditioned medium; NCM).

2.5

Patterns of exposure of DPSCs to CM and evaluation of cell viability by MTT assay

In a first series of experiments, DPSCs were exposed to CM1 and CM2 in order to examine how CM may influence their viability. First, DPSCs were seeded in 96-well plates (10 ⁴ cells/well) and left for 24 h to attach. After this period, the supernatant was replaced with different types and concentrations of CM1 and CM2 in order to screen the optimum conditions favoring cell viability, as follows:

• 1.

  Medium was replaced with CM1 (15% FBS) from each of the six collection periods (2, 4, 6, 8, 10, 12 d) at two different concentrations (50% and 100%). Dilution to obtain the 50% concentration was performed using normal CCM (15% FBS), so that the final FBS concentration remained 15%.

• 2.

  Medium was replaced with CM2 (0.5% FBS) from each of the six collection periods (4, 8, 12, 16, 20, 24 d) at two different concentrations (50% and 100%). Dilution to obtain the 50% concentration was performed using SDM (0.5% FBS), so that the final FBS concentration remained 0.5% (=CM2a).

• 3.

  Medium was replaced with CM2 that was previously enriched at 5% FBS, from each of the six collection periods (4, 8, 12, 16, 20, 24 d) at two different concentrations (50% and 100%). Dilution to obtain the 50% concentration was performed using SDM (5% FBS), so that the final FBS concentration was 5% (=CM2b). Actually, CM2b was collected under SDC (0.5% FBS), but was enriched at 5% FBS before being exposed again to the cells.

• 4.

  Medium was replaced with CM2 that was previously enriched at 15% FBS, from each of the six collection periods (4, 8, 12, 16, 20, 24 d) at two different concentrations (50% and 100%). Dilution to obtain the 50% concentration was performed using CCM (15% FBS), so that the final FBS concentration was 15% (=CM2c). Actually, CM2c was collected under SDC (0.5% FBS), but was enriched at 15% FBS before being exposed again to the cells.

Cells were exposed to all the above mentioned different conditions (screening experiment) and after incubation period of 24, 48 or 72 h, MTT assay was performed. At the end of each time-point 20 μl of MTT (5 mg/ml in PBS) 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 room temperature (RT). The absorbance was measured against blank (DMSO) at a wave-length of 545 nm and a reference filter of 630 nm by a microplate reader (Epock, Biotek, Biotek Instruments, Inc, Vermont, USA). Experiments were performed in six replicates and repeated three times.

2.6

Exposure of DPSCs to CM2 in the presence of TEGDMA and evaluation of cell viability by MTT assay

In the second series of experiments, DPSCs were exposed to various concentrations of TEGDMA (0.25–2 mM) in the presence or absence of different concentrations (25, 50 and 75%) of CM2, which proved to be the most effective in enhancing cell viability in the previous set of experiments. The aim of this second series of experiments was to assess whether CM2 could counteract the toxic influence of TEGDMA on DPSCs viability. DPSCs were seeded in 96-well plates (10 ⁴ cells/well) in CCM and they were incubated for 24 h to allow attachment. Afterwards, the medium was replaced with CCM enriched with three (25, 50 and 75%) concentrations CM2c (=medium collected under serum deprivation conditions and enriched with 15% FBS) and containing 4 concentrations (0.25, 0.5, 1 and 2 mM) of TEGDMA. In this experiment we used CM2 collected after the first 4 d of serum deprivation (CM2-4d), which was found to produce the best effect in the previous set of experiments. In control cultures CCM was replaced by fresh CCM containing the same concentrations of TEGDMA, so as the effects of CM on cell viability could be compared between these two types of cultures (+CM or −CM). TEGDMA was dissolved in DMSO in order to obtain different concentrations of stock solutions. TEGDMA stock solutions were added at a dilution of 1/400 into the respective media (+CM and −CM) in order to have 4 final concentrations of TEGDMA (0.25, 0.5, 1.0 and 2.0 mM). The final concentration of DMSO did not exceed 0.25% (v/v), which was found not to be toxic in cells in pilot experiments. Cells incubated with medium (+CM or −CM) containing 0.25% DMSO served as negative controls with respect to TEGDMA. MTT assay was performed at 24, 48 and 72 h, as already described. Experiments were performed in six replicates and repeated three times.

2.7

Exposure of DPSCs to CM2 in the presence of TEGDMA and evaluation of cell migration by the in vitro scratch assay

DPSCs were exposed to various concentrations of TEGDMA (0.25–2 mM) in the presence or absence (+CM or −CM) of 50% of CM2c (enriched with 15% FBS), collected at day 4 after induction of SD, which was found to be the most effective in enhancing cell viability, as assessed by the MTT assay. For qualitative assessment of DPSCs migratory potential, the in vitro scratch assay was used, as previously described . Briefly, DPSCs were seeded in 6-well plates (5 × 10 ⁵ cells/well) in CCM and after reaching 90% confluency, DPSCs were exposed to TEGDMA (0.25, 0.5, 1.0, 2.0 mM) in the presence or absence of 50% CM2c. After a 24 h exposure to the above factors (TEGDMA ± CM2c), an even scratch was performed on the monolayer by using a p200 pipette tip. Every 12 h and for a total period of 72 h the scratch was observed under an inverted microscope and photographs were taken in order to determine under which conditions cells migrated more rapidly in order to perform “wound closure”. Cultures exposed to the same concentrations of TEGDMA under the presence or absence of CM2 (+CM or −CM) were comparatively evaluated. All samples were run in triplicate and the experiments were repeated three times.

2.8

Exposure of DPSCs to CM2 in the presence of TEGDMA and evaluation of cell migration by transwell inserts assay

For the quantitative assessment of the migratory potential of DPSCs under the influence of the same concentrations of TEGDMA and CM2c used for the in vitro scratch assay, a more sophisticated test, based on the potential of cells to migrate through a porous membrane as a response to a chemical attractant was used. DPSCs were first cultured in 75 cm ² flasks with CCM (15% FBS) and when the confluency was about 70–80% the culture medium was changed with SDM (0.5% FBS) for 24 h. Afterwards, cells were transferred at 2 × 10 ⁵ /100 μl density onto the polyester transwell (PET) inserts of 24-well plates with 8 μm membrane pore size (Corning, Life Sciences, Ltd). Transwell inserts were previously coated with a collagen I solution (10 μg/cm ² ). Cells were seeded at the top of the membranes, whereas at the lower chamber of the 24 well plates either CCM (−CM) or CCM enriched with 50% CM2c (+CM), both containing different TEGDMA concentrations (0.5, 1.0, 2.0 mM) were added as chemo-attractants. After 24 h the medium was aspirated and the upper surface of each membrane of the transwell insert was gently swabbed in order to remove the cells that were not migrated through the membrane. Membranes containing the migrated cells were fixed with 10% neutral buffered formalin (NBF) for 30 min at RT and stained with crystal violet solution (0.5%) in order to be counted. Photographs of the stained cells were taken from at least 5 random fields of view (20×) for each membrane under an inverted microscope equipped with a digital camera (Zeiss Axiovert 40, Carl Zeiss micro imaging, GmbH, Göttingen, Germany). All samples were run in triplicate and the experiments were repeated three times.

2.9

Exposure of DPSCs to CM in the presence of low (‘subtoxic’) concentrations of TEGDMA and evaluation of the mineralization potential by Alizarin Red S assay

In this set of experiments DPSCs were induced for osteo/odontogenic differentiation in the presence of low (0.25 and 0.5 mM) concentrations of TEGDMA and CM2c (50%, collected at day 4 after SD), as described for the viability and migration experiments. Osteo/odontogenic differentiation was induced by CCM (−CM) or CCM enriched with 50% CM2c (+CM), 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) (=differentiation medium ± CM2c). Cells were treated for a total period of 30 d with the differentiation medium (±fresh CM2c) containing the different concentrations of TEGDMA (0.25, 0.5 mM) or DMSO-control (0.25%) being changed every 2 d (=induced -CM or +CM cultures). Cultures exposed to normal CCM or CCM enriched with 50% CM2c but without the osteogenic supplements for the same 4-week period served as negative control (=uninduced -CM or +CM cultures). Mineralization was assessed at each time-point (12, 18, 22 and 30 d after induction of differentiation) using the Alizarin Red S (AR-S) mineralization assay, as previously described . Briefly, cell cultures were washed twice with PBS (−) (without Ca ²⁺ and Mg ²⁺ ) 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 equipped with a digital camera (Zeiss Axiovert 40, Carl Zeiss microimaging, GmbH, Göttingen, Germany). 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 12 h at 37 °C and the OD _550nm was measured using a microplate reader (Epock, Biotek, Biotek instruments, Inc, Vermont, USA). Mineralized nodule formation was represented as nmol AR-S per μg of total cellular protein, determined by Bicinchoninic Acid (BCA) Protein assay. All samples were run in duplicate and the experiments were repeated three times.

2.10

ELISA for the presence of TGF-β1 and FGF-2 in CM

The concentrations of FGF-2 and TGF-β1, two of the most important factors implicated in cell proliferation, migration and differentiation were measured in CM2, collected under SDC (0.5% FBS) conditions, as well as in cultures grown under NSC (15% FBS) conditions and exposed to various concentrations of TEGDMA (0.25–2 mM), the latter in order to assess whether TEGDMA could be an additional factor to stimulate DPSCs autocrine/paracrine effects. For this purpose, sandwich ELISA kits were obtained from R&D Systems (Minneapolis, MN, USA) and assays were performed following the manufacturer’s instructions. Baseline values for TGF-β1 and FGF-2 contained in SDM (0.5% FBS) and CCM (15% FBS) were subtracted from the values measured in conditioned media at different time-points of CM2 (SDC) collection or under different TEGDMA concentrations respectively. ELISAs were performed in triplicate with two replicates each.

2.11

Statistics

Statistical analysis of the data was performed using one-way and two-way analysis of variance (ANOVA). The comparisons with the control were performed with Dunnett test (2-sided) and follow up comparisons between groups that differed from control were carried out using Least Significance Difference (LSD) control. Analysis was performed with IBM Statistics 19 and the level of statistical difference was 0.05 ( p < 0.05).