Influence of TEGDMA monomer on MMP-2, MMP-8, and MMP-9 production and collagenase activity in pulp cells

All chemicals used were obtained from Sigma-Aldrich (now Merck KGaA, Darmstadt, Germany) unless stated otherwise.

Pulp tissue was collected from five healthy third molar teeth extracted for orthodontic reasons. Informed consent was obtained under a protocol approved by the University of Pecs (Pecs, Hungary, under license No. PTE3026/2007). In line with patient confidentiality guidelines, all data were anonymized and study was performed in accordance with the ethical standards laid down in 1964 Declaration of Helsinki and its later amendments or comparable ethical standards.

Following extraction, pulp tissue was removed according to Sun et al. [25]. Through an explant method, cells were cultured in minimum essential medium Eagle-alpha modification (Alpha MEM) containing ultraglutamine 1, ribonucleosides, and deoxyribonucleosides (Lonza, Basel, Switzerland) with the addition of 10% fetal bovine serum (FBS, Euroclone, Milan, Italy) and antibiotics (100 U/ml penicillin, 100 μg/ml streptomycin, 2.5 μg/ml amphotericin B) and cultured in a humidified atmosphere containing 5% CO₂ at 37 °C. As the cultures reached 90% confluence, a passage was performed to plastic Petri dishes. Cell cultures were first washed with phosphate-buffered saline (PBS, 1.37 mM NaCl, 0.27 mM KCl, 0.43 mM Na₂HPO₄·7H₂O, 0.14 mM KH₂PO₄, pH 7.4) followed by a 10-min trypsin (0.25% trypsin + 0.02% ethylene-diamine-tetraacetic acid (EDTA); Gibco, Grand Island, NY, USA) digestion in a controlled, 37 °C, environment. After two or three passages, cells were seeded at an arbitrary density of 2 × 10⁴ cells/cm², based on previous experience on similar populations. Forty-eight hours prior to the start of TEGDMA exposure, the medium was exchanged to 2% FBS containing medium (without antibiotics) in order to decrease the potential signaling interference.

Cells were exposed to TEGDMA concentrations of 0.1, 0.2, and 0.75 mM for 24 h as suggested by our pilot viability studies. For preliminary viability testing, a 1-day exposure to concentrations of 0.1, 0.2, 0.75, 1.5, and 3 mM was carried out in order to find out if the concentration range has not yet cause significant cell death.

Water-soluble tetrazolium salts (WST-1) colorimetric assay was performed as an indicator of intracellular mitochondrial metabolism and hence viability. After TEGDMA exposure, the medium was removed and 200 μl of WST-1 reagent (Hoffmann-La Roche, Basel, Switzerland) was added in a 1:9 WST to 2% Alpha MEM medium ratio (180 μl of medium and 20 μl of WST dye). The cells were stored at 37 °C for 4 h followed by transfer to a 96-well plate. Absorbance was measured in 100 μl samples at 440 nm by a FluoStar Optima plate reader (BMG Labtech, USA).

EnzCheck Gelatinolytic/Collagenolytic activity assay kit (Molecular Probes, Eugene, OR, USA) was used to investigate the possible effect of various concentrations of TEGDMA monomers on enzyme activity in pulp cell lysates and media derived from cells. Following the manufacturer’s instructions, the highly labeled fluorescent gelatin substrate was mixed with reaction buffer in a final volume of 200 μl. Cell culture media or dilutions of mechanically homogenized cell extracts suspended in lysis buffer were collected into 96-well plates. Proteolysis was determined by a Promega Glo Max plate reader (Madison, Wisconsin, USA), operated at a fluorescent excitation maximum of 495 nm and emission maximum of 515 nm. Absolute enzyme activity values were plotted and analyzed.

After TEGDMA treatment, cells were harvested and lysed as described in previous studies [32]. Briefly, the pulp cells were collected in cold lysis buffer (50 mM Tris-base, pH 7.4, 10% glycerol, 150 mM NaCl, 1 mM EGTA, 1 mM Na-orthovanadate, 100 mM NaF, 5 μM ZnCl₂, 10 μg/ml aprotinin, 1 μg/ml leupeptin, 1 mM PMSF, 1% Triton X-100) and homogenized for 20 s. The homogenate was centrifuged at 4 °C and at 40,000×g for 30 min. Protein concentrations of the supernatants were measured (Lowry’s method, Detergent Compatible Protein Assay Kit, Bio-Rad, Hercules, CA, USA). Protein concentrates were then diluted to contain equal 30 μg of protein. Laemmli buffer (prepared from 25 ml 1 M Tris-HCl, pH 6.8, 40 ml glycerol, 8 g SDS, 10 ml 100 mM EGTA, 10 ml 100 mM EDTA, 1 ml 1% bromophenol blue, and distilled water to a total volume of 100 ml) was added followed by boiling for denaturation. Subsequently, 10% SDS-containing polyacrylamide gels were used to separate proteins based on molecular size. Proteins were then transferred to PVDF membranes (Hybond-P, GE Healthcare, UK) by the Trans-Blot Turbo system (Bio-Rad, Hercules, CA, USA). Nonspecific binding on the membrane was blocked by 3% nonfat dry milk in TBS-Tween (10 mM Tris-base, 150 mM NaCl, 0.2% Tween-20, pH 8.0). Rabbit polyclonal primary antibodies were added specific to MMP-8 (Thermo Fisher Scientific, Waltham, MA, USA); MMP-9 (Abcam, Cambridge, UK); p-(phospho-)ERK1/2 (Thr202/Tyr204), p-p38 (Thr180/Tyr182), and p-JNK (Thr183/Tyr185) (Cell Signaling Technology, Beverly, MA, USA); and rabbit monoclonal antibodies specific to MMP-2 (D4M2N, Cell Signaling Technology, Beverly, MA, USA) diluted 1:1000 in the blocking solution and then incubated overnight. Excess antibody was removed by five washes of TBS-Tween. Incubation with a horseradish-peroxidase (HRP)-conjugated polyclonal goat anti-rabbit secondary antibody (Pierce, Thermo Fischer Scientific, Rockford, IL, USA) diluted 1:10,000 in blocking solution followed. Enhanced chemiluminescent signal (Immobilon Western, Millipore Corporation, Billerica, MA, USA) was detected using a G:box gel documentation system (Syngene International Ltd, Bangalore, India). Membranes were then chemically stripped of antibodies (0.2 M glycin-HCl, 0.2% Tween-20, 0.05%, pH 2.5) and reprobed using β-actin, ERK1/2, p38, or JNK (Cell Signaling Technology, Beverly, MA, USA) rabbit polyclonal primary antisera as mentioned above to prove the absence of disparity in protein concentration among samples. Densitometry analysis was performed using the ImageJ software (National Institutes of Health, USA).

Pulp cells seated onto glass coverslips were exposed to the conditioned or control media and processed for immunocytochemistry. Briefly, a quick rinse in 37 °C PBS was followed by a 4% paraformaldehyde fixation in PBS at pH 7.4, 4 °C for 12 h. The fixative was removed by three changes of TBS (50 mM Tris-HCl, pH 7.4, 150 mM NaCl). Permeabilization was achieved by a 30-min wash with 0.1% Triton X-100 in TBS at 4 °C. Nonspecific binding sites were blocked by incubation in 5% nonfat dry milk in TBS at 4 °C for 1 h. The primary antibodies MMP-2 (D4M2N, Cell Signaling Technology, Beverly, MA, USA), MMP-8 (Thermo Fisher Scientific, Waltham, MA, USA), and MMP-9 (Abcam, Cambridge, UK) were diluted 1:100 in the blocking solution and incubated with the cells overnight at 4 °C. After five washes in TBS, Cy3-conjugated polyclonal donkey-anti-rabbit antibodies (Jackson Immuno Research, Cambridgeshire, UK) were diluted 1:200 in the blocking solution and added to the cells for overnight incubation at 4 °C. For each antigen respectively, control samples prepared by the omission of the primary antibodies produced no visible fluorescence signal using the same microscope. During the final five TBS washes, nuclei were counterstained with Hoechst 33342 (Calbiochem, La Jolla, CA, USA). Fluorescence microscopy images were obtained using an Olympus FV-1000 laser scanning confocal system (Olympus Europa, Hamburg, Germany). The presented single optical slice pictures were generated using a × 40 dry objective and are representative of series of four to five independent experiments with similar results. Determination of raw integrated density values was carried out using the ImageJ software (National Institutes of Health, USA).

WST-1, collagenolytic activity assay, western blot, and immunocytochemistry density data presented in diagrams were gathered in a series of four independent experiments; values shown are the means and standard deviations (± SD). Significance of differences was determined using one-way analysis of variance (ANOVA) test with the application of Tukey post hoc tests for multiple samples. P values ˂ 0.05 were considered to be significant. Significant differences considered relevant to major findings were marked in the graphs, and their corresponding P values were indicated in the figure legend.

The time- and dose-dependent effect of TEGDMA on cell viability was investigated by a WST-1 assay (Fig. 1). Results show, after 24 h, a significant decrease in viability upon exposure to 1.5 and 3 mM TEGDMA, while 0.1, 0.2, and 0.75 mM did not affect viability in a significant manner; 2nd- and 5th-day results proved to be erratic, and extermination of cells was apparent at 3 mM by the 2nd day and at 1.5 mM by the 5th day (data not presented). Therefore, the highest concentration and longest treatment time applicable for further experiments was decided to be 0.75 mM and 1 day, which are conditions not yet causing substantial cell death.

Compared with the control, pulp cell lysates showed after 1 day, a mild increase in total collagenase/gelatinase activity upon exposure to 0.1 and 0.2 mM TEGDMA (Fig. 2) (also from media upon 0.75 mM TEGDMA exposure). Longer exposures resulted in a stagnation and decrease in collagenase/gelatinase activity both from the lysates and medium for all groups including the control (results not presented).

TEGDMA exposure also led to increased levels of MMP-2, MMP-8, and MMP-9 in pulp cells, as detected by the western blot analysis (Fig. 3). The lowest TEGDMA concentration of 0.1 mM caused an increase in MMP-2 production only. A strong increase in all tested MMPs was seen after exposure to 0.2 mM TEGDMA; 0.75 mM TEGDMA increased MMP-2 and MMP-8 levels marginally, without influencing MMP-9 production. The presented photos are representative of series of four to five independent experiments with similar results.

Immunocytochemistry was also performed to further confirm the findings acquired by western blot and to investigate possible changes in antigen distribution. While untreated cells labeled weakly or not at all for the MMPs, exposure to 0.1, 0.2, and 0.75 mM TEGDMA resulted in increased MMP-2, MMP-8, and MMP-9 immunostaining in pulp cells (Figs. 4, 5, and 6) with the exception of the 0.75-mM treatment and MMP-2 levels. In the case of MMP-2 and MMP-8, the antigens showed a cytoplasmic distribution, with a grainier appearance for MMP-8. MMP-9 produced a grainy, cytoplasmic as well as a filamentous signal, the latter often in the vicinity of the cell membrane. There was no detectable difference in antigen localization between the control and treated pulp cells.

The identification of concurrent signaling pathway activation was also attempted. Western blotting (Fig. 7) showed after 1 day increased levels of both 44 and 42 kDa variants of phosphorylated (activated) ERK as well as p-JNK, for all tested TEGDMA concentrations. A considerable p38 activation could only be seen after exposure to 0.1 mM TEGDMA. After the removal of antibodies from the blots, ERK1/2, p38, and JNK proteins were detected on the corresponding membranes using the proper antisera.