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TEGDMA Causes Apoptosis in Primary Human Gingival Fibroblasts
1 Department of Conservative Dentistry & Periodontology Correspondence: +corresponding author, wgert{at}u.washington.edu
Previous in vivo studies have revealed that resins may generate a persistent inflammation of oral tissues and cell death as well. Apoptosis is an important regulated process that results in rapid cell death. This study tested the hypothesis that the comonomer triethyleneglycol-dimethacrylate (TEGDMA) causes apoptosis. The effects of TEGDMA on proliferation and apoptosis in primary oral fibroblasts were analyzed by light microscopy and flow cytometry (FACS; Annexin V-assay). TEGDMA at 5 and 7.5 mM inhibited proliferation after 24 hrs. No increased frequency of apoptosis or necrosis was observed with 1 mM or 2.5 mM TEGDMA after 24 hrs. Apoptosis and Annexin V-positive cells were observed with 5 mM and 7.5 mM TEGDMA by light microscopy after 24 hrs. A dramatic increase in apoptotic cells was detected by FACS after 24 hrs with 7.5 mM TEGDMA. Thus, TEGDMA was cytotoxic and "apoptotic" in a dose- and time-dependent manner.
Key Words: TEGDMA • apoptosis • necrosis • gingival fibroblasts
The content of the comonomer triethylene-glycoldimethacrylate (TEGDMA) in resin-modified dental materials may vary from 25% to 50%. Aqueous extracts of set resin specimens contain predominantly ethylene-glycol compounds, such as TEGDMA (Spahl et al., 1998). It was observed that TEGDMA is rapidly distributed in guinea pigs and mice after gastric application (Reichl et al., 2001). A study using high-resolution nuclear magnetic resonance (NMR) spectroscopy revealed that TEGDMA is distributed in various cell compartments, including the cytosol and the membrane lipid fraction (Engelmann et al., 2001). Recent studies provided evidence that TEGDMA causes large DNA sequence deletions in the genome of mammalian cells, as well as a quick and nearly complete depletion of the intracellular glutathione pool (Schweikl and Schmalz, 1999; Engelmann et al., 2001, 2002). Thus, released resinous compounds may cause a great variety of chemical-biological interactions, both in vitro and in vivo, which can result in inflammation and cell death (Geurtsen et al., 1998; Geurtsen, 2000). For instance, human pulps capped with a dentin adhesive and a composite resin initially revealed a neutrophilic infiltrate and death of odontoblasts, followed by a persistent inflammatory response (Hebling et al., 1999). Generally, two main types of cell death are differentiated, apoptosis and necrosis (Majno and Joris, 1995). Apoptosis is an active and physiological process characterized by various phenomena such as cell shrinkage. There is increasing evidence that the "apoptotic machinery" exists in cells all the time, but in a "switched off" state. A detrimental injury of the cell, e.g., due to a toxic substance, can quickly activate the apoptotic response, which rapidly causes cell death. The clearance of the remaining cell debris by phagocytes is also very quick, thus avoiding an acute inflammatory reaction (Hall PA, 1999). Recently, it was found that the CD31-mediated detachment of apoptotic leukocytes is disabled, making the cells susceptible to macrophage ingestion (Brown et al., 2002). Necrosis is generated by a massive, lethal injury of the cells. In contrast to apoptosis, necrosis generally sets off a tissue inflammation associated with clinical symptoms, which frequently leads to scar formation (Majno and Joris, 1995). Little is known about the type of cell death caused by toxic resinous dental materials. Some scientists examined whether eluates of denture-base acrylics induce apoptosis and/or necrosis in immortalized cells, such as U-937 human monoblastoid cells. Apoptosis and necrosis were caused by the non-analyzed extracts in a dose- and time-dependent manner (Cimpan et al., 2000). So far, no data are available in the literature about the potency of important individual resin compounds to generate apoptosis or necrosis in normal human cells. Therefore, it was the objective of the present study to test the hypothesis that TEGDMA causes cell death due to apoptosis in human gingival fibroblasts (HGF), depending on the concentration. Cell death was evaluated qualitatively and quantitatively by light microscopy and flow cytometry.
Tissue Collection and Cell Culture Human gingival fibroblasts (HGF) were obtained from biopsies of the attached gingiva of sound premolar and permanent molar teeth of healthy persons. Informed consent based on an appropriate protocol was obtained from the donors. The protocol was reviewed and approved by the Institutional Review Board. The biopsies were stored at 4°C for, at most, 24 hrs in Hanks’ salt solution supplemented with penicillin (100 U/mL), streptomycin (100 mg/mL), and amphotericin (2.5 mg/mL) (all from Biochrom KG, Berlin, Germany) prior to amplification. The gingival tissues were cut into 1- to 2-mm3 pieces, and then washed twice with Hanks’ salt solution. Thereafter, the cut biopsies were placed into 25-cm2 tissue culture flasks. The explants were incubated with culture medium consisting of Dulbecco’s modified Eagle’s medium (DMEM), 10 mM HEPES, glucose (4.5 g/L), NaHCO3 (3.7 g/L), penicillin (100 U/mL), streptomycin (100 mg/mL), and amphotericin (2.5 mg/mL) (all from Biochrom KG), supplemented with 10% heat-inactivated fetal calf serum (FCS) (PAN Systems, Aidenbach, Germany). The tissue samples were grown at 37°C in a humidified atmosphere of 10% carbon dioxide in air. When outgrowth of cells was observed, the medium was replaced twice weekly until cells reached confluence. Cells were detached from the monolayer by a brief treatment with trypsin-EDTA (0.25% trypsin, 0.02% EDTA) (Sigma, Deisenhofen, Germany), and re-cultured in 75-cm2 tissue flasks until confluent monolayers were re-obtained. Early passages were frozen in liquid nitrogen. Cell counts before plating revealed 95% to 98% cell viability, by the trypan blue exclusion test. Cells between the third and ninth passages were used for the experiments described below.
Exposure of HGF to TEGDMA
Cytotoxicity Assay After an incubation period of 24 hrs, the DNA content of the cells was determined with the use of the DNA-intercalating dye Hoechst 33342TM (Riedel de Haen, Seelze, Germany; working solution, 1 µg/mL in growth medium) (for details, see Geurtsen et al., 1999). The fluorescent intensity of the cells was evaluated in a cytofluor 2350 plate reader (Millipore Corporation, Bedford, MA, USA). All cytotoxicity experiments were run thrice at separate times with each 6 replicates, to ensure reproducibility.
Apoptosis Experiments
Annexin Assay Measured by FACS We evaluated redistribution of phosphatidylserine (PS) to the outer layer of the plasma membrane by incubating cells with the FLUOS-conjugated Annexin V. HGF with lost integrity of the plasma membrane (necrotic and "apoptotic necrotic" cells) were detected with PI. After incubation, adherent HGF were collected by trypsination and pooled with non-attached cells. The cells were harvested, washed, and stained with Annexin V-FLUOS and PI for 10 min at room temperature in the dark, according to the manufacturer’s instructions. After being stained, cells were analyzed by flow cytometry (FACS Calibur; Becton Dickinson, Heidelberg, Germany), with a 488-nm laser line for excitation. Green (FLUOS) fluorescence was collected between 505 and 545 nm, and red (PI) fluorescence between 605 nm and 635 nm. At least 20,000 cells were analyzed per sample. All experiments, which were run in duplicate, were repeated at least three times. Data analysis was performed with Cell Quest software version 3.1 (Becton Dickinson).
APOPercentageTM Apoptosis Assay After incubation of the HGF with various concentrations of TEGDMA (from 1 to 7.5 mM) for 24 hrs, the medium was removed, and fresh culture medium supplemented with APOPercentage Dye (Biocolor, Belfast, UK) was added to the flasks. Following one-hour incubation with the dye, cells were examined and photographed by means of an inverted microscope. Apoptotic cells appear intensely purple-red, whereas viable or necrotic cells remain unlabeled or pinkish. At least 1 x 106 cells were analyzed per sample.
Statistical Analysis
Cytotoxicity Assay In general, it was found that no concentration of TEGDMA affected cell growth after 4 hrs (data not shown). The effects of TEGDMA upon cell growth after 24 hrs are demonstrated in Fig. 1  . Incubation of the cells with TEGDMA concentrations of 1 mM and 2.5 mM resulted in cell growth of 96% compared with control assays (cell growth = 100%). However, the growth of HGF cells was significantly reduced, at concentrations of 5 mM and 7.5 mM, to 59% and 35% of controls (Fig. 1).
Apoptosis Experiments APOPercentageTM apoptosis assay HGF cells exposed to 5 mM and 7.5 mM TEGDMA for 24 hrs showed the typical morphological alterations indicative of apoptosis: reduced cell volume, and chromatin condensation. Further, apoptotic cells were rounded. Apoptotic cells are dyed red after 1 hr of in situ staining with APOPercentageTM (Figs. 2A-2E ).
FACS analysis A dot plot of green fluorescence (Annexin V-FLUOS) vs. red fluorescence (PI) showed three separate clusters: viable cells (lower left quadrant), apoptotic cells (lower right quadrant), "apoptotic necrotic" (upper right quadrant), plus necrotic cells (Figs. 3A-3F ).
Altogether, it was found that TEGDMA generated apoptosis and/or necrosis depending on treatment time and concentration (Figs. 3A-3F, 4  ). No apoptotic cells were detected after an incubation period of 4 hrs at any tested concentration (data not shown). Fig. 4 summarizes the results of the flow cytometry analysis after 24 hrs. At this treatment time, TEGDMA had no significant cytotoxic effect at lower concentrations (1 mM and 2.5 mM), as indicated by the number of vital cells (ca. 95% and 94%, respectively). An incubation of the HGF with TEGDMA concentrations of 5 mM and 7.5 mM caused increases of the apoptotic cells to  28% and 49%, respectively. Concentrations of 1 mM and 2.5 mM TEGDMA caused no significant increase of the "apoptotic necrotic"/necrotic cells ( 2% and 1%). However, a significant increase in the number of such cells could be observed by concentrations of 5 mM and 7.5 mM of TEGDMA ( 11% and 7%). The share of apoptotic cells, however, was always significantly higher than the percentage of "apoptotic necrotic"/necrotic cells (Fig. 4).
Analysis of our data provides the first evidence that severe chemical-biological interactions, such as cell growth inhibition, which are caused by elevated concentrations of TEGDMA in vitro, are predominantly due to apoptosis rather than to necrosis. The findings were confirmed by two different methods, the Annexin V-FLUOS/PI assay as well as the APOPercentageTM apoptosis assay, which yielded qualitative and quantitative data. Taken together, our experiments support the investigated hypothesis that TEGDMA can induce apoptosis in cultured primary human gingival cells in a dose- and time-dependent manner. This observation may be of considerable biological significance. During the past few years, numerous in vitro studies have addressed adverse cellular reactions caused by the most important comonomer TEGDMA, which frequently leaches from polymerized resins into aqueous environments in high quantities and can be found in all cell compartments (Spahl et al., 1998; Engelmann et al., 2001). Besides growth inhibition of various types of cultured primary and immortal cells of different origins, specific cellular injuries or a deleterious interference with important metabolic pathways was found (Geurtsen and Leyhausen, 2001). TEGDMA, for instance, induced large DNA sequence deletions and micronuclei in vitro (Schweikl and Schmalz, 1999; Schweikl et al., 2001). Recently, it was found that sub-lethal concentrations of this comonomer considerably reduce the heat-induced HSP72 expression. But HSP72 itself was not affected at the same time. Analysis of these data indicates an alteration of the cellular stress response without causing apparent changes in the cell metabolism (Noda et al., 2002). These findings supplement the observation that "sub-lethal" TEGDMA amounts can dramatically exhaust the cellular glutathione pool, which subsequently results in a rapid and significant decrease in intracellular detoxification potency. Simultaneously, most intracellular metabolic pathways are not perceptibly disturbed (Engelmann et al., 2001, 2002). There is an indication that this rapid and considerable glutathione exhaustion is accompanied by an intracellular increase of reactive oxygen species (ROS) (data not shown). Interestingly, it was observed that the depletion of glutathione and the increase of ROS play a critical role in the regulation of apoptosis (Hall AG, 1999). This hypothesis is substantiated by our findings. It may be concluded from recent experiments and the results of this study that TEGDMA at sub-lethal concentrations, and particularly at elevated quantities, initially depletes the glutathione pool and increases ROS concentration, which then generates apoptosis due to BCL2 overexpression or the activation of nuclear factor kappa B-dependent genes and DNA injury (Troyano et al., 2001; Agostini et al., 2002; Armstrong and Jones, 2002). Various observations corroborate this assumption: No apoptotic cells were observed after 4 hrs at any concentration, whereas abundant apoptotic cells were found after a treatment of 24 hrs at concentrations of 5 mM and 7.5 mM TEGDMA after 24 hrs. In contrast, however, a quick and dramatic decrease of the glutathione pool of human gingival fibroblasts within the short period of 2 hrs, even at the very low concentration of 0.5 mM TEGDMA, was determined in a preceding study (Engelmann et al., 2002). One main question arises at this point: Are there any in vivo data confirming these in vitro findings? Unfortunately, very little information is available about the in vivo effects of individual resin components. Studies in humans revealed death of odontoblasts and a persistent pulpal inflammation due to resin application (Hebling et al., 1999; Pereira et al., 2000). Noda et al. (2002) calculated that TEGDMA leaching from dentin adhesives might reach concentrations up to 4 mmol/L in the pulp. This concentration is within the range of our study. Apoptotic cells induced by TEGDMA undergo phagocytosis in vivo without subsequent acute inflammation and tissue alteration, whereas necrosis would generally induce a pronounced tissue inflammation in vivo (Majno and Joris, 1995). Altogether, our findings show that the evaluation of apoptosis contributes significant information for a more accurate in vitro assessment of the toxic potency of oral biomaterials, since apoptosis and necrosis have significantly different after-effects in vivo.
This study was supported by a grant from the Deutsche Forschungsgemeinschaft/German National Science Foundation (DFG) (LE 851/2-1).
* These authors contributed equally to this work;  Received for publication June 10, 2002. Revision received June 13, 2003. Accepted for publication June 27, 2003.
Journal of Dental Research, Vol. 82, No. 10,
814-818 (2003) This article has been cited by other articles:
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