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RESEARCH REPORT |
1 Department of Conservative Dentistry & Periodontology
2 Institute of Cell and Molecular Pathology, Medical Univ. Hannover, Germany; and
3 Department of Restorative Dentistry, School of Dentistry, University of Washington, Box 357456, Seattle, WA 98195-7456, USA;
+corresponding author, wgert{at}u.washington.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: TEGDMA • apoptosis • necrosis • gingival fibroblasts
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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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
TEGDMA was analyzed and investigated for purity by HPLC/GC/MS prior to application. The comonomer was dissolved in dimethyl sulfoxide (DMSO) (1 M/L stock solution), diluted at least 1:200 in culture medium, and tested within a concentration range of 1 to 7.5 mM. These dilutions contained a DMSO concentration not higher than 0.5%, which was non-toxic in HGF cultures (data not shown).
Cytotoxicity Assay
For cytotoxicity assays, 1 x 104 HGF in 200 µL DMEM per well were cultured in 96-well tissue culture plates and grown to sub-confluent monolayers for 48 hrs. TEGDMA concentrated between 1 mM and 7.5 mM was added to the monolayers by medium change. Control cultures were grown without TEGDMA.
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
For apoptosis experiments, 5 x 105 cells were placed into 75-cm2 tissue flasks and pre-cultured in a 10% CO2 atmosphere at 37°C. Monolayers of exponentially growing HGF (passages #4-9) were then exposed to 15 mL of culture medium containing various concentrations of TEGDMA. Treatment was stopped after 24 hrs. The control monolayers were grown without TEGDMA (negative control) or with 10 mM 5-fluorouracil (5-FU, a known inducer of apoptosis = positive control) under the same culture conditions (Nita et al., 1998). The potency of TEGDMA to generate apoptosis in primary oral human fibroblasts was also evaluated in preliminary experiments by DNA ladder formation (Paddenberg et al., 1996). A typical DNA fragmentation pattern was found (data not shown).
Annexin Assay Measured by FACS
This assay yields qualitative and quantitative data about the shares of the different types of cell death in an assay. Apoptotic, "apoptotic necrotic", and necrotic cells were labeled with Annexin V-FLUOS and PI (Annexin V-FLUOS Kit; Roche, Mannheim, Germany). The applied assay as well as its validation have been described in detail elsewhere (Vermes et al., 1995).
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
The APOPercentageTM apoptosis assay monitors the appearance of apoptosis in mammalian anchorage-dependent cells. For this method, a specially designed dye is used, which is selectively incorporated by cells undergoing apoptosis.
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
Data are presented as means ± standard deviation (SD). Statistical analysis was performed by ANOVA (Tukey tests), and p-values < 0.05 were considered "significant".
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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. 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).
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). 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).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received June 10, 2002; Last revision June 13, 2003; Accepted June 27, 2003
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REFERENCES |
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