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RESEARCH REPORT |
1 Department of Oral Health Science, Graduate School of Dental Medicine, Hokkaido University Graduate School of Dental Medicine, Kita 13, Nishi 7, Kita-ku, Sapporo 060-8586, Japan;
2 Department of Oral Rehabilitation, Medical College of Georgia, Augusta, GA 30912-1260; and
3 Department of Oral Functional Science, Hokkaido University Graduate School of Dental Medicine, Sapporo, Japan;
*corresponding author, nodam{at}den.hokudai.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: dental adhesive • monomers • cell stress
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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The biocompatibility of dentinal adhesives is related to resin component release and has been assessed in vitro (Camps et al., 1997; Yoshii, 1997; Bouillaguet et al., 1998, 2000; Geurtsen et al., 1998). Investigators have measured cellular growth, death, mitochondrial activity, protein synthesis, or other basic cellular metabolic activities as indicators of cellular response to resin components. However, the intracellular mechanisms altered by resin components are still not clear.
Intra-orally, cells are exposed to many stressors from materials or bacteria, or from mechanical or thermal sources. Heat shock proteins (HSPs) are multifunctional proteins known to be expressed where cells are exposed to stressors including elevated temperatures, chemicals, or bacterial products. HSPs play a key role as molecular chaperones (Bachelet et al., 1998; Fink, 1999; Yenari et al., 1999). They contribute to the correct folding of proteins that have lost or not yet acquired their normal three-dimensional structure. HSPs therefore help to protect cells against stress. HSP72 is a member of the HSP family. HSP72 is known as a stress-induced protein in mammalian cells and is therefore a logical indicator of cellular stress (Ohshima et al., 1997).
There is little information about the ability of resins to modulate the expression of HSPs. We hypothesized that if material components stress cells, this stress may be manifest by induction or modulation of HSP72 expression. To investigate this hypothesis, we exposed monocytes to HEMA or TEGDMA, major components of dentinal adhesives. The monocytes were considered a logical target because these cells orchestrate and amplify the inflammatory response to biomaterials (Auger and Ross, 1992).
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Monomers and Concentrations
2-Hydroxyethylmethacrylate (HEMA, Sigma, St. Louis, MO, USA, Lot 83H3503) and triethyleneglycol dimethacrylate (TEGDMA, Esschem Inc., Linwood, PA, USA, 419-50-05) were tested because they are major components of dentinal adhesives, and the release of these components from polymerized dentinal adhesives has been well-documented (Ferracane, 1994; Gerzina and Hume, 1996; Hanks et al., 1996).
To determine concentrations of resins for the HSP experiments, we incubated THP-1 cells with HEMA (0-40 mmol/L) or TEGDMA (0-3 mmol/L) for 2, 24, or 48 hrs, and measured succinic dehydrogenase (SDH) activity by the MTT assay (Wataha et al., 1992). All reagents used in the current experiment were chemical grade. After incubation with HEMA or TEGDMA, the cells (100,000 cells/mL, n = 8 for each time and concentration) were plated in 96-well plates. MTT (Sigma) solution was added at 1 mg/mL for 1 hr, after which the solution was removed. The cells were fixed with Tris-formalin (0.2 mol/L of Tris, 4% v/v formalin) and washed with double-distilled water. Finally, a dimethylsulfoxide solution (6.25% v/v DMSO in 0.1 mol/L NaOH) was added to dissolve the formazan, and the resulting solution was read in a spectrophotometer at 562 nm. Concentrations for the subsequent HSP experiments were chosen to be at or below toxic concentrations at 24 hrs. The concentrations used for HSP experiments were therefore 0, 2.5, 5, and 10 mmol/L for HEMA, and 0, 0.25, 0.5, and 1.0 mmol/L for TEGDMA.
Conditions of Heat Shock Stress and Recovery
First, the effects of HEMA and TEGDMA on cellular metabolic activity were determined with or without heat stress. THP-1 cells (100,000 cells/mL, n = 16 for each concentration and condition) were exposed to HEMA or TEGDMA for 7 hrs. Heat stress of 43°C for 1 hr in a water bath was applied to the appropriate groups. Heat stress conditions were selected according to other reports (Bachelet et al., 1998; Guzik et al., 1999). We measured SDH activity (see procedure above) to ensure that resin concentrations with or without heat stress were not acutely cytotoxic. We used ANOVA and Tukey multiple-comparison intervals (
= 0.05) to compare SDH activities at different concentrations. We repeated the entire experiment once to ensure reproducibility.
To measure HSP expression, we first added HEMA or TEGDMA to THP-1 cultures (5.0 x 106 cells/5 mL, n = 6), then applied 1 hr of heat stress. We allowed the culture to recover for 2, 4, or 6 hrs at 37°C so that there would be adequate time for HSP expression to occur (Polla et al., 1995; Bachelet et al., 1998; Guzik et al., 1999). Control groups received no heat stress, but remained at 37°C for an equivalent time (1 hr sham stress plus 6 hrs recovery). Cells were processed for SDS-PAGE and Western blotting for the measurement of HSP expression.
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)
To assess the expression of HSP72, 2.0 x 106 cells in each condition were washed three times with PBS and re-suspended into 500 µL of PBS. A 250-µL quantity of 2x SDS-sample buffer (0.125 M Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol) was added to 250 µL of the cell suspensions, and the samples were vortexed. Samples were boiled at 95°C for 4 min and immediately put on ice. They were aliquoted and stored at -20°C until use. The other 250-µL quantity of the cell suspensions was used for determination of the amount of protein (BCA assay, Pierce, Rockford, IL, USA) to apply in the SDS-PAGE. A 10-µg quantity of protein in each sample was electrophoresed on SDS-polyacrylamide slab gels (10%). Proteins were detected by silver stain (SILVER STAIN KITS, Sigma).
Immunoblotting
Proteins separated on SDS-polyacrylamide gels were transferred to a PVDF membrane (Immbolin-P, MILLIPORE Corp., Bedford, MA, USA) by wet electroblotting in a buffer containing 25 mM Tris, 192 mM glycine, 3.5 mM SDS, and 10% (v/v) methanol. The membrane was incubated in blocking buffer (5% [w/v] BSA in PBS) for 3 hrs. The membrane was then exposed to a mouse anti-human HSP72 antibody (diluted 1:2500, StressGen Biotechnologies Corp., Victoria, BC, Canada) for 18 hrs. HSP72 was detected by chemiluminescence by means of an ECF Western blotting Kit (Amersham Life Science Ltd, Buckingham, England). Chemifluorescence (excited at 450 nm) was detected at 560 nm with the Storm SystemTM and quantified with ImageQuantTM (Molecular Dynamics, Sunnyvale, CA, USA). The HSP expression was calculated as a percentage of the six-hour recovery of the no-resin stress control, because this HSP expression was maximal. The control for each time was compared with its HEMA or TEGDMA counterpart by ANOVA and Dunnett's multiple-comparison test ( = 0.05). Dunnett's test determined only if each concentration was different from its corresponding control.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). In the absence of heat stress, HEMA and TEGDMA had no significant effects on the SDH activity during the seven-hour exposure (ANOVA, p = 0.926 and 0.739, respectively). When 1 hr of heat stress was applied, there were no significant differences in SDH activity of the control cultures (without resins, Fig. 1, y axes, open vs. solid circles). For HEMA and TEGDMA, the combination of heat plus resins significantly depressed SDH activity about 15% at the highest concentrations only (vs. heat-stressed controls with no resin). At the highest concentrations, the depression of SDH activity was also significant with respect to unstressed, unexposed controls (two-tailed t test, p = 10-10 for HEMA, 10-7 for TEGDMA).
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show the effects of HEMA and TEGDMA on HSP72 expression in THP-1 (Fig. 2 is a typical SDS-PAGE and Western blot). There was no indication that HEMA alone (without heat stress) caused HSP72 expression (Fig. 3). However, the data clearly showed a significant drop in HSP72 expression 2, 4, and 6 hrs after heat stress plus HEMA exposure. HSP72 expression decreased in a dose-dependent fashion, falling at least 50% at 6 hrs after 10 mmol/L exposure to HEMA. The results for TEGDMA were similar to those for HEMA (Figs. 3 and 4), except that the suppression of the HSP72 expression by TEGDMA was more potent, suppressing the six-hour expression over 80% compared with controls not treated with TEGDMA. The TEGDMA effects were also dose-dependent, but occurred at doses one-tenth those of HEMA. Similar trends in HSP72 suppression were seen with HEMA and TEGDMA for the other recovery times (each relative to its corresponding control), although the trends were not always perfect.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). Although a synergism between the stress of the material and heat might have been expected to decrease SDH activity more than with the components alone, this result was not observed. In most cases, the combination of heat and resin was similar to heat or resin alone. The exceptions to this trend were HEMA and TEGDMA at their highest concentrations (Fig. 1). The lack of effect of heat on SDH activity indicates that these stress conditions do not alter the energy-producing capability of these cells. Based on the assumption that mitochondrial activity is central to the survival of the cell, the conditions of heat and chemicals applied in the current study appeared to be below lethal levels.
Exposure to chemicals and heat tends to cause errors in protein production or production of immature proteins in cells (Fink, 1999; Yenari et al., 1999). HSP72 has an essential role in assisting protein folding and refolding and is thought to aid in preventing or repairing protein errors under conditions of cellular stress (Polla et al., 1995). HSPs are induced by activation of heat shock factor in the cytoplasm, which is then transferred to the nucleus, where it binds heat shock elements, leading to transcription and synthesis of HSP72 and other HSPs (Yenari et al., 1999). In the current THP-1 model, heat stress of 43°C for 1 hr induced HSP72 fully at 4 and 6 hrs, with minimal expression at 2 hrs (Figs. 2, 3). These results agree with those previously published (Samali and Cotter, 1996; Bachelet et al., 1998), indicating that the THP-1 monocytes successfully modeled the heat shock response.
HEMA and TEGDMA significantly suppressed HSP72 even at apparently sublethal concentrations (Fig. 3). Both resins affected the monocytes similarly. The sublethal suppression of other cellular functions, such as cytokine secretion, has been observed previously with these resins in both the THP-1 and peripheral blood monocyte model (Rakich et al., 1999; Heil et al., 2002). The ability of these resins to potently inhibit transcription at levels that leave cellular metabolism intact may compromise the ability of the monocytes to respond adequately to heat or other biological stress in the pulpal tissues. This possibility is supported by reports that have shown that HEMA and TEGDMA rapidly traverse dentin (Gerzina and Hume, 1996; Bouillaguet et al., 1998) and the use of dentin bonding agents directly on exposed pulp (Costa et al., 2000). These results also support the importance of measuring more than cellular metabolism to assess cellular response to restorative material components.
The current study does not address the mechanism by which HEMA and TEGDMA modulate HSP72 expression. Recent results show that HEMA and TEGDMA damage the nucleus (Schweikl et al., 2001) and therefore may alter the activity of the heat shock element and the levels of HSP. Alternatively, HEMA and TEGDMA could inhibit phosphorylation of the heat shock factor, thereby limiting levels of HSP. The role of these or other mechanisms for HSP72 inhibition cannot be determined from the current data. Furthermore, it is currently not known if HEMA and TEGDMA modulate the expression of other HSPs.
HEMA and TEGDMA modulated heat-induced HSP72 at concentrations from 2.5 to 10 mmol/L for HEMA and 0.25 to 1 mmol/L for TEGDMA. Although these concentrations appear higher than would be observed clinically, closer analysis supports their relevance to the clinical situation. Dentinal adhesive primers are mostly HEMA, and pure HEMA is approximately 8 mol/L. If used in a direct pulp-capping procedure, then this concentration is clearly higher than those used in the current study. However, the primer is more often used on intact dentin. HEMA has been shown to have a dilution factor of 1000-5000 times across 0.5 mm of dentin with a 10-cm H2O back-pressure (Bouillaguet et al., 1996). Thus, the concentration of HEMA diffusing across the dentin would be on the order of 1.5-8 mmol/L. These concentrations are similar to those used in the current study. TEGDMA is a component of many dentinal bonding resins in approximately 50% concentration. Since pure TEGDMA has a 3.8 mol/L concentration, many bonding resins would have approximately a 2 mol/L concentration of TEGDMA. The dilution of TEGDMA across 0.5 mm of dentin has been shown to be approximately 500 times (Hanks et al., 1994). Thus, the concentration of TEGDMA reaching the pulp would be on the order of 4 mmol/L, again within the range used in the current study. Although there are many factors to consider when converting the in vitro results of the current study to an in vivo situation, the concentrations of monomers used in the current study are plausible.
In summary, the current study has shown that several components released from dentinal adhesives can significantly modulate the expression of HSP72 at sublethal concentrations in monocytes. The response can be enhanced or suppressed, depending on the component, its concentration, and time after heat stress.
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ACKNOWLEDGMENTS |
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Received June 20, 2001; Last revision February 11, 2002; Accepted February 13, 2002
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