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Activation of Adenosine-receptor-enhanced iNOS mRNA Expression by Gingival Epithelial Cells
S. Murakami*,
N. Yoshimura,
H. Koide,
J. Watanabe,
M. Takedachi,
M. Terakura,
M. Yanagita,
T. Hashikawa,
T. Saho,
Y. Shimabukuro and
H. Okada
Department of Periodontology, Division of Oral Biology and Disease Control, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan;
Correspondence: *corresponding author, ipshinya{at}dent.osaka-u.ac.jp
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ABSTRACT
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A series of reports has revealed that adenosine has a plethora of biological actions toward a large variety of cells. In this study, we investigated the influence of adenosine receptor activation on iNOS mRNA expression in human gingival epithelial cells (HGEC) and SV-40-transformed HGEC. HGEC expressed adenosine receptor subtypes A1, A2a, and A2b, but not A3 mRNA. Ligation of adenosine receptors by a receptor agonist, 2-chloroadenosine (2CADO), enhanced iNOS mRNA expression by both HGEC and transformed HGEC. In addition, the adenosine receptor agonist enhanced the production of NO2-/NO3-, NO-derived stable end-products. An enhanced expression of iNOS mRNA and NO2-/NO3- was also observed when SV40-transformed HGEC were stimulated with CPA or CGS21680, A1- or A2a-selective adenosine receptor agonists, respectively. These results provide new evidence for the possible involvement of adenosine in the regulation of inflammatory responses by HGEC in periodontal tissues.
Key Words: periodontal disease • gingival epithelial cells • inflammation, adenosine • iNOS
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INTRODUCTION
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Nitric oxide (NO), a signaling and effector molecule, has diverse biological potencies, such as neurotransmission (Shibuki and Okada, 1991), vasodilation (Palmer et al., 1988), immune modulation (Moilanen and Vapaatalo, 1995), and bactericidal action in infection (Nathan and Hibbs, 1991; MacMicking et al., 1995; Wei et al., 1995), which are both beneficial and detrimental to the host. Thus, it has been documented that NO takes part in both host defense and pathophysiological events of various diseases (Nathan and Hibbs, 1991; Janoff et al., 1997; Salzman et al., 1998).
In NO synthase (NOS), the expression of iNOS is induced by stimulation with various cytokines as well as by bacterial infection (Nathan, 1992). Interestingly, it has also been reported that iNOS expression was observed in gingival epithelial basal layers of non-inflamed and inflamed periodontal tissues, and a higher density level of iNOS was found in basal keratinocytes in the inflamed tissues (Kendall et al., 2000).
A series of reports has revealed that adenosine, an endogenous nucleoside, has a plethora of biological actions toward a large variety of cells and can modulate the various functions of cells involved in inflammatory responses (Cronstein et al., 1991, 1993). Furthermore, recent studies have demonstrated that stimulation of the adenosine receptor activated NO production by macrophages (Hasko et al., 1996), cardiac myocytes (Ikeda et al., 1997), and vascular endothelial cells (Li et al., 1995). However, very little is known about the regulatory effects of adenosine on NO production by human gingival epithelial cells (HGEC).
In this study, we investigated whether stimulation of the adenosine receptor activated iNOS mRNA expression in HGEC and production of the NO stable end-metabolites, nitrate and nitrite.
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MATERIALS & METHODS
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Reagents
We commercially obtained the following reagents: recombinant human IL-1β (Genzyme Corporation, Cambridge, MA, USA); the adenosine receptor agonist, 2-chloroadenosine (2CADO) (Research Biochemicals International [RBI], Natick, MA, USA); N6-cyclopentyladenosine (CPA) (RBI); CGS-21680 hydrochloride (RBI); and the adenosine receptor antagonist, xanthine amine congener (XAC) (RBI).
Human Gingival Epithelial Cells (HGEC)
HGEC were established as described below. All human subjects who participated in this study provided informed consent to a protocol that was reviewed and approved by the Institutional Review Board of the Osaka University Graduate School of Dentistry. Gingival specimens, obtained during periodontal surgery, were minced and treated with 0.4% dispase II (Boehringer Mannheim GmbH, Mannheim, Germany) overnight at 4°C. The epidermal sheet was separated and trypsinized with 0.05% Trypsin-EDTA (Life Technologies, Rockville, MD, USA) so that single cells would be dispersed. The cells were then seeded and subcultured in a 25-cm2 flask (Corning, NY, USA). The HGEC were grown in Humedia KB2 (Kurabo, Osaka, Japan) with a final concentration of 0.5 µg/mL hydrocortisone, 10 µg/mL insulin, 0.4% v/v bovine pituitary extract, 0.1 ng/mL hEGF, 50 µg/mL gentamycin, and 50 ng/mL amphotericin B. HGEC were passaged by trypsinization and used in the experiments at passages 1 to 3. In this study, we reproduced the identical experiments by using 3 or 5 cell cultures of HGEC isolated from different patients. In addition, several lines of cultured primary HGEC were transformed with SV-40 T-antigen (Prasad et al., 1992), and 1 clone, epi 4, was finally established.
Purification of Peripheral Blood Mononuclear Cells and Polymorphonuclear Leukocytes
We collected peripheral blood mononuclear cells and granulocytes from healthy donors by density gradient centrifugation using a Histopaque-1077 (density of 1.077 g/mL; Sigma Diagnostics, St. Louis, MO, USA). Mononuclear leukocytes were isolated from the interface and then washed twice. Similarly, granulocytes were separated by density gradient centrifugation with a Histopaque-1077. The interface containing the mononuclear leukocytes and the Histopaque was removed, and the granulocytes were further isolated by dextran sedimentation and then treated for 30 min with an ice-cold isotonic NH4Cl solution (155 mM NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA, pH7.4) for lysis of the contaminating erythrocytes. The remaining granulocytes were washed twice. The granulocytes had a purity of > 98% and consisted mainly of neutrophils (> 95%) as judged by standard Wright-Giemsa staining techniques.
Detection of iNOS mRNA and Adenosine Receptor Subtypes in HGEC by Reversetranscription-Polymerase Chain-reaction (RT-PCR)
Total RNA was isolated from each cell by RNAzolTM (Cinna/Biotecx Laboratories, Inc., Friendswood, TX, USA) according to the manufacturer's instructions. The precipitated RNA was re-dissolved in 0.1% diethylpyrocarbonate-treated distilled water (DEPC-treated H2O).
cDNA synthesis and amplification via semi-quantitative PCR were performed according to the methods described by Murakami et al. (1994). To generate cDNA for PCR analysis, we prepared a 40-µL cDNA synthesis reaction mixture for each RNA sample and incubated it at 37°C for 60 min. The 40-µL cDNA synthesis reaction mixture contained 5.2 µL of DEPC-treated H2O, 4 µL of 10x PCR buffer II (100 mM Tris-HCl, pH 8.3, 500 mM KCl; Perkin-Elmer Cetus, Norwalk, CT, USA), 6 µL of 25 mM MgCl2, 4 µL each of 10 mM deoxynucleotide-triphosphates (Takara Shuzo Co. Ltd., Kyoto, Japan), 0.4 µL of 20 U/mL RNase inhibitor (Perkin-Elmer Cetus), 1 µL of 50 U/mL M-MLV reverse transcriptase, and 4 µL of 0.25 µg/mL RNA sample. After incubation, all samples were heated to 94°C for 5 min for inactivation of the reverse transcriptase.
Oligonucleotide PCR primers specific for iNOS, adenosine A1, A2a, A2b, and A3 receptor subtypes, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA were synthesized by Clontech Laboratories, Inc. (Palo Alto, CA, USA). The sequences of the sense primers were: 5'-ATG GAA CAT CCC AAA TAC GA-3', 5'-AGT ACT ATG GGA AGG AGC TGA AGA T-3', 5'-ACG CCC CTC TCT CTG GCT CAT GTA CCT-3', 5'-GTG CCA CCA ACA ACT GCA CAG AAC-3', 5'-CAC CAC CTT CTA TTT CAT TGT CTC T-3', and 5'-TGA AGG TCG GAG TCA ACG GAT TTG GT-3'. Those of the antisense primers were: 5'-GTC GTA GAG GAC CAC TTT GT-3', 5'-GGT AGT TAA CTC CTA GTG GAG GGA C-3', 5'-TCA TCA GGA CAC TCC TGC TCC ATC C-3', 5'-CTG ACC ATT CCC ACT CTT GAC ATC-3', 5'-GGT ACT CTG AGG TCA GTT TCA TGT T-3', and 5'-CAT GTG GGC CAT GAG GTC CAC CAC-3'. We amplified the cDNA samples by adding them to a PCR reaction mixture which included 10 mM Tris-HCl buffer (pH 8.3), 1.5 mM MgCl2, 50 mM KCl, 0.15 mM dNTP mixture, 1.25 U AmpliTaq GoldTM (Perkin-Elmer, Foster City, CA, USA), and 0.2 µM sense and antisense oligonucleotide primers. The PCR reaction mixture was subjected to amplification for different numbers of cycles in a DNA Thermal cycler 480 (Perkin-Elmer Cetus Co., Emeryville, CA, USA). After initial denaturation at 94°C for 4 min, each cycle consisted of 94°C for 45 sec, 60°C for 45 sec, and 72°C for 2 min. PCR products were analyzed by electrophoresis at 100 volts for 30 min on a 1.5% TAE agarose gel (NIPPON GENE Co., Ltd., Toyama, Japan) containing 0.5 µg/mL ethidium bromide. The expression of each mRNA was then quantitated by image analysis (NIH Image). The results were represented as the ratio of each mRNA level to GAPDH mRNA level at the most optimal and unsaturated cycle. The mean ratio of each mRNA expression in the reproduced identical experiments was arithmetically calculated.
A3 Receptor Nested PCR
The PCR products from the A3 receptor reaction were re-amplified with the use of nested amplification primers: 5'-AAG TCA TAA AAA GGC AGC TGT AGA A-3'. The PCR conditions were not changed for the re-amplification.
Determination of NO2-/NO3-
Transformed HGEC (epi 4) were seeded at a density of 105/well in 24-well culture plates (Corning) and grown to subconfluence. The cells were treated with adenosine receptor agonists, and the supernatants were harvested after incubation for 24 hrs. Supernatants were treated for enzymatic reduction by nitrate reductase from NO3- to NO2- and then measured as NO2- (designated NO2-/NO3-). NO2- was determined by means of a NO2-/NO3- assay kit-F (Dojindo Laboratories, Kumamoto, Japan), according to the manufacturer's instructions. Freshly prepared 2,3-diaminonaphthalene was added and mixed immediately. After a 15-minute incubation at room temperature, the reaction was terminated with NaOH.
Formation of 2,3-diaminonaphthoriazole, a fluorescent product, was measured by means of an MTP32 (Corona Electric Co. Ltd., Ibaragi, Japan) fluorescent microplate reader with excitation set at 365 and emission read at 450 nm.
We then calculated nitrite levels by first subtracting the value of the blank from the experimental samples and then determining the value using a standard curve for nitrite.
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RESULTS
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Expression of Adenosine A1, A2a, and A2b, but not A3 Receptor mRNA, was Detected in HGEC
Recent studies have demonstrated that at least 4 isoforms exist in the adenosine receptor. To examine the mRNA expression of adenosine receptor subtypes in HGEC, we performed RT-PCR analysis. As shown in Fig. 1A , RNA transcripts for A1, A2a, and A2b adenosine receptor subtypes were observed in HGEC, but A3 receptor subtype mRNA was not detected, while all 4 isotype receptors were seen in mononuclear leukocytes and granulocytes. For further verification that A3 receptor mRNA could not be detected in HGEC, a second round of PCR amplification with nested primers was performed (Fig. 1B). Even though this more sensitive method of visualizing the A3 messenger was used, the expression of A3 adenosine receptor mRNA was not found in HGEC, despite the presence of A3 mRNA in mononuclear leukocytes and granulocytes (Fig. 1C).
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Figure 1. Demonstration of adenosine receptor subtypes. (A) Expression of adenosine receptor subtype mRNA in HGEC. We performed RT-PCR analysis to examine adenosine receptor mRNA of the A1, A2a, A2b, and A3 subtypes in HGEC, mononuclear leukocytes, and granulocytes. Representative results of 1 of 3 identical experiments are shown. The number of PCR cycles is shown above each lane. The mean ratios ± SD, which were determined as described in MATERIALS & METHODS, of A1, A2a, A2b, and A3 subtype expression were 2.0 ± 0.4, 13.2 ± 3.0, 8.9 ± 1.4, and 0.0 ± 0.0, respectively. (B) Examination of adenosine receptor A3 subtype mRNA expression in HGEC by nested PCR. Nested amplification primers for the A3 receptor subtype were designed within the primers for the A3 receptor. (C) Detection of adenosine receptor A3 subtype mRNA expression by nested PCR. To detect the adenosine receptor A3 subtype mRNA in HGEC, mononuclear leukocytes, and granulocytes, we performed RT-PCR for 40 cycles and then carried out re-amplification using nested primers. Results of 1 representative experiment from among 3 identical experiments are shown. The number of PCR cycles is shown above each lane.
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A Non-selective Adenosine Receptor Agonist, 2CADO, Enhanced the Expression of iNOS mRNA by HGEC and Transformed HGEC
It has been shown that iNOS expression is regulated by several factors, including cytokines such as IL-1β, TNF , IFN , and IL-8, as well as by LPS or a combination of these factors. To confirm whether iNOS mRNA in HGEC was elicited, we performed RT-PCR analysis. As shown in Fig. 2A, activation of HGEC with a combination of IL-1β and TNF induced an increase of iNOS mRNA in HGEC. When HGEC were stimulated with 2CADO, which is known to bind to both A1 and A2 receptors, increased iNOS mRNA was observed (Fig. 2A).
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Figure 2. Regulation of iNOS expression. (A) Ligation of adenosine receptor with 2CADO resulted in an increase of iNOS mRNA expression in HGEC. HGEC were cultured (1 x 106/well in a 60-mm culture dish) with or without 2CADO (100 µM) for 6 hrs at 37°C. HGEC were also cultured (1 x 106/well in a 60-mm culture dish) in the presence of IL-1β (25 U/mL) plus TNF (10 ng/mL) as a positive control. RT-PCR was then carried out for detection of iNOS and GAPDH mRNA expression in HGEC as described in MATERIALS & METHODS. Results of 1 representative experiment from among 5 identical experiments are shown. The number of PCR cycles is shown above each lane. The mean ratios ± SD, which were determined as described in MATERIALS & METHODS, of iNOS mRNA expression in untreated, IL-1β plus TNF-treated, and 2CADO-treated HGEC were 0.11 ± 0.16, 0.85 ± 0.25, and 0.64 ± 0.34, respectively. (B) Adenosine receptor antagonist abrogated 2CADO-induced iNOS expression in SV-40-transformed HGEC. SV-40-transformed HGEC (epi 4) were cultured (1 x 105/well in 24-well plates) in the presence or absence of 2CADO (100 µM) with or without XAC (10 µM), an adenosine receptor antagonist, for 2.5 hrs at 37°C, and then RT-PCR was carried out for the detection of iNOS and GAPDH mRNA expression in epi 4. Results of 1 representative experiment from among 3 identical experiments are shown. The number of PCR cycles is shown above each lane. The mean ratios ± SD, which were determined as described in MATERIALS & METHODS, of iNOS mRNA expression in untreated, 2CADO-treated, 2CADO plus XAC-treated, and XAC-treated epi4 were 0.02 ± 0.04, 0.98 ± 0.36, 0.07 ± 0.01, and 0.01 ± 0.02, respectively.
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The effect of 2CADO on the expression of iNOS mRNA in the transformed HGEC, epi 4, was then examined. As shown in Fig. 2B , ligation of adenosine receptor with 2CADO resulted in an increase of iNOS mRNA in transformed HGEC as well as in HGEC. The enhanced iNOS mRNA in response to 2CADO stimulation was inhibited by XAC, an antagonist of the adenosine receptor (Fig. 2B).
A1 and A2a Selective Adenosine Receptor Agonists (CPA and CGS21680) Increased iNOS mRNA Expression by HGEC
It is regarded that 2CADO binds to both the A1 and A2 subtype receptors. To examine which receptor subtype(s) account for the iNOS mRNA elevation by HGEC, we introduced the A1 selective agonist, CPA, and the A2a selective agonist, CGS21680. As shown in Fig. 3, both CPA and CGS21680 up-regulated iNOS mRNA expression by epi 4, transformed HGEC.
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Figure 3. Effects of CPA and CGS21680 on iNOS mRNA expression by HGEC. SV-40-transformed HGEC were cultured (1 x 105/well in 24-well plates) with or without 2CADO (100 µM), CPA (50 µM), or CGS21680 (10 µM), and then RT-PCR was carried out for detection of iNOS and GAPDH mRNA expression. Results of 1 representative experiment from among 3 identical experiments are shown. The number of PCR cycles is shown above each lane. The mean ratios ± SD, which were determined as described in MATERIALS & METHODS, of iNOS mRNA expression in untreated, 2CADO-treated, CPA-treated, and CGS21680-treated epi 4 were 0.09 ± 0.15, 1.42 ± 0.35, 0.81 ± 0.26, and 0.79 ± 0.42, respectively.
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Adenosine Receptor Agonists Increased NO Production in Transformed HGEC
NO is a gas mediator and short-lived molecule. To address whether stimulation of the adenosine receptor can lead to production of NO, we measured NO2- and NO3- (NO2-/NO3-), which are stable end-products of NO, in the culture supernatants of transformed HGEC (epi 4) after stimulation with adenosine receptor agonists. As shown in Fig. 4, a significant increase in NO2-/NO3- production was found in the culture supernatant of not only 2CADO- but also of CPA- and CGS21680-activated transformed HGEC. There was no significant difference in NO2-/NO3- production between adenosine analogues examined. Furthermore, 2CADO-enhanced NO2-/NO3- production was inhibited by 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine hydrochloride, an iNOS-selective inhibitor (data not shown).
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Figure 4. Effects of 2CADO, CPA, and CGS21680 on NO production in HGEC. SV-40-transformed HGEC (1 x 105/well in 24-well plates) were cultured with or without 2CADO (100 µM), CPA (50 µM), or CGS21680 (10 µM), and then NO2-/NO3- in the culture supernatants was measured as described in MATERIALS & METHODS. Results of 1 representative experiment from among 3 identical experiments are shown. Statistical analysis was performed by a one-way analysis of variance (ANOVA) with Scheffé's multiple-comparison test to a significance level of p < 0.05. Asterisks (*) indicate significant (p < 0.05) difference as compared with untreated cells.
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DISCUSSION
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Adenosine is a widely distributed substance that is released by B-cells (Resta et al., 1998), as well as by fibroblasts and endothelial cells (Cronstein et al., 1991) under various physiological conditions. It has also been revealed that adenosine levels are elevated in inflammatory lesions (Cronstein et al., 1993) and that adenosine-regulating agents modulate the inflammation (Cronstein et al., 1991, 1993). We recently reported that adenosine regulates the various cellular functions of human gingival fibroblasts, suggesting a possible effect of adenosine as an immunomodulator of inflammatory periodontal lesions (Murakami et al., 2000, 2001). In the present study, it was demonstrated that ligation of adenosine receptors by a receptor agonist enhanced iNOS mRNA expression as well as production of NO2-/NO3- by HGEC. These findings suggest a possible mechanism by which adenosine receptor activation may result in the control of inflammatory responses in periodontal tissues.
We found that HGEC expressed A1, A2a, and A2b, but not A3 adenosine receptor mRNA (Fig. 1), and that 2CADO, CPA (an A1-specific agonist), and CGS21680 (an A2a-specific agonist) increased iNOS mRNA expression by HGEC (Fig. 3). These findings suggest that signal(s) transmitted via at least the A1 and A2a adenosine receptor subtypes may play an important role in the up-regulation of iNOS mRNA expression by HGEC. Since stimulation of HGEC with 2CADO did not generate the production of IL-1β, IL-6, IL-8, IFN, or TNF (data not shown), which are known to induce NO production by various cell types, it is unlikely that 2CADO-induced iNOS mRNA expression is cytokine-mediated.
Recent reports have indicated that granulocytes migrate across the epithelium and then release 5'-AMP during acute intestinal inflammation, with a subsequent conversion of 5'-AMP to adenosine by 5'-nucleotidase located on epithelial surfaces (Strohmeier et al., 1997), which may function in an anti-inflammatory manner. Since it is well-known that granulocytes similarly migrate across the gingival epithelium into the gingival sulcus, HGEC may also be involved in adenosine accumulation in periodontal tissues following direct interactions with inflammatory cells.
Adenosine and adenosine receptor agonists are known to bind separately to adenosine receptors, which are coupled to G-protein, leading to the modulation of adenyl cyclase, which increases or decreases cAMP levels depending on the receptor subtypes and cell types (Sunahara et al., 1996). On the other hand, cAMP has been documented to mediate the activation of NF- B (Beg et al., 1993), which is associated with iNOS expression (Chartrain et al., 1994). Further, the permeable cAMP analogue and PGE1, which activates adenylate cyclase, were found to augment iNOS mRNA expression in HGEC (data not shown). In addition, we confirmed that stimulation of HGEC with 2CADO increased intracellular cAMP levels (data not shown). These findings suggest that adenosine-dependent iNOS expression is due, in part, to cAMP elevation through NF-B activation in HGEC.
It is reasonable to speculate that up-regulation of iNOS mRNA in HGEC with an adenosine receptor agonist may prevent the actions of periodontopathic bacteria. Thus, it is plausible that the present observations provide evidence for the utility of adenosine receptor agonists with antibacterial host-modulating drugs. In this study, we demonstrated that adenosine receptor agonists, in concentrations ranging from 10 µM to 100 µM, induced NO production by HGEC. Furthermore, we confirmed that 20 µM of 2CADO was also able to induce significant NO2-/NO3- production (data not shown). Although the concentrations used in our study were high, recent in vitro estimation of adenosine levels has shown that 30 µM can be reached at the receptor level during ischemia (Pedata et al., 2001). Thus, it can be speculated that a very high concentration of adenosine may be applied at the receptor level in an in vivo situation. Further investigation with regard to the functions of adenosine on HGEC through distinct adenosine receptor subtypes, which may transduce different intracellular messages, will provide greater insight into the biology of adenosine and the pharmacology of adenosine receptor agonists in inflammatory responses in periodontal tissues.
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ACKNOWLEDGMENTS
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This work was supported by Grants-in-Aid from the Ministry of Education, Science and Culture (No. 11470461, No. 12672034, and No. 13307061). M.Y. is a recipient of the Japan Society for the Promotion of Science.
Received for publication May 11, 2001.
Revision received November 13, 2001.
Accepted for publication February 12, 2002.
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Journal of Dental Research, Vol. 81, No. 4,
236-240 (2002)
DOI: 10.1177/154405910208100403
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ABSTRACT
INTRODUCTION