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RESEARCH REPORT |
Department of Oral Health, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8525, Japan;
* corresponding author, wyobou{at}md.okayama-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: dietary cholesterol • serum lipids • periodontitis • cell proliferation • apoptosis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Topical application of lipopolysaccharide (LPS) and/or proteases has been widely used for the study of periodontitis in rats. LPS from Escherichia coli (0.1-10 µg/mL) induces keratinocyte proliferation in vitro (Meghji et al., 1996), and, in vivo, a one-time application of E. coli LPS (5 mg/mL) has been demonstrated to increase cell-proliferative activity in the rat junctional epithelium (Takata et al., 1997). Following the application of E. coli LPS (25 µg/µL) and Streptomyces griseus proteases (2.25 U/µL) to rat gingival sulcus, apical migration of the junctional epithelium has been demonstrated to occur simultaneously with the apoptosis of periodontal ligament fibroblasts, following basal cell proliferation (Ekuni et al., 2005). Changes in cell proliferation and apoptosis may provide information valuable for assessing the progression of periodontitis.
A histological investigation revealed that high dietary cholesterol can induce a disturbance of periodontal ligament fibers and thickening of the cementum in the rat periodontium (Ueno, 1965). However, it is unclear how high dietary cholesterol affects cell behavior in periodontal lesions. The purpose of this study was to investigate the effects of high dietary cholesterol on cell proliferation and apoptosis in rat periodontitis.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental Design
Rats were randomly divided into 4 groups of 8 rats. During the experimental period, the first 2 groups were fed with a regular diet for 8 wks, and were treated with pyrogen-free water (Control group) or periodontitis-inducing agents (Periodontitis group) administered into gingival sulcus for 4 wks prior to the end of the experimental period. The remaining 2 groups were fed with a diet containing 1% cholesterol (w/w) and 0.5% cholic acid (w/w) (Oriental Yeast Co., Tokyo, Japan) for 8 wks, and were treated with pyrogen-free water (Cholesterol group) or periodontitis-inducing agents (Combination group) for 4 wks prior to the end of the experimental period. Periodontitis was induced in the Periodontitis and Combination groups by a combination of a 25 µg/µL E. coli LPS (Sigma Chemical Co., St. Louis, MO, USA) suspension in pyrogen-free water and 2.25 U/µL proteases from Streptomyces griseus (Sigma Chemical Co.) (Ekuni et al., 2003). Depending upon group, LPS (0.5 µL x 3 times) and proteases (0.5 µL x 3 times) or pyrogen-free water (0.5 µL x 6 times) was introduced daily by micropipette into the palatal gingival sulcus of both maxillary first molars within 1 hr of intraperitoneal anesthesia with sodium pentobarbital (0.5 mL/kg body weight).
Lipid Assays
Blood samples were collected at 8 wks directly from the hearts of 24-hour-fasted animals. Blood was allowed to clot at room temperature for 1 hr, and serum was separated by centrifugation at 1500 x g for 15 min. Levels of total serum cholesterol, high-density lipoprotein (HDL) cholesterol, and triglycerides were determined with the use of an enzymatic commercial kit (Cholesterol E-test Wako kit; Wako Pure Chemical Industries, Ltd., Osaka, Japan).
Histological and Immunohistochemical Analysis
After the experimental period, rats were killed by intracardiac perfusion of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) under intraperitoneal anesthesia. Following initial fixation, the maxillary molar regions were resected from each rat. Tissues were decalcified with 10% tetrasodium-EDTA aqueous solution (pH 7.3) for 14 days at 4°C. The decalcified tissue blocks were embedded in paraffin, and sections (thickness, 4 µm) were stained with hematoxylin and eosin or other stains, as described below.
Proliferating cell nuclear antigen (PCNA) was stained with the use of a commercial kit (Histofine Simple Stain MAX PO; Nichirei Co., Tokyo, Japan) (Tomofuji et al., 2003). Monoclonal antibody against PCNA was diluted at 1/200 in phosphate-buffered saline, and the color was developed with 3-3'-diamino benzidine tetrahydrochloride. Sections were counterstained with Mayer’s hematoxylin.
The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling (TUNEL) method was used for the in situ detection of nuclear DNA fragmentation (apoptotic cells) (Jarnbring et al., 2002). Briefly, the deparaffinized sections were incubated with 2% H2O2 for 30 min and treated with 0.02 mg/mL proteinase K for 10 min at room temperature. After being rinsed, the sections were incubated with a mixture of terminal deoxynucleotidyl transferase enzyme and fluorescein-deoxyuridine triphosphate for 60 min at 37°C.
All histometric analyses described below were performed under a magnification of 200x (measurement of distances) or 400x (cell count) by one examiner, blinded to treatment assignment. Tissue sections stained with hematoxylin and eosin were used for measurement of the linear distance from the cemento-enamel junction (CEJ) to the apical portion of the junctional epithelium, from the CEJ to the alveolar bone crest, and from the most apical portion of the junctional epithelium to the most apical portion of the destroyed collagen fibers in the connective tissue (Ekuni et al., 2003, 2005). Polymorphonuclear leukocytes and blood vessels per standard area [0.05 mm (depth) x 0.1 mm] were counted in the connective tissue subjacent to the junctional epithelium (Ekuni et al., 2003).
We used the tissue sections stained with PCNA and TUNEL to evaluate cell-proliferative activity and apoptosis, respectively (Norata et al., 2002). PCNA-positive fibroblasts, TUNEL-positive fibroblasts, and total fibroblasts were counted in standard areas (0.1 mm x 0.1 mm each) within the gingiva (the connective tissues subjacent to the junctional epithelium) and periodontal ligament (Ekuni et al., 2005). PCNA-positive basal cells and total basal cells of the basement membrane of the junctional epithelium (0.1 mm) were also counted (Takata et al., 1997).
Statistical Analysis
Means of histological data were calculated for each rat. One-way ANOVA and Tukey’s method were performed with the use of a statistical software package (SPSS 10.0J for Windows; SPSS Japan, Tokyo, Japan).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Changes in serum lipids, including an increase in total cholesterol and a decrease in HDL cholesterol, were evident in the Cholesterol and Combination groups (Table 1
). There were no differences in serum triglyceride levels among the 4 groups.
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). In the Combination group, more PCNA-positive basal cells and TUNEL-positive fibroblasts were visible than in the Control group in the junctional epithelium and periodontal ligament, respectively (Fig. 2).
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). In the Periodontitis and Combination groups, the increase in the linear distance between the CEJ and the most apical portion of the junctional epithelium was greater than in the Cholesterol group, and significantly more polymorphonuclear leukocytes, blood vessels, and advanced collagen destruction were present. There were significant differences in polymorphonuclear leukocyte density between the Cholesterol and Combination groups.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Rats fed with a high-cholesterol diet showed apical migration of the junctional epithelium and bone resorption. This was observed not only in those rats treated with periodontitis-inducing agents, but also in those rats treated with water. Even where few bacterial pathogens are present in the gingival sulcus, feeding a high-cholesterol diet can initiate periodontitis.
In the current study, high dietary cholesterol induced an increase in total serum cholesterol and a decrease in serum HDL cholesterol. Such a hypercholesterolemic condition can induce a marked increase in the production of Interleukin-1 beta and Tumor Necrosis Factor-alpha (Han et al., 2002) and an increase in macrophage infiltration (Hosoyamada et al., 2002), and can affect T-cell-mediated immune functions (Han et al., 2003). Such a response in the periodontium could contribute to periodontitis progression (Doxey et al., 1998).
In those rats fed cholesterol and treated water, the periodontal ligament exhibited cell proliferation rather than apoptosis, while dietary cholesterol and treatment with periodontitis-inducing agents induced apoptosis rather than cell proliferation. These results suggest that LPS and proteases may have a more detrimental effect on periodontal ligament fibroblast populations than does cholesterol.
Gingival fibroblast proliferation and apoptosis did not increase following the consumption of high dietary cholesterol and/or topical application of LPS and proteases. Gingival fibroblasts may be relatively resistant to the induction of proliferation and apoptosis by bacterial pathogens or high dietary cholesterol. This is in agreement with the results of an in vitro study in which human gingival fibroblasts exhibited a response to Staphylococcus epidermidis peptidoglycan and muramyldipeptide comparable with, or slightly less than, that of human periodontal ligament fibroblasts (Hatakeyama et al., 2003).
The apical migration of the junctional epithelium in those rats fed cholesterol, but not treated with periodontitis-inducing agents, occurred to a lesser degree than in those rats fed a regular diet and treated with periodontitis-inducing agents. One possible reason for this difference between the 2 groups is that LPS and proteases, but not high dietary cholesterol, can induce periodontal ligament fibroblast apoptosis and collagen destruction (Ekuni et al., 2005). The increase in apoptotic periodontal ligament fibroblasts induces the detachment of connective tissue from tooth surfaces (Sakai et al., 1999). The junctional epithelium would migrate apically to these areas (Ekuni et al., 2005).
In this study, LPS from E. coli and proteases from Streptomyces griseus were applied to rat gingivae. These bacterial species are not generally considered periodontal pathogens, and this is a potential limitation of this study. However, the application of E. coli LPS and Streptomyces griseus protease provides us a rat periodontitis model with high reproducibility (Ekuni et al., 2003, 2005). In addition, the use of commercial products ensures that experimental conditions will be more uniform.
In conclusion, high dietary cholesterol can initiate periodontitis and augment the inflammatory responses induced by bacterial pathogens. Analysis of these data supports high dietary cholesterol being a risk factor for periodontitis progression.
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ACKNOWLEDGMENTS |
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Received October 12, 2004; Last revision April 23, 2005; Accepted April 26, 2005
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REFERENCES |
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