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RESEARCH REPORTS |
1 Department of Restorative Dentistry and Endodontology, Osaka University Graduate School of Dentistry, 1–8 Yamadaoka, Suita, Osaka 565-0871, Japan; and
2 Department of Microbiology, School of Dentistry, Aichi-Gakuin University, Nagoya, Aichi 464-8650, Japan;
* corresponding author, noiri{at}dent.osaka-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: Porphyromonas gingivalis fimbriae • localization • human periodontal pockets • plaque-free zone • immunohistochemistry
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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P. gingivalis appears to form small aggregates distributed throughout human periodontal pockets (Noiri et al., 1997) and is associated with plaque-biofilm formation at the bottoms of human periodontal pockets in the so-called ‘plaque-free zone’ (PFZ) (Noiri and Ebisu, 2000). However, the distribution of P. gingivalis-carrying fimbriae in situ has not yet been examined.
The objective of the present study was to investigate the expression and localization of P. gingivalis fimbriae in human periodontal pockets.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). Nine teeth were extracted for examination under a transmission electron microscope (TEM) (Table 1). The criteria for all subjects and specimens have been described (Noiri et al., 1997). Informed consent was obtained from all patients according to the protocol approved by the Ethics Committee of the Osaka University Graduate School of Dentistry.
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). Mass cultures were grown anaerobically at 37°C as described previously (Yoshimura et al., 1984).
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). A fimbriae-deficient mutant from strain ATCC 33277, KDP98 (Watanabe-Kato et al., 1998), was kindly provided by Prof. Koji Nakayama (Nagasaki University Graduate School of Dentistry, Japan). Negative control samples obtained from periodontally healthy extracted teeth, and their surrounding periodontal tissues were prepared.
Immunohistochemical Procedure and Image Analysis
After separating them from their surrounding periodontal tissue, we carefully extracted the 11 selected sites, immediately fixed the specimens using microwave irradiation in 2% paraformaldehyde and 2.5% glutaraldehyde, and immersed them in the same fixative (Noiri et al., 1997, 2001) (Table 1). All specimens were dehydrated through a graded polyhydroxy dimethacrylate resin (LR-White®, London Resin Co. Ltd., London, UK) series, embedded in the same resin, and polymerized under ultraviolet light for 5–7 days at 4°C. The resin blocks were trimmed, and the subgingival parts of the tooth were hollowed out. Serial sections 2.5 µm thick were cut on a rotary microtome (2065 SUPER CUT, Leica Instruments GmbH, Nussloch, Germany) with the use of a tungsten-carbide knife (16 cm/dTC, Leica Instruments GmbH), and either stained by the Brown and Brenn-modified Gram-staining procedure or subjected to the alkaline-phosphatase-conjugated streptavidin biotin method (Noiri et al., 1997, 2001). The results of both methods were observed under a light microscope (Optiphot-2, Nikon, Tokyo, Japan).
Computer image analysis was carried out on the light microscopic images according to a previously described method (Noiri et al., 2001), and the immunolocalizations were then analyzed in human periodontal pocket specimens.
Preparation for Immuno-transmission Electron-microscopic Observation
The 9 teeth extracted were immediately fixed in 4% paraformaldehyde in 0.1 M sodium cacodylate (pH 7.4), 0.1% glutaraldehyde, for 2 hrs at 4°C (Table 1). The resin blocks were prepared by the methods described above. Parts of the PFZ were cut away from the blocks, and ultrathin sections (70 nm thick) were placed on Ni grids.
The sections were blocked with 20% normal goat serum in 0.1% bovine serum albumin (BSA) solution for 30 min, and then incubated with the anti-fimbriae serum diluted 1:100 in 0.1 M Tris-HCl-buffered saline (TBS) (pH 7.4) containing 1% BSA for 3 hrs at 4°C. After being washed, the samples were reacted with goat anti-rabbit IgG conjugated with 5 nm colloidal gold particles (GAR-G5; Amersham International plc, Buckinghamshire, UK), diluted 1:50 in 0.1% BSA-TBS. Subsequently, all the samples on the grids were fixed in 1% glutaraldehyde in a 0.1 M cacodylate buffer (pH 7.4), and stained with 1% aqueous uranyl acetate for 10 min and 1% lead acetate for 5 min. All samples were examined under a TEM (H-7500; Hitachi, Tokyo, Japan).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). The anti-fimbriae serum reacted with almost all P. gingivalis strains except for KDP98, a strain deficient in fimbriae, although strains W50 and W83 were previously reported to be afimbriate in biochemical tests (Suzuki et al., 1988). None of the other 5 bacterial species examined for cross-reactivity was labeled with the anti-fimbriae serum. In healthy periodontal tissues (negative controls), no background or any reaction with anti-P. gingivalis and anti-fimbriae was observed.
Localization of P. gingivalis-carrying Fimbriae in situ in Human Periodontal Pockets
Positive reactions with the 2 antisera were found in 6 of the 11 samples examined: 1, 3, 7, 8, 10, and 11 (Figs. 1A, 1B). Positive reactions with anti-P. gingivalis serum were distributed throughout all the separate sites (Fig. 1A). Interestingly, within the pocket epithelial cells, no positive immunoreactions with either antiserum were detected. Fimbriae were detected in the deep pocket zone of the cementum-attached plaque area in all 6 positive samples (Fig. 1B). In 4 of 6 positive samples, positive reactions were detected at the cementum site of the cementum-attached plaque area. In the other 2 samples, fimbriae were observed at the superficial site in the deep pocket zone. In the epithelium-associated plaque area, positive reactions with the anti-fimbriae serum were detected in only 2 of the 6 positive samples (Nos. 3 and 10).
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). The gold particles were distributed along the fimbriae. Positive reactions with the anti-fimbriae serum were detected in the PFZ in 6 of 9 samples examined. All positive reactions detected were observed on P. gingivalis attached to the dental cuticle, and fimbriae did not attach directly to the acellular cementum. Small numbers of bacteria, including different morphotypes, formed micro-colonies on the dental cuticle (DC) covering the acellular cementum (Fig. 2B). P. gingivalis carrying immunogold-labeled fimbriae was observed as small aggregates, as well as single cells, in close contact with the DC (Figs. 2C, 2D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Little attention has been paid to the DC on periodontitis-affected teeth, despite the fact that it possibly plays a key role in apical epithelial migration, progressive bacterial adhesion to the root surface, and protection of the exposed root (Friedman et al., 1993). We speculate that apical epithelial migration will occur by two pathways. The first is that bacteria inhabiting the bottoms of periodontal pockets and/or their toxins directly affect the junctional epithelium and periodontal ligament. The second is that subgingival bacteria attached to the pocket epithelium invade the gingiva, and then bacterial toxins and noxious products are transmitted via the gingival tissue and cause damage, eventually followed by deepening of the periodontal pockets. It is strongly suspected that P. gingivalis-carrying fimbriae, heavily located in the PFZ, could directly exhibit their pathogenicity on the junctional epithelium, because fewer samples were positive in the epithelium-associated plaque area, and no positive immunoreactions with the anti-fimbriae serum were observed within the pocket epithelial cells.
The origin of the DC on periodontitis-affected cementum remains unclear. Kobayashi and Rose (1979) noted that the DC was a layer composed of highly condensed protein, since it was completely digested with the proteolytic enzymes trypsin and protease. Previous studies have suggested that the structure consists of anionic polymers, including glycoproteins (Friedman et al., 1993), or serum proteins, mainly albumin, IgA, and IgG, in the gingival fluid (Eide et al., 1984; López et al., 1990; Abbas et al., 1991). In contrast, purified P. gingivalis fimbriae bound strongly to glyco-conjugates, such as albumin-fucosylamide, through lectin-like interaction with carbohydrate (Sojar et al., 2004). P. gingivalis adherence to the DC might depend on P. gingivalis fimbriae and serum protein-carbohydrate interactions. The DC may also contain bacterial components (Eide et al., 1984). It has been suggested that bacterial adherence to the exposed root surface is mediated by the DC (Carrassi et al., 1989; Vrahopoulos et al., 1992; Friedman et al., 1993). A few studies have reported that bacterial adherence to cementum without prior subgingival pellicle formation could not be demonstrated (Carrassi et al., 1989; Friedman et al., 1993). P. gingivalis cells were detected in the PFZ (Mordan et al., 1999; Noiri and Ebisu 2000), and P. gingivalis-carrying fimbriae were also observed in the same zone in this study, indicating that fimbriae may play a crucial role in bacterial adherence to the DC. Results from the present study suggest that P. gingivalis fimbriae may possibly adhere to the DC rather than to other substances and organisms at the bottoms of human periodontal pockets. However, it remains unknown how the fimbriae mediate bacterial adherence to the DC.
P. gingivalis has been considered to be a late colonizer and to bind to Gram-positive facultative bacteria that have already colonized as the first bacteria (Kolenbrander et al., 2002). However, at the bottoms of periodontal pockets, P. gingivalis appeared to play a role as an early colonizer in biofilm formation, although several types of obligate anaerobes were observed.
In conclusion, we demonstrated that P. gingivalis fimbriae mediate adherence to the root surface at the bottoms of human periodontal pockets, rather than to the pocket epithelium.
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ACKNOWLEDGMENTS |
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Received January 8, 2004; Last revision September 9, 2004; Accepted September 21, 2004
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REFERENCES |
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