Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Language：English   Publishing type：Research paper (scientific journal)  

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

With the rising number of older adults residing at home, there is a growing need for risk assessment and patient management in home nursing. This study aims to develop point-of-care test (POCT) reagents that can aid in risk assessment and home care, especially in settings with limited resources. Our focus was on creating a C-reactive protein (CRP) POCT, which can accurately diagnose clinically significant judgment values in home nursing. Additionally, we assessed the utility of the HemoCue WBC DIFF system in providing differential counts of white blood cells (WBC). These performances were compared with a laboratory test using blood samples from patients with pneumonia. The CRP POCT showed a comparable result to that of a laboratory method, with an average kappa index of 0.883. The leukocyte count showed good agreement with the reference method. While the correlation coefficients for both neutrophil and lymphocyte counts were deemed acceptable, it was observed that the measured values tended to be smaller in cases where the cell count was higher. This proportional error indicates a weak correlation with the neutrophil-to-lymphocyte ratio. CRP POCT and WBC counts provided reliable and accurate judgments. These tools may benefit risk management for older adults at home, patients with dementia who cannot communicate, and those living in depopulated areas.

DOI： 10.3390/diagnostics13142407.

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)  

Hydroxyapatite formation on endodontic hydraulic calcium silicate cements (HCSCs) plays a significant role in sealing the root canal system and elevating the hard-tissue inductivity of the materials. This study evaluated the in vivo apatite-forming ability of 13 new-generation HCSCs using an original HCSC (white ProRoot MTA: PR) as a positive control. The HCSCs were loaded into polytetrafluoroethylene tubes and implanted in the subcutaneous tissue of 4-week-old male Wistar rats. At 28 days after implantation, hydroxyapatite formation on the HCSC implants was assessed with micro-Raman spectroscopy, surface ultrastructural and elemental characterization, and elemental mapping of the material-tissue interface. Seven new-generation HCSCs and PR had a Raman band for hydroxyapatite (v1 PO43- band at 960 cm-1) and hydroxyapatite-like calcium-phosphorus-rich spherical precipitates on the surfaces. The other six HCSCs with neither the hydroxyapatite Raman band nor hydroxyapatite-like spherical precipitates did not show calcium-phosphorus-rich hydroxyapatite-layer-like regions in the elemental mapping. These results indicated that 6 of the 13 new-generation HCSCs possessed little or no ability to produce hydroxyapatite in vivo, unlike PR. The weak in vivo apatite-forming ability of the six HCSCs may have a negative impact on their clinical performance.

DOI： 10.3390/jfb14040213

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.jbc.2023.104760

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)  

Calcium salt precipitation induced by intracanal medicaments contributes to the formation of apical hard tissue during apexification. This study compared the calcium salt-forming ability of a new calcium silicate-based intracanal medicament (Bio-C Temp) with that of two commercial calcium hydroxide pastes (Calcipex Plane II and Vitapex) in a rat subcutaneous implantation model. Polytetrafluoroethylene tubes containing each of the three materials were subcutaneously implanted in 4-week-old male Wistar rats. After 28 days, the composition and amount of calcium salts formed at the material-tissue interface were assessed using micro-Raman spectroscopy, X-ray diffraction, and elemental mapping. The tested materials produced white precipitates that had Raman spectra with peaks corresponding to hydroxyapatite and calcite. X-ray diffraction detected hydroxyapatite formation on Calcipex Plane II and Vitapex implants, as well as calcite formation on all three materials. Elemental mapping revealed that Bio-C Temp generated significantly smaller calcium- and phosphorus-rich calcified regions within the subcutaneous connective tissue than Vitapex. These results indicate that Bio-C Temp produced less calcium salt in rat subcutaneous tissue than Vitapex, although all materials formed hydroxyapatite and calcite in rat subcutaneous tissue. Bio-C Temp could be less effective than Vitapex in promoting apical hard tissue formation during apexification.

DOI： 10.3390/dj11040091

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)  

INTRODUCTION: Prostaglandin E2 (PGE2) exerts biological actions through its transport pathway involving intracellular synthesis, extracellular transport, and receptor binding. This study aimed to determine the localization of the components of the PGE2-transporting pathway in human dental pulp and explore the relevance of PGE2 receptors (EP2/EP4) to angiogenesis and dentinogenesis. METHODS: Protein localization of microsomal PGE2 synthase (mPGES), PGE2 transporters [(multidrug resistance-associated protein-4 (MRP4) and prostaglandin transporter (PGT)], and EP2/EP4 was analyzed using double immunofluorescence staining. Tooth slices from human third molars were cultured with or without butaprost (EP2 agonist) or rivenprost (EP4 agonist) for 1 week. Morphometric analysis of endothelial cell filopodia was performed to evaluate angiogenesis, and real-time polymerase chain reaction was performed to evaluate angiogenesis and odontoblast differentiation markers. RESULTS: MRP4 and PGT were colocalized with mPGES and EP2/EP4 in odontoblasts and endothelial cells. Furthermore, MRP4 was colocalized with mPGES and EP4 in human leukocyte antigen-DR-expressing dendritic cells. In the tooth slice culture, EP2/EP4 agonists induced significant increases in the number and length of filopodia and mRNA expression of angiogenesis markers (vascular endothelial growth factor and fibroblast growth factor-2) and odontoblast differentiation markers (dentin sialophosphoprotein and collagen type 1). CONCLUSIONS: PGE2-producing enzyme (mPGES), transporters (MRP4 and PGT), and PGE2-specific receptors (EP2/EP4) were immunolocalized in various cellular components of the human dental pulp. EP2/EP4 agonists promoted endothelial cell filopodia generation and upregulated angiogenesis- and odontoblast differentiation-related genes, suggesting that PGE2 binding to EP2/EP4 is associated with angiogenic and dentinogenic responses.

DOI： 10.1016/j.joen.2023.01.009

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1002/osi2.1172

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

The oral cavity is the second most colonized site of Helicobacter pylori after the stomach. This study aimed to compare the genetic relatedness between gastric and oral H. pylori in Japanese patients with early gastric cancer through multilocus sequence typing (MLST) analysis using eight housekeeping genes. Gastric biopsy specimens and oral samples were collected from 21 patients with a fecal antigen test positive for H. pylori. The number of H. pylori allelic profiles ranged from zero to eight since the yield of DNA was small even when the nested PCR was performed. MLST analysis revealed that only one patient had a matching oral and gastric H. pylori genotype, suggesting that different genotypes of H. pylori inhabit the oral cavity and gastric mucosa. The phylogenetic analysis showed that oral H. pylori in six patients was similar to gastric H. pylori, implying that the two strains are related but not of the same origin, and those strains may be infected on separate occasions. It is necessary to establish a culture method for oral H. pylori to elucidate whether the oral cavity acts as the source of gastric infection, as our analysis was based on a limited number of allele sequences.

DOI： 10.3390/ijms24032211

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Ascorbic acid (AA; vitamin C) plays a crucial role in the biosynthesis and secretion of collagen to produce the organic matrix of hard tissues. Nevertheless, the detailed mechanism by which AA induces reparative dentinogenesis is still unknown. This study aimed to investigate the pathway and function of AA during wound healing in a rat pulpotomy model. Sodium-dependent vitamin C transporter (SVCT) 2 and glucose transporter (GLUT) 1 were detected in odontoblasts, endothelial cells, and nerve fibers in normal pulp tissues. SVCT2 and GLUT1 were also expressed in odontoblast-like cells in pulpotomized tissues of Wistar rats, and immunopositive cells of SVCT2 were significantly increased at 5 days after pulpotomy (p < 0.05). By contrast, osteogenic disorder Shionogi (ODS) rats, which cannot generate AA, also expressed SVCT2 and GLUT1 in normal and wound healing conditions. However, in ODS rats, when compared with the AA-addition group, the formation of dentin bridges in the AA-loss group was not evident, a layer of osteopontin was significantly increased beneath the wound surface (p < 0.05), and alpha smooth muscle actin at the odontoblast-like cells observed along this layer was significantly increased (p < 0.05), but not Nestin. Moreover, the amounts of type 1 collagen generated in the reparative dentin and beneath the wound healing site were significantly diminished (p < 0.05). Macrophages expressing CD68 and CD206 increased beneath the wound site. Hence, AA may be involved in odontoblast-like cell differentiation and anti-inflammatory response during dental pulp wound healing. Our results provide new insights into the function of AA through SVCT2 and GLUT1 in reparative dentinogenesis and may help in developing new therapeutic targets for dental pulpal disease.

DOI： 10.1038/s41598-023-28197-9

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Authorship：Last author   Language：Japanese   Publishing type：Research paper (scientific journal)  

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)  

The coronavirus disease pandemic has afforded dental professionals an opportunity to reconsider infection control during treatment. We investigated the efficacy of combining extraoral high-volume evacuators (eHVEs) with preprocedural mouth rinsing in reducing aerosol contamination by ultrasonic scalers. A double-masked, two-group, crossover randomized clinical trial was conducted over eight weeks. A total of 10 healthy subjects were divided into two groups; they received 0.5% povidone-iodine (PI), essential oil (EO), or water as preprocedural rinse. Aerosols produced during ultrasonic scaling were collected from the chest area (PC), dentist's mask, dentist's chest area (DC), bracket table, and assistant's area. Bacterial contamination was assessed using colony counting and adenosine triphosphate assays. With the eHVE 10 cm away from the mouth, bacterial contamination by aerosols was negligible. With the eHVE 20 cm away, more dental aerosols containing bacteria were detected at the DC and PC. Mouth rinsing decreased viable bacterial count by 31-38% (PI) and 22-33% (EO), compared with no rinsing. The eHVE prevents bacterial contamination when close to the patient's mouth. Preprocedural mouth rinsing can reduce bacterial contamination where the eHVE is positioned away from the mouth, depending on the procedure. Combining an eHVE with preprocedural mouth rinsing can reduce bacterial contamination in dental offices.

DOI： 10.3390/ijerph19106048

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Antimicrobial mouthwash improves supragingival biofilm control when used in conjunction with mechanical removal as part of an oral hygiene routine. Mouthwash is intended to suppress bacterial adhesion during biofilm formation processes and is not aimed at mature biofilms. The most common evidence-based effects of mouthwash on the subgingival biofilm include the inhibition of biofilm accumulation and its anti-gingivitis property, followed by its cariostatic activities. There has been no significant change in the strength of the evidence over the last decade. A strategy for biofilm control that relies on the elimination of bacteria may cause a variety of side effects. The exposure of mature oral biofilms to mouthwash is associated with several possible adverse reactions, such as the emergence of resistant strains, the effects of the residual structure, enhanced pathogenicity following retarded penetration, and ecological changes to the microbiota. These concerns require further elucidation. This review aims to reconfirm the intended effects of mouthwash on oral biofilm control by summarizing systematic reviews from the last decade and to discuss the limitations of mouthwash and potential adverse reactions to its use. In the future, the strategy for oral biofilm control may shift to reducing the biofilm by detaching it or modulating its quality, rather than eliminating it, to preserve the benefits of the normal resident oral microflora.

DOI： 10.3390/antibiotics1106072719106048

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Language：English   Publishing type：Research paper (scientific journal)  

Laminin, a basement membrane heterotrimeric glycoprotein composed of α/β/γ subunits, has important tissue-specific functions in the control of cellular behavior. Our recent study showed the colocalization of CD163+ M2-like macrophages with Schwann cells in human dental pulp, leading us to hypothesize that the laminin isoform of Schwann cells is associated with CD163 expression. The present study investigated the distribution of laminin isoforms in human dental pulp and the underlying mechanisms that affect macrophage phenotypes. Immunofluorescence analysis indicated that blood vessels were exclusively positive for laminin α4 and α5, whereas laminin α2 was associated with Schwann cells. Unexpectedly, laminin α3/laminin-332 (α3β3γ2) was detected on lymphatic vessels. In intact and carious teeth, CD163+ cells were associated with laminin α2, whereas CD206 single-positive cells were present inside, outside, and along blood vessels. In vitro incubation of THP-1 macrophages in plates coated with laminin-211/511 or its functionally analogous E8 fragments of α-chain (E8-α) indicated that cell shapes differed between macrophages grown on laminin-211/E8-α2 and macrophages grown on laminin-511/E8-α5. Laminin-211/E8-α2-coated plates upregulated CD163 expression, compared with laminin-511/E8-α5-coated plates. Integrin α3- and integrin α6-neutralizing Abs altered the shape of THP-1 macrophages and upregulated mRNA levels of CD206 and CD163 in macrophages grown on laminin-511; the neutralizing Abs did not affect macrophages grown on laminin-211. These findings suggest that laminin isoforms differentially regulate macrophage behavior via distinct integrin-laminin affinities. Of note, laminin-332 is expressed by pulpal lymphatic vessels, the existence of which has been debated; laminin-211 might have a role in maintaining CD163 expression on macrophages.

DOI： 10.4049/immunohorizons.2100110

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We performed a comprehensive microbiome analysis of root caries lesions using 22 teeth extracted from patients with severe periodontitis. The carious lesions were mechanically collected and cryo-pulverized following tooth extraction. Differences in the microbiome were compared between independent lesions at the supragingival site (SG) and lesions extending beyond the gingival margin (GCB). DNA was extracted and the microbiome was characterized on the basis of the V3-V4 hypervariable region of the 16S rRNA gene using paired-end sequencing on an Illumina MiSeq device. The microbiota in root caries lesions showed compositionally distinct microbiota depending on the location. The most abundant OTUs in the SG group were Streptococcus (26.0%), Actinomyces (10.6%), and Prevotella (7.6%). GCB presented Prevotella (11.1%) as the most abundant genus, followed by Fusobacterium (9.6%) and Actinomyces (8.7%). The SG group showed a lack of uniformity in microbiota compared with the GCB group. The bacterial profiles of GCB varied considerably among patients, including periodontal pathogens such as Porphyromonas, Selenomonas, Filifactor, Peptococcus, and Tannerella. Periodontal pathogens inhabit root caries lesions that extend beyond the gingival margin. This study provides a new perspective for elucidating the microbial etiology of root caries.

DOI： 10.3390/microorganisms9112349

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Language：English   Publishing type：Research paper (scientific journal)  

Resin monomers and polymerisation initiators have been shown to be cytotoxic for pulp cells and to disturb odontoblast differentiation. This study aimed to compare the effect of a resin-modified calcium silicate cement (TheraCal LC; TC) and a resin-free calcium silicate cement (ProRoot MTA; PR) on pulpal healing after pulpotomy. Pulpotomy was performed on the maxillary first molars of 8-week-old rats using either PR or TC. After 1, 3, 7, 14 and 28 days, pulpal responses were assessed by micro-computed tomography, haematoxylin-eosin staining and immunostaining against CD68, which is a pan-macrophage marker. The results showed that pulpotomy with TC induced persistent infiltration of inflammatory cells, including CD68-positive macrophages, and delayed the formation of reparative dentin as compared with that with PR, although both materials allowed pulpal healing over the long term. Therefore, resin-modified TC was not as biocompatible nor bioinductive as resin-free PR when applied on the healthy pulp of rat molars.

DOI： 10.1111/aej.12568

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Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

This study compared the apatite-forming ability (AFA) levels of flowable and putty formulations of Nishika Canal Sealer BG Multi (F-NBG and P-NBG, respectively) and attempted to clarify the cause of differences in the AFA levels of F-NBG and P-NBG. NBG samples were aged in simulated body fluid (SBF) or 1-, 5-, or 10-g/L bovine serum albumin-containing SBF (BSA-SBF) and analyzed in terms of their ultrastructures, elemental compositions, and Raman spectra to identify apatite formation. The phosphate ion consumption rates of NBG samples in the media were evaluated as an indicator of apatite growth. The original elemental composition, calcium ion release, and alkalizing ability levels of F-NBG and P-NBG were also evaluated. Apparent apatite formation was detected on all NBG samples except F-NBG aged in 10-g/L BSA-SBF. P-NBG consumed phosphate ions faster than F-NBG. As-prepared P-NBG showed more silicon elements on its surface than as-prepared F-NBG. P-NBG released more calcium ions than F-NBG, although their alkalizing ability levels did not differ statistically. In conclusion, the AFA of P-NBG was greater than that of F-NBG, probably because of the greater ability of P-NBG to expose silanol groups on the surface and release calcium ions.

DOI： 10.3390/app11198969

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Language：English   Publishing type：Research paper (scientific journal)  

This study was designed to investigate the effects of Sake Lees extracts (SLE, Sake Kasu) on the functional activity of odontoblastic cells and tooth pulp of the rats. For in vitro studies, a rat clonal odontoblast-like cell line, KN-3 cells were cultured. SLE significantly decreased KN-3 cell proliferation, but showed no significant cytotoxicity. SLE effects on several protein productions of KN-3 cells were compared with PBS. SLE and PBS increased alkaline phosphatase (ALP), dentin sialoprotein (DSP), and osterix in a day-course dependent manner, while SLE increased the induction of ALP on day 9-21 and DSP on day 15-21. SLE also increased Runx2 expression on day 3 and 9 compared to PBS. Alizarin Red stainings revealed that SLE showed a subtle increase in mineralization of KN-3 cells on day 15 and 21. A histological investigation was conducted to assess if SLE induced reparative dentin formation after direct capping at the exposed tooth pulp in rats, suggesting that SLE could increase the reparative dentin formation more than PBS. These findings suggest that Sake Lees could have functional roles in the alterations of odontoblastic activity, which might influence the physiology of the tooth pulp.

DOI： 10.1007/s10266-021-00654-9

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

OBJECTIVE: Bioceramic-containing root canal sealers promote periapical healing via Ca2+ and OH- release and apatite formation on the surface. This study aimed to compare Ca2+ and OH- release and in vivo apatite formation of three bioceramic-containing root canal sealers: EndoSequence BC sealer (Endo-BC), MTA Fillapex (MTA-F), and Nishika Canal Sealer BG (N-BG). MATERIALS AND METHODS: Polytetrafluoroethylene tubes filled with sealers were immersed in distilled water for 6 and 12 h and for 1, 7, 14, and 28 days to measure Ca2+ and OH- release. Additionally, tubes filled with sealers were implanted in the backs of rats for 28 days, and in vivo apatite formation was analyzed using an electron probe microanalyzer. RESULTS: Endo-BC released significantly more Ca2+ than the other sealers at 6 and 12 h and 1 day. Ca2+ release was significantly lower from N-BG than from Endo-BC and MTA-F at 14 and 28 days. OH- release was significantly higher from Endo-BC than from the other sealers throughout the experiment, except at 1 day. OH- release was lower from N-BG than from MTA-F at 6 h and 7 days. Only Endo-BC implants exhibited apatite-like calcium-, phosphorus-, oxygen-, and carbon-rich spherulites and apatite layer-like calcium- and phosphorus-rich, but radiopaque element-free, surface regions. CONCLUSIONS: Ca2+ and OH- release is ranked as follows: Endo-BC > MTA-F > N-BG. Only Endo-BC demonstrated in vivo apatite formation. CLINICAL RELEVANCE: Endo-BC could promote faster periapical healing than MTA-F and N-BG.

DOI： 10.1007/s00784-021-04118-w

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Other Link： https://link.springer.com/article/10.1007/s00784-021-04118-w/fulltext.html

Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)  

This study aimed to evaluate the anticariogenic biofilm activity of a novel zinc-containing glass ionomer cement, Caredyne Restore (CR), using a flow-cell system that reproduces Stephan responses. Streptococcus mutans biofilms were cultured on either CR or hydroxyapatite (HA) discs mounted on a modified Robbins device. The media were allowed to flow at a speed of 2 mL/min for 24 h while exposed to an acidic buffer twice for 30 min to mimic dietary uptake. Acid exposure enhanced biofilm inhibition in the CR group, which showed 2.6 log CFU/mm2 in viable cells and a 2 log copies/mL reduction in total cells compared to the untreated group after 24 h of incubation, suggesting enhanced anticariogenic activity due to the release of fluoride and zinc ions. However, there was no difference in the number of viable and total cells between the two experimental groups after 24 h of incubation in the absence of an acidic environment. The anticariogenic biofilm activity of CR occurs in acidic oral environments, for example in the transient pH drop following dietary uptake. CR restorations are recommended in patients at high risk of caries due to hyposalivation, difficulty brushing, and frequent sugar intake.

DOI： 10.3390/antibiotics10080977

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Language：Japanese   Publisher：日本歯科医学会  

【研究目的】根尖性歯周炎の治療の原則は、根管系に侵入した感染源を除去することである。外科的歯内療法の一つ、歯根端切除術は手術用顕微鏡下で行うことで、感染経路の特定と除去を高い精度で行うことが可能となり、成功率が飛躍的に上昇した。一方で基礎疾患を背景に炎症そのものが治療抵抗性を獲得する可能性が指摘されており、感染源の除去を主目的として行われてきた標準治療に加え、直接炎症を制御する新規医療技術の開発が必要となる。自己血製剤であるConcentrated Growth Factor(CGF)は高い治癒組織再生および骨再生誘導能力を有する。本研究は、CGFを歯根端切除術に併用することで、骨、歯周組織の再生、臨床症状の改善に対する治療効果を検証することを目的として、東北大学を主施設に東京医科歯科大学、大阪大学および新潟大学で実施している多施設共同臨床研究について報告する。【研究方法】自己血製剤を使用する本術式は、第3種再生医療等技術に該当し、認定再生医療等技術委員会での審査および承認後に研究を開始した。臨床研究の実施にあたっては、大学間の共通実施プロトコールの作成や症例選択基準および除外基準の設定後、手術件数を各施設6例(CGF併用3例、コントロール3例)として実施した。【結果】現在、予定症例数である24例の歯根端切除術はすべて終了している。現在、経過の大半の臨床データは取得済みであり、順次解析に移行している。【考察】第3種再生医療等技術および多施設共同臨床研究の実施にあたって、いくつかの課題が明らかになったが、現在のところ順調に研究が進捗している。今後、患者の創傷治癒能力を考慮した歯内療法の確立、または再生医療等技術の歯内療法への普及について、さらに検討を進めていきたい。(著者抄録)

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Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Oral biofilms are associated with caries, periodontal diseases, and systemic diseases. Generally, antimicrobial therapy is used as the first line of treatment for infectious diseases; however, bacteria in biofilms eventually develop antibiotic resistance. This study aimed to apply our in situ biofilm model to verify whether an arginine preparation is useful for plaque control. Ten healthy subjects who did not show signs of caries, gingivitis, or periodontitis were recruited. The dental biofilms from the subjects were obtained using our oral device before and after gargling with arginine solution for 4 weeks. We found that 8% arginine solution significantly increased the concentration of ammonium ions (NH4+) in vitro and in vivo in saliva (p &lt; 0.05) and decreased the proportions of the genera Atopobium and Catonella in vivo. However, the viable count was unaffected by the mouthwash. Further, oral populations of the genera Streptococcus and Neisseria tended to increase with the use of arginine. Therefore, we concluded that using an 8% arginine solution decreased the NH4+ concentration in the oral cavity without affecting the number of viable bacteria, and that the diversity of oral bacterial flora changed. We suggest that arginine might help prevent mature biofilm formation.

DOI： 10.3390/pharmacy9010018

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PORTFOLIO  

Dental biofilm present on the tooth surface is associated with oral diseases, such as dental caries and periodontal disease. Because bacterial numbers rapidly increase in saliva during sleep, oral care before sleeping is recommended for the prevention of chronic oral diseases. However, temporal circadian changes in the quantity and quality of dental biofilms are poorly understood. This study aimed to investigate the impacts of sleeping on dental biofilm amounts and compositions by using an in situ model. The use of this in situ model enabled us to investigate dental biofilm formed in the oral cavity and to perform a quantitative analysis. Subjects began wearing oral splints in the morning or before sleeping, and biofilm samples were collected at 8, 16, and 24 h after the subjects began wearing oral splints; these samples were then used in various experiments. No significant changes in the numbers of biofilm-forming bacteria were caused by sleep. However, the relative abundances of genera related to periodontitis (i.e., Fusobacterium and Prevotella) increased after awakening. In conclusion, the numbers of biofilm-forming bacteria were not affected by sleep, and the abundances of obligate anaerobes increased after sleep. This research may aid in defining efficacious preventive oral care.

DOI： 10.1038/s41598-020-80541-5

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Language：English   Publishing type：Research paper (scientific journal)  

Dysbiosis of the oral microbiome is associated with diseases such as periodontitis and dental caries. Because the bacterial counts in saliva increase markedly during sleep, it is broadly accepted that the mouth should be cleaned before sleep to help prevent these diseases. However, this practice does not consider oral biofilms, including the dental biofilm. This study aimed to investigate sleep-related changes in the microbiome of oral biofilms by using 16S rRNA gene sequence analysis. Two experimental schedules-post-sleep and pre-sleep biofilm collection-were applied to 10 healthy subjects. Subjects had their teeth and oral mucosa professionally cleaned 7 days and 24 h before sample collection. Samples were collected from several locations in the oral cavity: the buccal mucosa, hard palate, tongue dorsum, gingival mucosa, tooth surface, and saliva. Prevotella and Corynebacterium had higher relative abundance on awakening than before sleep in all locations of the oral cavity, whereas fluctuations in Rothia levels differed depending on location. The microbiome in different locations in the oral cavity is affected by sleep, and changes in the microbiome composition depend on characteristics of the surfaces on which oral biofilms form.

DOI： 10.1371/journal.pone.0259850

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The purpose of this study was to develop a high-frequency wave therapy model in rats and to investigate the influence of high-frequency waves on root canal treatment, which may provide a novel strategy for treating apical periodontitis. Root canal treatments with and without high-frequency wave irradiation were performed on the mandibular first molars of 10-week-old male Wistar rats. The mesial roots were evaluated radiologically, bacteriologically, and immunohistochemically. At 3 weeks after root canal treatment, lesion volume had decreased significantly more in the irradiated group than in the non-irradiated group, indicating successful development of the high-frequency therapy model. The use of high-frequency waves provided no additional bactericidal effect after root canal treatment. However, high-frequency wave irradiation was found to promote healing of periapical lesions on the host side through increased expression of fibroblast growth factor 2 and transforming growth factor-β1 and could therefore be useful as an adjuvant nonsurgical treatment for apical periodontitis.

DOI： 10.1371/journal.pone.0239660

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)  

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© 2020, The Author(s). When regenerative endodontic procedures (REPs) are performed on immature teeth diagnosed with pulp necrosis and apical periodontitis, various healing patterns occur. Furthermore, infected immature teeth with endodontic disorders often exhibit some remnant pulp and apical tissue. Therefore, this study investigated the impact of remnant healthy or fully functional pulp and apical tissue on healing patterns after REPs. Simulated REPs were performed on non-infected immature rat molars with different amounts of remnant pulp and apical tissue. Healing patterns in these teeth were assessed after 28 days. Teeth with 0.81–0.91 mm of remnant pulp healed with pulp-like tissue, dentin, and osteodentin-like dentin-associated mineralized tissue (OSD-DAMT); teeth with 0.60–0.63 mm of remnant pulp healed with pulp-like tissue and OSD-DAMT; teeth with 0.13–0.43 mm of remnant pulp healed with periodontal ligament (PDL)-like tissue, OSD-DAMT, and cementum-like dentin-associated mineralized tissue (CEM-DAMT); and teeth with disorganization of pulp and apical tissues at 0.15–0.38 mm beyond the root apex healed with PDL-like tissue, CEM-DAMT, and intracanal bone (IB). Loss of Hertwig’s epithelial root sheath was observed with IB formation. These results showed that four distinct healing patterns occurred after REPs, depending on the preoperative amount of remnant healthy pulp and apical tissue.

DOI： 10.1038/s41598-020-78022-w

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<title>Abstract</title><sec>
<title>Background</title>
Sulfated vizantin, a recently developed immunostimulant, has also been found to exert antibiofilm properties. It acts not as a bactericide, but as a detachment-promoting agent by reducing the biofilm structural stability. This study aimed to investigate the mechanism underlying this activity and its species specificity using two distinct ex vivo oral biofilm models derived from human saliva.


</sec><sec>
<title>Results</title>
The biofilm, composed mainly of the genus <italic>Streptococcus</italic> and containing 50 μM of sulfated vizantin, detached significantly from its basal surface with rotation at 500 rpm for only 15 s, even when 0.2% sucrose was supplied. Expression analyses for genes associated with biofilm formation and bacterial adhesion following identification of the <italic>Streptococcus</italic> species, revealed that a variety of <italic>Streptococcus</italic> species in a cariogenic biofilm showed downregulation of genes encoding glucosyltransferases involved in the biosynthesis of water-soluble glucan. The expression of some genes encoding surface proteins was also downregulated. Of the two quorum sensing systems involved in the genus <italic>Streptococcus</italic>, the expression of <italic>luxS</italic> in three species, <italic>Streptococcus oralis</italic>, <italic>Streptococcus gordonii</italic>, and <italic>Streptococcus mutans</italic>, was significantly downregulated in the presence of 50 μM sulfated vizantin. Biofilm detachment may be facilitated by the reduced structural stability due to these modulations. As a non-specific reaction, 50 μM sulfated vizantin decreased cell surface hydrophobicity by binding to the cell surface, resulting in reduced bacterial adherence.


</sec><sec>
<title>Conclusion</title>
Sulfated vizantin may be a candidate for a new antibiofilm strategy targeting the biofilm matrix while preserving the resident microflora.


</sec>

DOI： 10.1186/s12866-020-02033-w

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Other Link： http://link.springer.com/article/10.1186/s12866-020-02033-w/fulltext.html

Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Following antimicrobial administrations in oral environments, bacteria become exposed to a sub-minimum inhibitory concentration (sub-MIC), which can induce in vitro single-species biofilms. This study explored the effects of chlorhexidine gluconate (CHG) at a sub-MIC on in vitro multi-species biofilms comprising Streptococcus mutans, Streptococcus oralis and Actinomyces naeslundii. CHG at a sub-MIC was found to induce in vitro biofilm growth, although the bacterial growth was not significantly different from that in the control. The gene transcription related to S. mutans multi-species biofilm formation with CHG at a sub-MIC was significantly higher than that of the control, but this was not found in S. mutans single-species biofilms. The bio-volume of extracellular polysaccharides with CHG at a sub-MIC was significantly higher than that of the control. This suggests that CHG at a sub-MIC may promote the development of multi-species biofilms by affecting the gene transcription related to S. mutans biofilm formation.

DOI： 10.1080/08927014.2020.1739271

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OBJECTIVES: To determine glucose transporter 1 (GLUT1) and runt-related transcription factor 2 (RUNX2) expression during reparative dentinogenesis after pulpotomy with mineral trioxide aggregate (MTA) capping. SUBJECTS AND METHODS: Eight-week-old male Wistar rats were used. Pulp of the upper left first molar was exposed and capped with MTA. The upper right first molar of the same animal was used as a control. After collecting molars at various time points, GLUT1, RUNX2 and mammalian target of rapamycin (MTOR) were examined by immunohistochemistry. mRNA levels of Slc2a1 (encoding GLUT1), Runx2, Nestin and Mtor were determined by real-time PCR. RESULTS: Pulp exhibited progressive formation of reparative dentine lined with GLUT1- and MTOR-immunoreactive odontoblast-like cells at 5 days after pulpotomy. RUNX2 was detected in nuclei of most pulp tissue cells at day 5 after pulpotomy. Double immunofluorescence staining revealed GLUT1 immunoreactivity on odontoblast-like cells positive for Nestin or RUNX2, 5 days after pulpotomy. Slc2a1, Runx2, Nestin and Mtor mRNA levels were significantly upregulated on days 3-5 after pulpotomy. CONCLUSIONS: After rat molar pulpotomy, dental pulp induced formation of reparative dentine with colocalization of GLUT1 and Nestin or RUNX2. Moreover, mRNA levels of Slc2a1, Runx2, Nestin and Mtor were significantly upregulated in pulpotomized dental pulp.

DOI： 10.1111/odi.13230

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

目的:機能性糖脂質ビザンチン(Viz-S)は,Streptococcus mutansバイオフィルムを易剥離性に変化させることで抗バイオフィルム作用を示す.本研究では,培養条件のうち,スクロース濃度を変化させたときのS.mutansの抗バイオフィルム効果,各種バイオフィルム形成関連遺伝子の転写量ならびにグルコシルトランスフェラーゼ(GTF)の発現について解析を行った.材料と方法:実験1・スクロース濃度を変化させたときのS.mutansの抗バイオフィルム効果;0.2,0.4,0.8%および1.6%のスクロース含有Brain Heart Infusion培地に,0,10,50μmol/lおよび75μmol/lのViz-Sを添加した.S.mutans UA159株を24時間嫌気培養することで,in vitroバイオフィルムモデルを作製した.リン酸緩衝生理食塩水(PBS)で2回洗浄したときの残存バイオフィルム量をクリスタルバイオレット法(CV法)により定量した.実験2・遺伝子発現動態の解析;0,10μmol/lおよび50μmol/lのViz-S存在下で24時間培養後のバイオフィルム形成菌を回収し,mRNAを抽出後,cDNAを合成した.バイオフィルム形成関連遺伝子の転写量をReal-time PCR法で解析した.実験3・ウエスタンブロッティング解析;in vitroバイオフィルムモデルを作製後,バイオフィルム形成菌と上清をそれぞれ回収した.タンパク質を抽出しSDS-PAGEを行った.疎水性膜に転写し,菌体結合型GTF(CA-GTF)抗体および遊離型GTF(CF-GTF)抗体を反応させた後,酵素標識二次抗体を反応させた.化学発光法により可視化し,タンパクの発現量を比較した.成績:10μmol/l Viz-S群のバイオフィルムは,PBSによる2回の洗浄により構造は変化しなかったが,50μmol/l Viz-S群は0.8%までのスクロース含有条件下においてバイオフィルム構造が93%減少した.1.6%のスクロース含有条件では32%のバイオフィルムが残存した.75μmol/l Viz-S群は,全スクロース濃度においてバイオフィルム形成が阻害された.10μmol/l Viz-S群は,すべての条件下において,遺伝子転写量の有意な変化はなかった.50μmol/l Viz-S群のgtfBおよびgtfC遺伝子の転写は,0.4%以上のスクロース含有条件で,コントロール群(Viz-S非含有)と比較して有意に増加したが,gtfDの転写は,すべてのスクロース含有群で0.46〜0.66倍に減少した.ウエスタンブロッティング解析で50μmol/l Viz-S群のGTFBおよびGTFCの産生量を比較したところ,コントロール群と比較して0.2%および0.4%スクロース含有条件下で有意に低下し,0.8%および1.6%スクロース含有条件下で有意に増加した.一方,GTFDは,コントロール群と比較して1/20以下に産生量が減少した.結論:50μmol/lのViz-Sは口腔粘膜への為害性が低く,殺菌ではない機序でS.mutansのバイオフィルムを剥離した.その機序の一つは,GTFDのタンパク発現を低下させることによるバイオフィルムの構造安定性の低下であった.75μmol/lのViz-Sはバイオフィルム形成を抑制した.(著者抄録)

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Macrophages are immune cells with high plasticity that perform many functions related to tissue injury and repair. They are generally categorized as 2 functional phenotypes: M1 (proinflammatory) and M2 (anti-inflammatory and prohealing). To investigate the role of macrophages in human dental pulp, we examined the localization and distributional alterations of macrophages in healthy dental pulp as well as during the reparative process of pulp capping with mineral trioxide aggregate (MTA) and in cariously inflamed pulp of adult human teeth. We also quantified the populations of M1/M2 macrophages in healthy dental pulp by flow cytometric analysis. CD68+CD86+ cells (M1 phenotype) and CD68+CD163+ cells (M2 phenotype) were 2.11% ± 0.50% and 44.99% ± 2.22%, respectively, of 2.96% ± 0.41% CD68+ cells (pan-macrophages) in whole healthy dental pulp. Interestingly, M2 phenotype macrophages were associated with Schwann cells in healthy pulp, during mineralized bridge formation, and in pulp with carious infections in vivo. Furthermore, the M2 macrophages associated with Schwann cells expressed brain-derived neurotrophic factor (BDNF) under all in vivo conditions. Moreover, we found that plasma cells expressed BDNF. Coculture of Schwann cells isolated from human dental pulp and human monocytic cell line THP-1 showed that Schwann cells induced M2 phenotypic polarization of THP-1 cell-derived macrophages. The THP-1 macrophages that maintained contact with Schwann cells were stimulated, leading to elongation of their cell shape and expression of M2 phenotype marker CD163 in cocultures. In summary, we revealed the spatiotemporal localization of macrophages and potent induction of the M2 phenotype by Schwann cells in human dental pulp. M2 macrophages protect neural elements, whereas M1 cells promote neuronal destruction. Therefore, suppressing the neurodestructive M1 phenotype and maintaining the neuroprotective M2 phenotype of macrophages by Schwann cells may be critical for development of effective treatment strategies to maintain the viability of highly innervated dental pulp.

DOI： 10.1177/0022034519894957

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INTRODUCTION: Pulp capping materials allow healing of injured pulp with a layer of reparative dentin. Glucose is needed to cure the injured area. Glucose is transported by glucose transporter (Glut) 2 and Glut4, which are transmembrane proteins that act as gatekeepers. We hypothesized that the transport of glucose via Glut2/Glut4 might contribute to the production of a dentin bridge during wound healing. Therefore, we explored Glut2 and Glut4 expression during reparative dentinogenesis after mineral trioxide aggregate capping. METHODS: The upper left first molar of 8-week-old Wistar rats underwent pulpotomy with mineral trioxide aggregate. At 1, 3, 5, 7, and 14 days after treatment, localization and colocalization of Glut2, Glut4, nestin (odontoblast marker), and antiendothelial cell antigen 1 (RECA-1; endothelial cell marker) were analyzed with immunohistochemical staining. Messenger RNA expression levels of Slc2a2 (encoding Glut2), Slc2a4 (encoding Glut4), Igf-1r (encoding insulinlike growth factor 1 receptor), and nestin were analyzed in the extracted teeth using real-time polymerase chain reaction. RESULTS: Glut2 and Glut4 were localized within odontoblasts and endothelial cells in normal control teeth. Three days after pulpotomy, Glut2- and Glut4-positive cells were detected; 7 days after pulpotomy, immunoreactivity for Glut2 and Glut4 was confined to newly differentiated odontoblastlike cells arranged beneath reparative dentin. Messenger RNA expression levels of Slc2a2 and Slc2a4 were significantly up-regulated after pulpotomy. CONCLUSIONS: Glut2 and Glut4 regulate glucose transport during wound healing beneath the injured area. This may contribute to the development of new vital pulp therapy for patients with deep caries.

DOI： 10.1016/j.joen.2019.10.003

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Streptococcus pneumoniae is often isolated from patients with community-acquired pneumonia. Antibiotics are the primary line of treatment for pneumococcal pneumonia; however, rising antimicrobial resistance is becoming more prevalent. Hinokitiol, which is isolated from trees in the cypress family, has been demonstrated to exert antibacterial activity against S. pneumoniae in vitro regardless of antimicrobial resistance. In this study, the efficacy of hinokitiol was investigated in a mouse pneumonia model. Male 8-week-old BALB/c mice were intratracheally infected with S. pneumoniae strains D39 (antimicrobial susceptible) and NU4471 (macrolide resistant). After 1 h, hinokitiol was injected via the tracheal route. Hinokitiol significantly decreased the number of S. pneumoniae in the bronchoalveolar lavage fluid (BALF) and the concentration of pneumococcal DNA in the serum, regardless of whether bacteria were resistant or susceptible to macrolides. In addition, hinokitiol decreased the infiltration of neutrophils in the lungs, as well as the concentration of inflammatory cytokines in the BALF and serum. Repeated hinokitiol injection at 18 h intervals showed downward trend in the number of S. pneumoniae in the BALF and the concentration of S. pneumoniae DNA in the serum with the number of hinokitiol administrations. These findings suggest that hinokitiol reduced bacterial load and suppressed excessive host immune response in the pneumonia mouse model. Accordingly, hinokitiol warrants further exploration as a potential candidate for the treatment of pneumococcal pneumonia.

DOI： 10.1371/journal.pone.0240329

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Language：Japanese   Publisher：The Japanese Society of Conservative Dentistry  

<p> Purpose: The purpose of this study was to elucidate the mechanism of preventing <i>Streptococcus mutans</i> from bacterial adhesion by sulfated vizantin (Viz-S). The change of surface property and transcriptions of genes related to bacterial adhesion were analyzed.</p><p> Methods: Bacterial suspension of <i>S. mutans</i> in phosphate buffer saline was reacted with Viz-S at the concentration of 0, 50 and 75 μmol/<i>l</i> for 10 min, respectively. 200 μ<i>l</i> of n-Hexadecane was added to 1 m<i>l</i> of each bacterial suspension, and the solution was stirred vigorously for 1 min. The hydrophobicity was assessed by the microbial adhesion to hydrocarbon test (MATH test). ZsGreen-tagged <i>S. mutans</i> was added in a brain-heart infusion broth (BHI) supplemented with 0.2, 0.4, 0.8 and 1.6% sucrose containing Viz-S at the concentration of 0, 50 and 75 μmol/<i>l</i>. Equal volumes of absorbance-adjusted suspensions were inoculated into a coverglass chamber coated with human saliva, followed by incubation for 1 h at 37°C statically. The number of microorganisms adhered on the surface was analyzed using a confocal laser scanning microscope. The transcriptions of genes related to bacterial adhesion in <i>S. mutans</i> incubated in the absence or presence of either 50 or 75 μmol/<i>l</i> Viz-S for 4 h were investigated using real-time polymerase chain reaction.</p><p> Results: The hydrophobicity was 57.5±13.4 for the control group, 29.4±10.4 for the 50 μmol/<i>l</i> group and 14.9±7.0 for the 75 μmol/<i>l</i> group. Adherence of <i>S. mutans</i> in the experimental group was significantly decreased in comparison with that of the control group, showing a decrease ratio of 1/8 to 1/10 for the 50 μmol/<i>l</i> group and 1/40 to 1/55 for the 75 μmol/<i>l</i> group, respectively. The expression of <i>gtfD</i> was significantly downregulated in the presence of Viz-S, with 50 μmol/<i>l</i> Viz-S by 0.38 to 0.5-fold, and 75 μmol/<i>l</i> Viz-S by 0.33 to 0.36-fold. The expression of genes that encode bacterial surface protein was not significantly changed.</p><p> Conclusion: 75 μmol/<i>l</i> Viz-S inhibits the bacterial adhesion of <i>S. mutans</i> by the mechanisms of altered surface property and the downregulation of <i>gtfD</i> gene transcription.</p>

DOI： 10.11471/shikahozon.63.173

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INTRODUCTION: This study aimed to develop a technique to create sequential slices, allowing the fluorescent visualization of bacterial viability in all parts of an infected dentin. METHODS: Cylindrical dentin blocks were prepared from freshly extracted human teeth with a single-rooted canal. Each block was immersed in 5% sodium hypochlorite (NaOCl) and 17% EDTA for 5 minutes before being infected with Enterococcus faecalis. The bacteria were allowed to develop inside dentin specimens for 28 days under anaerobic conditions. The specimens were exposed in 2% NaOCl for either 2 minutes or 20 minutes at 20°C, 37°C, and 45°C, respectively. After staining with calcein AM (Thermo Fisher Scientific, Waltham, MA) and propidium iodide, the samples were cryoembedded, mounted on an adhesive film, and sectioned at a thickness of 10 μm along the running of the dentinal tubules. Stacks of fluorescent images were collected in the z dimension using confocal laser scanning microscopy, and the maximum affected distance from a root canal was measured from the 3-dimensional reconstructed image. The reliability of this technique was verified by comparison with a dye bleaching test. RESULTS: Horizontal sequential sections preserving 3-dimensional bacterial distribution and their viabilities could be made without decalcification. The treatment time contributed to the penetration of NaOCl into dentinal tubules, whereas temperature did not significantly affect the penetration. The judgment by confocal laser scanning microscopic analysis was consistent with that of a dye bleaching test. CONCLUSIONS: The horizontal sectioning method has the advantage of creating sequential sections, allowing information to be imaged at every portion.

DOI： 10.1016/j.joen.2019.06.004

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AIM: To explore the expression profile of CD45+/pro-collagen I+ fibrocytes in intact dental pulps as well as during wound healing in adult dental pulp tissue. METHODOLOGY: A total of 16 healthy permanent teeth were obtained from young patients (18 to 25 years) undergoing orthodontic treatment. Routine pulp capping with mineral trioxide aggregate (MTA) was performed under local anaesthesia to induce a mineralized barrier at the exposed surface. Teeth were extracted from patients after 7, 14 and 35 days. Sections of the extracted teeth were prepared and stained for various markers using indirect immunofluorescence. Fibrocytes were counted, and the data were statistically evaluated using the Dunnett test. RESULTS: In uninflammed pulp tissue, a pro-collagen I-positive reaction was detected in odontoblasts, as well as in perivascular cells. Most of the CD45-positive cells were negative for pro-collagen I in normal pulp tissue, whereas CD45+/pro-collagen I+ fibrocytes were detected 7 days after injury. At day 14, fibrocytes were recognized under the fibrous matrix in contact with MTA and had infiltrated into regions of new capillary formation, where the fibrocytes were positively stained for vascular endothelial growth factor. By 35 days, fibrocytes were few, coincident with the formation of dentine bridges. The number of fibrocytes peaked 7 days post-injury and decreased at 14 days. CONCLUSIONS: The presence of fibrocytes in human pulp wound healing was observed. The spatiotemporal distribution of fibrocytes suggests that fibrocytes are involved in the early stages of pulp wound healing, specifically by contributing to new blood vessel formation.

DOI： 10.1111/iej.12940

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Background: Bacterial biofilms that develop on root surfaces outside apical foramens have been found to be associated with refractory periapical periodontitis. However, several other factors cause endodontic failures apart from extraradicular biofilms. The aim of this study was to identify the factors causing endodontic failures in general practices in Japan. Methods: Patients diagnosed as having refractory periapical periodontitis by general practitioners and who requested endodontic treatment at Osaka University Dental Hospital were selected by checking medical records from April 2009 to March 2013. Factors causing endodontic failures were identified. Results: A total of 103 teeth were selected, and 76 teeth completed root-canal treatment. Tooth extractions were required for 18 teeth after or without endodontic treatment. Six teeth required apicoectomy after endodontic treatment. One tooth needed hemisection. One tooth needed intentional replantation. One tooth needed adhesion and replantation. The main causes of treatment failure were open apices (24 teeth), perforation (18 teeth), and root fracture (13 teeth). In six teeth with open apices that required apicoectomy or extraction, extraradicular biofilms may have been related to endodontic failure. Conclusions: Most endodontic cases diagnosed with refractory periapical periodontitis by general practitioners were compromised by any other factors rather than extraradicular biofilms.

DOI： 10.1186/s12903-018-0530-6

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Background: Chlorhexidine gluconate (CHG) has been proven to be effective in preventing and controlling biofilm formation. At the same time, an increase in calculus formation is known as one of considerable side effects. The purpose of this study was to investigate whether mineral deposition preceding a calculus formation would occur at an early stage after the use of CHG using an in vitro saliva-related biofilm model. Methods: Biofilms were developed on the MBEC™ device in brain heart infusion (BHI) broth containing 0.5% sucrose at 37 °C for 3 days under anaerobic conditions. Biofilms were periodically exposed to 1 min applications of 0.12% CHG every 12 h and incubated for up to 2 days in BHI containing a calcifying solution. Calcium and phosphate in the biofilm were measured using atomic absorption spectrophotometry and a phosphate assay kit, respectively. Morphological structure was observed using a scanning electron microscope (SEM), and chemical composition was analyzed with an electron probe microanalyzer (EPMA). Results: The concentrations of Ca and Pi following a single exposure to CHG increased significantly compared with the control. Repeatedly exposing biofilms to CHG dose-dependently increased Ca deposition, and the amount of Ca was five times as much as that of the control. Pi levels in CHG-treated biofilms were significantly higher than those from the control group (p &lt
0.05)
however, the influence of the number of exposures was limited. Analyses using an SEM and EPMA showed many clusters containing calcium and phosphate complexes in CHG-treated biofilms. Upon composition analysis of the clusters, calcium was detected at a greater concentration than phosphate. Conclusions: Findings suggested that CHG may promote mineral uptake into the biofilm soon after its use. It is necessary to disrupt the biofilm prior to the start of a CHG mouthwash in order to reduce the side effects associated with this procedure. The management of patients is also important.

DOI： 10.1186/s12903-018-0517-3

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nature Publishing Group  

Root canal treatment is performed to treat apical periodontitis, and various procedures and techniques are currently used. Although animal models have been used in the developmental research of root canal treatment, little of this research has used small animals such as rats, because of their small size. In this study, root canal treatment was performed on the rat mandibular first molar, which had four root canals, using a microscope, and the therapeutic effect was evaluated bacteriologically, radiologically and histopathologically. By performing root canal treatment, the level of bacteria in the mesial root of the treated teeth was reduced by 75% compared with the control. Additionally, the volume of the periapical lesions of the treated teeth as measured by micro-computed tomography decreased significantly 2 weeks after the root canal treatment when compared with the control. Histological evidence of healing was observed in the treatment group 8 weeks after root canal treatment. These results suggest that a root canal treatment model using rats can be used in developmental research for novel methods of root canal treatment.

DOI： 10.1038/s41598-017-03628-6

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Porhyromonas gingivalis, a causative bacterium of periodontitis, is implicated in the etiology of rheumatoid arthritis (RA), mainly because of expressing peptidyl arginine deiminase (PAD) that generates RA-related autoantigens. However, compared with other periodontopathic bacteria, the precise role of P. gingivalis in RA is largely unknown. We found that orally administered P. gingivalis changed the gut microbiome with concomitant elevation of serum endotoxin and inflammatory markers, and impairment of the gut barrier function. Based on findings showing a relationship between gut microbiota and RA, we investigated whether the change of gut microbiota induced by P. gingivalis and Prevotella intermedia, another periodontopathic bacterium without PAD, is associated with collagen-induced arthritis (CIA). DBA/1J mice were orally administered with or without bacteria followed by induction of CIA. P. gingivalis, but not P. intermedia, administration significantly aggravated arthritis with increased interleukin-17 levels in sera and culture supernatants, increased Th17 cell proportions among mesenteric lymphocytes, and a significant change in the gut microbiome. However, P. gingivalis administration did not elevate the level of anti-citrullinated protein antibody. These results suggest a unique role of P. gingivalis in the link between periodontitis and RA by affecting the gut immune system and the gut microbiota composition.

DOI： 10.1038/s41598-017-07196-7

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AimTo evaluate the Ca2+-releasing, alkalizing and apatite-like surface precipitate-forming abilities of a prototype tricalcium silicate cement, which was mainly composed of synthetically prepared tricalcium silicate and zirconium oxide radiopacifier.
MethodologyThe prototype tricalcium silicate cement, white ProRoot MTA (WMTA) and TheraCal LC (a light-cured resin-modified calcium silicate-filled material) were examined. The chemical compositions were analysed with a wavelength-dispersive X-ray spectroscopy electron probe microanalyser with an image observation function (SEM-EPMA). The pH and Ca2+ concentrations of water in which the set materials had been immersed were measured, and the latter was assessed with the EDTA titration method. The surface precipitates formed on the materials immersed in phosphate-buffered saline (PBS) were analysed with SEM-EPMA and X-ray diffraction (XRD). Kruskal-Wallis tests followed by Mann-Whitney U-test with Bonferroni correction were used for statistical analysis (=0.05).
ResultsThe prototype cement contained Ca, Si and Zr as major elemental constituents, whereas it did not contain some metal elements that were detected in the other materials. The Ca2+ concentrations and pH of the immersion water samples exhibited the following order: WMTA=prototype cement&gt;TheraCal LC (P&lt;0.05). All three materials produced Ca- and P-containing surface precipitates after PBS immersion, and the precipitates produced by TheraCal LC displayed lower Ca/P ratios than those formed by the other materials. XRD peaks corresponding to hydroxyapatite were detected in the precipitates produced by the prototype cement and WMTA.
ConclusionThe prototype tricalcium silicate cement exhibited similar Ca2+-releasing, alkalizing and apatite-like precipitate-forming abilities to WMTA. The Ca2+-releasing, alkalizing and apatite-like precipitate-forming abilities of TheraCal LC were lower than those of the other materials.

DOI： 10.1111/iej.12737

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DOI： 10.26420/jdentoraldisord.2017.1073

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Mineral trioxide aggregate (MTA) is a commonly used dental pulp-capping material with known effects in promoting reparative dentinogenesis. However, the mechanism by which MTA induces dentine repair remains unclear. The aim of the present study was to investigate the role of prostaglandin E2 (PGE2) in dentine repair by examining the localisation and mRNA expression levels of its transporter (Pgt) and two of its receptors (Ep2 and Ep4) in a rat model of pulpotomy with MTA capping. Ep2 expression was detected in odontoblasts, endothelial cells, and nerve fibres in normal and pulpotomised tissues, whereas Pgt and Ep4 were immunolocalised only in the odontoblasts. Moreover, mRNA expression of Slco2a1 (encoding Pgt), Ptger2 (encoding Ep2), and Ptger4 (encoding Ep4) was significantly upregulated in pulpotomised dental pulp and trigeminal ganglia after MTA capping. Our results provide insights into the functions of PGE2 via Pgt and Ep receptors in the healing dentine/pulp complex and may be helpful in developing new therapeutic targets for dental disease.

DOI： 10.1038/s41598-017-07167-y

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Introduction: Myofibroblasts express alpha smooth muscle actin (alpha-SMA) and play a critical role in wound healing. Myofibroblast differentiation is controlled by the joint actions of transforming growth factor beta 1 (TGF-beta 1) and the extradomain A fibronectin splice variant (EDA-FN). Currently, the contribution of myofi-broblasts to dental pulp healing is unknown. Therefore, we analyzed expressional characteristics of alpha-SMA positive cells and investigated TGF-beta 1, EDA-FN, and alpha-SMA expression levels after pulpotomy to better understand dental pulp healing. Methods: The maxillary first molars of 8-week-old Wistar rats were pulpotomized with mineral trioxide aggregate. After 1 to 14 days, localization and colocalization of alpha-SMA, rat endothelial cell antigen-1 (as a marker of endothelial cells), neuron-glial antigen 2 (as a marker of perivascular cells), prolyl-4-hydroxylase (P4H, as an additional marker of myofibroblasts), and EDA-FN were analyzed using immunohistochemistry and double immunofluorescence. Time-course changes in the messenger RNA expression levels of TGF-beta 1, EDA-FN, and alpha-SMA were evaluated using quantitative real-time polymerase chain reaction analysis. Results: Spindle-shaped alpha-SMA positive cells transiently appeared after pulpotomy. These cells initially emerged in the pulp core on day 3 and then accumulated at the wound site by day 5. These cells were isolated from rat endothelial cell antigen-1 positive cells and did not express neuron-glial antigen 2 but did express P4H. The messenger RNA levels of TGF-beta 1, EDA-FN, and alpha-SMA were significantly up-regulated after pulpotomy. EDA-FN and a-SMA were colocalized at the wound sites on day 5. Conclusions: In association with up regulation of TGF-beta 1 and EDA-FN expression, alpha-SMA and P4H double-positive cells accumulated at the wound sites after pulpotomy. This suggests that myofibroblasts participate in dental pulp healing.

DOI： 10.1016/j.joen.2017.02.018

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Eikenella corrodens 1073 was found to show hemolytic activity when grown on sheep blood agar. A high and dose-dependent hemolytic activity was detected in the cell envelope fraction, which was further purified by ion-exchange and gel-filtration chromatography. Consequently, a 65-kDa protein with hemolytic activity was obtained, suggesting that this protein might be a hemolysin. Its N-terminal amino acid sequence was nearly identical to that of X-prolyl aminopeptidase from E. corrodens ATCC 23834. To confirm that X-prolyl aminopeptidase functions as a hemolytic factor, we expressed the hlyA gene, encoding X-prolyl aminopeptidase, in Escherichia coli. After induction with isopropyl -D-1-thiogalactopyranoside, a protein of about 65 kDa was purified on a Ni column, and its hemolytic activity was confirmed. Meanwhile, a strain with a disrupted hlyA gene, which was constructed by homologous recombination, did not show any hemolytic activity. These results suggested that X-prolyl aminopeptidase might function as a hemolysin in E. corrodens.

DOI： 10.1080/09168451.2017.1295807

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An ideal antibiofilm strategy is to control both in the quality and quantity of biofilm while maintaining the benefits derived from resident microflora. Vizantin, a recently developed immunostimulating compound, has also been found to have antibiofilm property. This study evaluated the influence on biofilm formation of Streptococcus mutans in the presence of sulfated vizantin and biofilm development following bacterial adhesion on a hydroxyapatite disc coated with sulfated vizantin. Supplementation with sulfated vizantin up to 50 mu M did not affect either bacterial growth or biofilm formation, whereas 50 mu M sulfated vizantin caused the biofilm to readily detach from the surface. Sulfated vizantin at the concentration of 50 mu M upregulated the expression of the gtfB and gtfC genes, but downregulated the expression of the gtfD gene, suggesting altered architecture in the biofilm. Biofilm development on the surface coated with sulfated vizantin was inhibited depending on the concentration, suggesting prevention from bacterial adhesion. Among eight genes related to bacterial adherence in S. mutans, expression of gtfB and gtfC was significantly upregulated, whereas the expression of gtfD, GbpA and GbpC was downregulated according to the concentration of vizantin, especially with 50 mu M vizantin by 0.8-, 0.4-, and 0.4-fold, respectively. These findings suggest that sulfated vizantin may cause structural degradation as a result of changing gene regulation related to bacterial adhesion and glucan production of S. mutans. (C) 2016 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2016.10.021

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nature Publishing Group  

Numerous studies on oral biofilms have been performed in vitro, although it is difficult to mimic the oral environment. Here we used an in situ model to conduct a quantitative analysis and comprehensive identification of bacterial communities over time by performing deep sequencing of 16S rRNA genes. We show here that the number of viable bacteria in supragingival biofilms increased in two steps. Using scanning and transmission electron microscopy, as well as confocal laser scanning microscopy, we detected gram-positive cocci during the first 8 h. The biofilm was subsequently covered with a thick matrix-like structure composed of different bacterial morphotypes that diversified as the number of bacteria increased. Streptococcus accounted for 420% of the population until 16 h, and obligate anaerobes such as Fusobacterium, Prevotella and Porphyromonas predominated after 48 h, and this increase was statistically significant after 96 h (Po0.05). Together, our data demonstrate that an initial population of facultative anaerobic bacteria was replaced with a population of gram-negative anaerobic bacteria during oral biofilm formation. This study, therefore, contributes to a comprehensive understanding of the composition of the bacterial microbiota involved in the health of the human oral cavity.

DOI： 10.1038/npjbiofilms.2016.18

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Microbes commonly adhere to surfaces, aggregate in self-produced extracellular polymeric substances (EPS) and live in biofilms. Periodontitis is a serious oral infection that is initiated by the formation of biofilms by Porphyromonas gingivalis. EPS act as a barrier that protects biofilmforming cells against sources of stress, including those induced by host immune cells and antimicrobial agents. Therefore, drugs intended to kill such micro-organisms cannot be used for the treatment of biofilm infections. Our previous studies revealed that subminimal inhibitory concentrations (subMIC) of two macrolide antibiotics (azithromycin, AZM and erythromycin, ERY) reduced P. gingivalis biofilms. Furthermore, we demonstrated that the Bacillus subtilis sinR orthologue (PGN_0088) inhibits the synthesis of carbohydrates that are components of EPS in P. gingivalis biofilms. Here, we constructed a novel sinR mutant from P. gingivalis ATCC 33277 and reveal that the increased abundance of carbohydrate in EPS of the mutant led to a reduced infiltration rate of AZM and FRY through EPS, and consequently elevated biofilm resistance to these macrolides. Detailed elucidation of the interaction between the product of the sinR gene and EPS will assist in the development of novel approaches that target EPS to prevent and inhibit the formation of biofilms.

DOI： 10.1099/mic.0.000013

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Scanning electron microscopy (SEM) has been successfully used to image biofilms because of its high resolution and magnification. However, conventional SEM requires dehydration and metal coating of biological samples before observation, and because biofilms consist mainly of water, sample dehydration may influence the biofilm structure. When coated with an ionic liquid, which is a kind of salt that exists in the liquid state at room temperature, biological samples for SEM observation do not require dehydration or metal coating because ionic liquids do not evaporate under vacuum conditions and are electrically conductive. This study investigates the ability of ionic liquids to allow SEM observation of Streptococcus mutans biofilms compared with conventional coating methods. Two hydrophilic and two hydrophobic ionic liquids, all of which are electronic conductors, are used. Compared with samples prepared by the conventional method, the ionic-liquid-treated samples do not exhibit a fibrous extracellular matrix structure and cracking on the biofilm surface. The hydrophilic ionic liquids give clearer images of the biofilm structure than those of the hydrophobic ionic liquids. This study finds that ionic liquids are useful for allowing the observation of biofilms by SEM without preparation by dehydration and metal coating.

DOI： 10.1186/s13568-015-0097-4

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Although extraradicular biofilm formation is related to refractory periapical periodontitis, the mechanism of extraradicular biofilm development, as well as its effect on periapical lesions, is unknown. Therefore, we aimed to develop an in vivo extraradicular biofilm model in rats and to identify and quantify extraradicular biofilm-forming bacteria while investigating the effect of extraradicular biofilms on periapical lesions. Periapical lesions were induced by exposing the pulpal tissue of the mandibular first molars of male Wistar rats to their oral environment. Four weeks later, gutta-percha points were excessively inserted into the mesial root canals of the right first molars (experimental sites) but not the left first molars (control sites). After 6 and 8 weeks of pulp exposure, the presence of extraradicular biofilms was confirmed histomorphologically, and biofilm-forming bacteria were identified by using classical culture methods. The biofilms were observed in the extraradicular area of the experimental sites. Similar species were detected both inside and outside the root canals. The bacterial count, quantified by real-time PCR assays, in the extraradicular area gradually increased in the experimental sites until 20 weeks after pulp exposure. After 8 weeks of pulp exposure, the periapical lesion volume that was measured by micro-computed tomography was significantly larger in the experimental sites than in the control sites (P &lt; 0.05 by Welch's t test). These results suggest that we developed an extraradicular biofilm model in rats and that extraradicular biofilms affect developing periapical lesions.

DOI： 10.1128/AEM.00421-14

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Aims The aim of this study was to investigate the effects of tea catechin epigallocatechin gallate (EGCg) on established biofilms and biofilm formation by Porphyromonas gingivalis, a major pathogen of periodontal disease. Methods and Results Biofilm cell survival was measured using adenosine triphosphate (ATP) bioluminescence. In the presence of EGCg, the ATP level in cells of established biofilms was significantly decreased compared to the controls (P&lt;0 center dot 0001). Transmission electron microscopy revealed that EGCg damaged the cell membrane and cell wall of P.gingivalis. Confocal laser-scanning microscopy revealed that the proportion of dead cells was higher in biofilms treated with EGCg. Moreover, the effects of subminimal inhibitory concentrations (MICs) of EGCg on P.gingivalis biofilm formation were dose-dependent (P&lt;0 center dot 0001). Conclusion Our results suggest that EGCg destroys established P.gingivalis biofilms and inhibits biofilm formation. Significance and Impact of the Study Development of chemical control agents against oral biofilms is necessary, because oral biofilms can be only removed using mechanical debridement. This article indicates that EGCg may represent a novel antibiofilm agent that prevents infections involving bacterial biofilms such as periodontitis.

DOI： 10.1111/jam.12458

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Previously, we reported that biofilm formation of Eikenella corrodens is regulated by autoinducer-2 (AI-2), based on observations that biofilm-forming efficiency of δluxS mutant was greater than that of the wild type (Azakami etal., J. Biosci. Bioeng., 102, 110-117, 2006). To determine whether the AI-2 molecule affects biofilm formation directly, we added purified AI-2 to luxS mutant and wild-type E.corrodens and compared biofilm formations by using a static assay. Results indicated that biofilm formation in E.corrodens was enhanced by the addition of AI-2. We also compared the biofilms formed by flow cell system for the luxS mutant and the wild type by using scanning electron microscopy and confocal laser scanning microscopy. The number of viable bacteria in the luxS mutant biofilm was dramatically reduced and more sparsely distributed than that of the wild type, which suggested that AI-2 might enhance the mature biofilm. Conversely, further analysis by modified confocal reflection microscopy indicated that the wild-type biofilm was matured earlier than that of the luxS mutant, and became thinner and more sparsely distributed with time. These data suggest that LuxS may facilitate the maturation and detachment of biofilm in E.corrodens. © 2013 The Society for Biotechnology, Japan.

DOI： 10.1016/j.jbiosc.2013.03.013

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Chlorhexidine (CHX) gluconate effectively reduces the viability of biofilm-forming bacteria, such as Porphyromonas gingivalis. However, it is impossible to completely remove biofilms. The goal of the present study was to assess the potential pathogenicity of residual P. gingivalis biofilms in vitro after treatment with CHX gluconate. Scanning and transmission electron microscopy and confocal laser imaging revealed that treatment with CHX gluconate disrupted individual biofilm-forming P. gingivalis cells but did not destroy the biofilms. The volumes of the protein and carbohydrate constituents in the residual biofilms were not significantly different from those of the controls. The physical resistance of the residual biofilms to ultrasonication was significantly higher than that of controls. The volume of P. gingivalis adherent to the residual biofilms was higher than that to saliva-coated wells. These findings suggest that although CHX gluconate caused disruption of biofilm-forming cells, the constituents derived from disrupted cells were maintained in the biofilms, which sustained their external structures. Moreover, the residual biofilms could serve as a scaffold for the formation of new biofilms. © 2013 Eur J Oral Sci.

DOI： 10.1111/eos.12050

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Biofilm-forming cells are distinct from well characterized planktonic cells and aggregate in the extracellular matrix, the so-called extracellular polymeric substances (EPS). The sinR gene of Bacillus subtilis encodes a transcriptional regulator that is known to be involved in the biosynthesis of EPS in biofilms. Porphyromonas gingivalis inhabits the subgingival and extraradicular biofilm of humans and is one of the primary pathogens that cause progressive marginal and refractory apical periodontitis. Furthermore, P. gingivalis possesses PGN_0088, which encodes a putative ortholog of B. subtilis sinR. Here, we investigated the role of PGN_0088 (sinR) on biofilm formation. P. gingivalis strains formed biofilms on saliva-coated glass surfaces in phosphate buffered saline. Quantitative analysis indicated that the biofilm of the sinR null mutant consisted of dense exopolysaccharide. Microscopic observations showed that the increased levels of exopolysaccharide produced by the sinR mutant changed the morphology of the EPS to a mesh-liked structure. Furthermore, physical analyses suggested that the enrichment of exopolysaccharide in the EPS enhanced the resistance of the biofilm to hydrodynamic shear force. The results presented here demonstrate sinR plays important roles in the ability of P. gingivalis strain ATCC 33277 to act as a negative mediator of exopolysaccharide accumulation and is indirectly associated with the structure of the EPS and the force of its adhesion to surfaces.

DOI： 10.1371/journal.pone.0056017

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Eikenella corrodens produces autoinducer-2 (AI-2) in the mid log phase, and AI-2 activity decreases dramatically during the stationary phase. We investigated the mechanism underlying this decrease in AI-2 activity. To analyze the mechanism, we extracted and purified AI-2 from the supernatant of mid-log-phase culture. Simultaneously, the stationary-phase culture supernatant was fractionated by ammonium sulfate precipitation. On incubating purified AI-2 and 4-hydroxy-5-methyl-3(2H)- furanone (MHF) with each fraction, the 30% fraction decreased both AI-2 and MHF activities. The data suggest that AI-2 and MHF were rendered inactive in the same manner. Heat and/or trypsin treatment of the 30% fraction did not completely arrest AI-2 inactivation, suggesting that partially heat-stable proteins are involved in AI-2 inactivation. We observed that an enzyme converted MHF to another form. This suggests that E. corrodens produces an AI-2 inactivating enzyme, and that AI-2 can be degraded or modified by it.

DOI： 10.1271/bbb.130047

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Aims: To investigate the effects of the combined application of an N-acyl homoserine lactone (HSL) analog and antibiotics on biofilms of Porphyromonas gingivalis, a major pathogen of periodontal disease. Methods and Results: Antibiotics used were cefuroxime, ofloxacin and minocycline. A flow-cell model was used for biofilm formation. Samples were divided into four groups: control, analog-treated, antibiotic-treated and combined application groups. Biofilm cell survival was determined using adenosine triphosphate (ATP) bioluminescence and confocal laser microscopy (CLSM). In the combined application group, the ATP count in biofilm cells was significantly decreased compared with the antibiotic-treated group (Games-Howell test, P &lt; 0 05). A combination of cefuroxime and the analog was most effective against the P. gingivalis biofilm. CLSM observations revealed that the proportion of dead cells was highest in the combined application group. Conclusions: The combined application of the N-acyl HSL analog and antibiotics was effective at reducing the viability of P. gingivalis cells in biofilms. Significance and Impact of the Study: The combined application of the N-acyl HSL analog and antibiotics may be successful for eradicating infections involving bacterial biofilms, such as periodontitis.

DOI： 10.1111/j.1365-2672.2011.05194.x

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Antibiotic resistance of biofilm-grown bacteria contributes to chronic infections, such as marginal and periapical periodontitis, which are strongly associated with Porphyromonas gingivalis. Concurrent azithromycin (AZM) administration and mechanical debridement improve the clinical parameters of periodontal tissue in situ. We examined the in vitro efficacy of AZM against P. gingivalis biofilms. The susceptibilities of adherent P. gingivalis strains 381, HW24D1, 6/26, and W83 to AZM, erythromycin (ERY), ampicillin (AMP), ofloxacin (OFX), and gentamicin (GEN) were investigated using a static model. The optical densities of adherent P. gingivalis cells were significantly decreased by using AZM and ERY at sub-MIC levels compared with those of the controls in all the strains tested, except for the effect of ERY on strain W83. AMP and OFX inhibited P. gingivalis adherent cells at levels over their MICs, and GEN showed no inhibition in the static model. The effects of AZM and ERY against biofilm cells were investigated using a flow cell model. The ATP levels of P. gingivalis biofilms were significantly decreased by AZM at concentrations below the sub-MICs; however, ERY was not effective for inhibition of P. gingivalis biofilm cells at their sub-MICs. Furthermore, decreased density of P. gingivalis biofilms was observed three-dimensionally with sub-MIC AZM, using confocal laser scanning microscopy. These findings suggest that AZM is effective against P. gingivalis biofilms at sub-MIC levels and could have future clinical application for oral biofilm infections, such as chronic marginal and periapical periodontitis.

DOI： 10.1128/AAC.05169-11

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Chronological gene expression patterns of biofilm-forming cells are important to understand bioactivity and pathogenicity of biofilms. For Porphyromonas gingivalis ATCC 33277 biofilm formation, the number of genes differentially regulated by more than 1.5-fold was highest during the growth stage (312/2,090 genes), and some pathogen-associated genes were time-dependently controlled.

DOI： 10.1128/AEM.00746-11

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The periodontopathogenic bacterium Eikenella corrodens has an N-acetyl-D-galactosamine (GalNAc)-specific lectin, that contributes significantly to the pathogenicity of the bacterium. Recently, we reported that plasmid-mediated genomic recombination enhances the activity of this lectin. In this study, we investigated the effects of genomic recombination on certain virulence factors. Introduction of the recombinase gene resulted in hemolysis and significantly increased bacterial adhesion to epithelial cells. It was suggested that the enhanced adhesion was attributable to increased lectin activity due to genomic recombination, because it was inhibited by the addition of GalNAc. In contrast, invasion of the epithelial cells was remarkably reduced by genomic recombination. Although we assumed that this decrease in invasion resulted from a loss of type-IV pili, the phase variant did not show any decrease in invasion activity. This suggests that type-IV pili do not contribute to the invasive ability of E. corrodens. Our results suggest that genomic recombination enhances the pathogenicity of E. corrodens.

DOI： 10.1271/bbb.100866

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Eikenella corrodens is a periodontopathogenic bacterium that forms biofilm even by itself. In this study, we investigated the inhibitory effects of catechins on E. corrodens biofilm formation. Biofilm formation was inhibited by the addition of 1 mM of the catechins with the pyrogallol-type B-ring and/or the galloyl group. The catechins with the galloyl group were effective at smaller doses than those with only the pyrogallol-type B-ring. An inhibitory effect was observed even when these catechins and gallic acid were added at subminimal inhibitory concentration (MIC) or at concentrations that showed no bactericidal effect. These results suggest that some catechins at sub-MIC might inhibit biofilm formation. No inhibitory effect of catechins at sub-MIC on biofilm formation was observed in the luxS deletion mutant. Our studies suggest that some species of catechins with the galloyl group affect autoinducer 2-mediated quorum sensing and thereby inhibit biofilm formation by E. corrodens.

DOI： 10.1271/bbb.100499

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The Gram-negative anaerobic bacterium Porphyromonas gingivalis is a major pathogen in periodontal disease, one of the biofilm-caused infectious diseases. The bacterium possesses potential virulence factors, including fimbriae, proteinases, hemagglutinin, lipopolysaccharide (LPS), and outer membrane vesicles, and some of these factors are associated with biofilm formation; however, the precise mechanism of biofilm formation is still unknown. Colonial pigmentation of the bacterium on blood agar plates is related to its virulence. In this study, we isolated a nonpigmented mutant that had an insertion mutation within the new gene PGN_1251 (gtfB) by screening a transposon insertion library. The gene shares homology with genes encoding glycosyltransferase 1 of several bacteria. The gtfB mutant was defective in biosynthesis of both LPSs containing O side chain polysaccharide (O-LPS) and anionic polysaccharide (A-LPS). The defect in the gene resulted in a complete loss of surface-associated gingipain proteinases, strong autoaggregation, and a marked increase in biofilm formation, suggesting that polysaccharide portions of LPSs influence attachment of gingipain proteinases to the cell surface, autoaggregation, and biofilm formation of P. gingivalis.

DOI： 10.1128/IAI.00071-10

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P&gt;Aim
To investigate the antibacterial effects of 12-methacryloyloxydodecylpyridinium bromide (MDPB), an antibacterial monomer synthesized by combining quaternary ammonium with a methacryloyl group, against three anaerobes associated with endodontic infections using planktonic and biofilm cells.
Methodology
The antibacterial activity of unpolymerized MDPB against Enterococcus faecalis, Fusobacterium nucleatum and Prevotella nigrescens was examined by agar-disc diffusion tests and determination of the minimum inhibitory/bactericidal concentrations (MIC/MBC). Rapid killing effects of MDPB against three bacteria in planktonic form were examined by a cell number counting method, and those against biofilm cells were assessed by a viability staining method.
Results
MDPB demonstrated inhibition against all of the bacteria tested by agar-disc diffusion tests. The MIC/MBC values of MDPB for the three anaerobes were much smaller than those of other resin monomers, although greater compared with those of cetylpyridinium chloride or chlorhexidine diacetate for E. faecalis and F. nucleatum. Significant reduction in viable planktonic cells was obtained by contact with 250 mu g mL-1 of MDPB for 20 s (P &lt; 0.05, Fisher&apos;s PLSD tests), and 40 s contact with 500 mu g mL-1 or 20 s contact with 1000 mu g mL-1 of MDPB resulted in more than 90% killing. Biofilm cells of all species were completely killed by application of 1000 mu g mL-1 of MDPB for 60 s.
Conclusion
MDPB was found to have strong antibacterial effects against E. faecalis, F. nucleatum and P. nigrescens, and such effects were rapidly exhibited even against biofilm cells, suggesting the usefulness of application of MDPB to resin-based materials for root canal filling.

DOI： 10.1111/j.1365-2591.2010.01716.x

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Background and Objective:
The gram-negative anaerobic rod Porphyromonas gingivalis in oral biofilms is a primary etiological agent of periodontal disease. Biofilm formation of various gram-negative bacteria is regulated by a quorum-sensing circuit that relies on N-acyl homoserine lactones (HSLs). Some synthetic N-acyl HSL analogues act as quorum-sensing inhibitors and suppress biofilm formation in Pseudomonas aeruginosa. Development of chemical control agents against oral biofilms is necessary, because until now, biofilms have been removed only by mechanical debridement. The present study investigated the effect of N-acyl HSL analogues on P. gingivalis biofilm formation, with the aim of developing new drugs that inhibit oral biofilm formation.
Material and Methods:
A flow-cell model was used for P. gingivalis biofilm formation. Seventeen synthetic N-acyl HSL analogues were quantitatively assessed by spectrophotometry. The effects of three antagonistic compounds against P. gingivalis biofilm formation were further examined by confocal laser scanning microscopy, and investigated for primary attachment using spectrophotometry and phase contrast microscopy.
Results:
Ten out of 17 analogues affected P. gingivalis biofilm formation. Three out of 10 analogues significantly decreased biofilm-forming cells (p &lt; 0.05), and these biofilm structures were less well formed three-dimensionally. There were no quantitative or qualitative differences in cell attachment between the control and the three analogue-treated groups.
Conclusion:
Three synthetic N-acyl HSL analogues inhibited biofilm formation in P. gingivalis. We suggest that these analogues influence the development stage of P. gingivalis biofilm formation.

DOI： 10.1111/j.1600-0765.2009.01228.x

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Pyogenic liver abscess (PLA) formation is thought to originate from the transmission of infection via three major routes including the biliary tract, portal vein and hepatic artery. However, about 50% of PLA cases are considered to be cryptogenic. Here we report an unusual autopsy case of PLA associated with periodontopathic bacterial infection. A 59-year-old female suddenly developed cardiopulmonary arrest and died. Despite macroscopic and microscopic examinations, the infectious routes and source of infection were unidentified, and the case appeared to be cryptogenic. Since this patient had suffered severe periodontitis for a long period of time, we investigated the involvement of periodontal infection in PLA formation by performing immunohistochemical analyses. We identified several periodontopathic bacterial species in the PLA of this patient, including Fusobacterium nucleatum, Treponema denticola, Prevotella intermedia and Porphyromonas gingivalis. Thus, we demonstrate here that periodontal infection is a potential source of infection in the formation of PLA.

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With the development of dental laser delivery systems that can enter into the root canals, it is possible to use Er:YAG lasers to remove the residual biofilm associated with infected root canals. We examined their effects against biofilms made of Actinomyces naeslundii, Enterococcus faecalis, Lactobacillus casei, Propionibacterium acnes, Fusobacteriurn nucleatum, Porphyromonas gingivalis, or Prevotella nigrescens in vitro. After Er: YAG laser irradiation with energy densities ranging between 0.38-0.98 J/cm(2), the biofilm samples on hydroxyapatite disks were quantitatively and morphologically evaluated. The Er:YAG laser was effective against biofilms of 6 of the bacterial species examined, with the exception of those formed by L. casei. After irradiation, the numbers of viable cells in the biofilms were significantly decreased, whereas atrophic changes in bacterial cells and reductions in biofilm cell density were seen morphologically. Er: YAG lasers might be suitable for clinical application as a suppressive and removal device of biofilms in endodontic treatments.

DOI： 10.1016/j.joen.2008.04.010

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Quorum sensing (QS) is a process by which bacteria communicate using secreted chemical signaling molecules called autoinducers (AIs). By this process, many bacterial species modulate the expression of a wide variety of physiological functions in response to changes in population density. In this study, the periodontal pathogen Eikenella corrodens was observed to secrete type 2 signaling molecules. An ortholog of luxS, the gene required for AI-2 synthesis in Vibrio harveyi, was isolated from the E. corrodens genome. A V. harveyi bioassay showed luxS functionality in E. corrodens and the ability of luxS to complement the luxS-negative phenotype of Escherichia coli DH5 alpha. AI activity was detected in the supernatant, and the maximum expression of AI-2 was observed during the late exponential phase. To determine the potential role of luxS in the colonization processes, an E. corrodens luxS mutant was constructed and tested for its capacity to form an in vitro biofilm on a polystyrene surface. The biofilm forming efficiency of the luxS mutant was approximately 1.3-fold greater than that of the wild type. These data suggest that a LuxS-dependent signal plays a role in the biofilm formation by E. corrodens.

DOI： 10.1263/jbb.102.110

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC GENERAL MICROBIOLOGY  

Eikenella corrodens belongs to a group of periodontopathogenic bacteria and forms unique corroding colonies on solid medium due to twitching motility. It is believed that an N-acetyl-D-galactosamine (GalNAc)-specific lectin on the cell surface contributes significantly to its pathogenicity and can be estimated by its haemagglutination (HA) activity. Recently, a plasmid, pMU1, from strain 1073 has been found; this plasmid affects pilus formation and colony morphology. To identify the gene involved in these phenomena, ORF 4 and ORFs 5-6 on pMU1 were separately subcloned into a shuttle vector, and the resultant plasmids were introduced into E corrodens 23834. Transformants with the ORF 4 gene, which is identified to be a homologous gene of the type IV pilin gene-specific recombinase, lost their pilus structure and formed non-corroding colonies on a solid medium, whereas transformants with ORFs 5-6 exhibited the same phenotype as the host strain 23834. Southern analysis showed that the introduction of the ORF 4 gene into strain 23834 resulted in genomic recombination at the type IV pilin gene locus. The hybridization pattern of these transformants was similar to that of strain 1073. These results suggest that ORF 4 on pMU1 encodes a site-specific recombinase and causes genomic recombination of the type IV pilin gene locus. Furthermore, the introduction of ORF 4 into strain 23834 increased GalNAc-specific HA activity to a level equivalent to that of strain 1073. Although the morphological colony changes and loss of pilus structure are also observed in phase variation, genomic recombination of the type IV pilin gene locus did not occur in these variants. Moreover, an increase was not observed in the GalNAc-specific HA activity of these variants. These results suggested that the loss of pilus structure, the morphological change in colonies and the increase in HA activity due to plasmid pMU1 might be caused by a mechanism that differs from phase variation, such as a genomic recombination of the type IV pilin gene locus.

DOI： 10.1099/mic.0.28490-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

Eikenella corrodens is known not only as one of the periodontopathogenic bacteria but also as a pathogen associated with many infectious diseases of humans. Dental plaque is a complex biofilm community comprised of many bacterial species. E. corrodens has a lectin on its cell surface that is thought to be involved in its pathogenicity. In this study, we found that E. corrodens forms a biofilm on a polystyrene surface. A biofilm was formed at the bottom of the wells in microtiter plates after 24 h. Microcolonies were observed as the amount of biofilm became larger. When anaerobic respiration was repressed due to nitrate limitation, the biofilm formed only at the air-water interface. Strain 1073 and HU, which have higher lectin activity, formed a biofilm more effectively than other strains. Biofilm formation was repressed by the addition of N-acetyl-D-galactosamine. These results suggest that the lectin on the surface of E. corrodens might be involved in biofilm formation.

DOI： 10.1271/bbb.70.441

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Bacterial biofilms have been found to develop on root surfaces outside the apical foramen and be associated with refractory periapical periodontitis. However, it is unknown which bacterial species form extraradicular biofilms. The present study aimed to investigate the identity and localization of bacteria in human extraradicular biofilms. Twenty extraradicular biofilms, used to identify bacteria using a PCR-based 16S rRNA gene assay, and seven root-tips, used to observe immunohistochemical localization of three selected bacterial species, were taken from 27 patients with refractory periapical periodontitis. Bacterial DNA was detected from 14 of the 20 samples, and 113 bacterial species were isolated. Fusobacterium nucleatum (14 of 14), Porphyromonas gingivalis (12 of 14), and Tannellera forsythensis (8 of 14) were frequently detected. Unidentified and uncultured bacterial DNA was also detected in 11 of the 14 samples in which DNA was detected. In the biofilms, P. gingivalis was immunohistochemically detected in all parts of the extraradicullar biofilms. Positive reactions to anti-F. nucleatum and anti-T. forsythensis sera were found at specific portions of the biofilm. These findings suggested that P. gingivalis, T. forsythensis, and F. nucleatum were associated with extraradicular biofilm formation and refractory periapical periodontitis.

DOI： 10.1128/AEM.71.12.8738-8743.2005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL MUNKSGAARD  

The participation of bacterial biofilms in the over-filled gutta-percha points associated with refractory periapical periodontitis has recently been reported. This study investigated the initial biofilm-forming ability of root canal isolates (Enterococcus faecalis, Streptococcus sanguis, Strep. intermedius, Strep. pyogenes, Staphylococcus aureus, Fusobacterium nucleatum, Propionibacterium acnes, Porphyromonas gingivalis and Prevotella intermedia) on gutta-percha points in vitro. Each bacterial strain was suspended in 100% cell culture medium or in culture medium containing 4.5, 45 or 90% (vol/vol) serum. The bacterial suspensions were then co-incubated anaerobically with gutta-percha points for 7 d. The gutta-percha points were processed for scanning electron microscopic observation and examined for biofilm presence and thickness. E. faecalis, Strep. sanguis, Strep. intermedius, Strep. pyogenes and Staph. aureus biofilms were generated on the surfaces of the specimens incubated in culture medium supplemented with 45 or 90% (vol/vol) serum. The E. faecalis and Strep. sanguis biofilms were significantly thicker than those of Strep. intermedius, Strep. pyogenes and Staph. aureus. No biofilms were detected on the specimens incubated with F. nucleatum, Prop. acnes, Porph. gingivalis and Prev. intermedia. These findings suggest that Gram-positive facultative anaerobes have the ability to colonize and form extracellular matrices on gutta-percha points, while serum plays a crucial role in biofilm formation.

DOI： 10.1111/j.1600-0722.2004.00165.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INT AMER ASSOC DENTAL RESEARCHI A D R/A A D R  

Fimbriae, which are involved in adherence, constitute an important pathogenic factor of Porphyromonas gingivalis. In vivo, however, the distribution of P. gingivalis- carrying fimbriae is unknown. The localization of P. gingivalis-carrying fimbriae was examined in situ. From 19 patients with severe periodontitis and P. gingivalis, we obtained 20 teeth with periodontal tissue attached, with and without immunolocalized fimbriae. Eleven teeth were subjected to light microscopy, 9 to electron microscopy. In 6 of the 11 samples examined, we detected positive reactions with an anti-P. gingivalis- fimbriae serum, located in the cementum-attached plaque area in the deep pocket zones. In the so-called 'plaque-free zones', P. gingivalis- carrying fimbriae were immunocytochemically observed to reside in contact with the dental cuticle in 6 of the 9 samples examined. These findings suggest that P. gingivalis- carrying fimbriae are strongly related to adherence to the root surface at the bottoms of human periodontal pockets.

DOI： 10.1177/154405910408301210

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INT AMER ASSOC DENTAL RESEARCHI A D R/A A D R  

Cyclosporin A ( CsA) might induce immune response alterations in periapical lesions and modify bone remodeling. This study determined the changes that occur in the periapical lesions of rats during CsA administration and after CsA withdrawal. After the induction of periapical lesions, the animals were treated with CsA ( 0- 20 mg/ kg/ day) for 4 wks. Lesion volumes were measured by computed tomography. Histological observations and immunohistochemical evaluations were performed with anti- CD3 and anti- CD25 antibodies. CsA administration reduced lesion volumes, and the lesions significantly expanded after CsA withdrawal. CsA inhibited the proliferation and activation of T- cells at lesion sites. The effects of CsA on T- cells were dose-dependent up to 10 mg/ kg/ day, after which no significant difference was evident. These results suggest that CsA inhibits periapical destruction by interfering with T- cell function in periapical lesions.

DOI： 10.1177/154405910408300905

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OPERATIVE DENTISTRY INC  

This study examined the hypothesis that experimental primer containing the antibacterial monomer 12-methacryloloxydodecylpyridinium bromide (MDPB), which was previously reported to show bactericidal effects in vitro, inhibits bacteria in cavities under in vivo conditions. The number of bacteria resulting from applying primer solution to cavities in dog teeth infected with Streptococcus mutans was determined. The infected cavities were also restored using primer and the pulp response was histopathologically examined after 7, 30 and 75 days. No bacteria were recovered after applying the experimental primer, although the bactericidal effects of the proprietary primer were insignificant. Restoration with the experimental primer resulted in little or no pulpal inflammation for all periods; whereas, mild to moderate inflammatory response was observed when using proprietary primer. These results indicate that the experimental primer containing MDPB could exhibit in vivo antibacterial effects, suggesting its possible clinical benefit.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Porphyromonas gingivalis, an oral bacterium, might play a role in the pathogenesis or progression of adult periodontitis. In this study, we isolated from P. gingivalis a putative glycosyltransferase gene, designated gtfA, which had a consensus domain for glycosyltransferase in its N terminus. GtfA consisted of 248 amino acids and its predicted molecular mass was 28 kDa; however, as the molecular mass of endogenous GtfA, protein was around 40 kDa, this suggested that GtfA had undergone some posttranslational modifications. To reveal the role of the gtfA gene in P. gingivalis, we established gtfA-deficient strains by allelic replacement. Morphologically, gtfA-deficient P. gingivalis lacked mature fimbriae. gtfA-deficient P. gingivalis also showed a very low ability for autoaggregation, and its ability to attach to epithelial cells was severely impaired. Thus, the results indicate that the gtfA gene is required for P. gingivalis autoaggregation as well as attachment to epithelial cells. These results suggest that GtfA might have an important role in the pathogenicity of P. gingivalis by regulating adhesion.

DOI： 10.1128/IAI.72.5.2698-2702.2004

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INT AMER ASSOC DENTAL RESEARCHI A D R/A A D R  

Ethyleneglycol dimethacrylate monomers have been previously reported to stimulate the growth of certain caries-associated bacteria on the basis of turbidity measurements. To elucidate the detail of this effect, we examined the influence of resin monomers on the growth of Streptococcus sobrinus and Streptococcus sanguis by determination of bacterial numbers (colony-forming units), morphological observation, and chemical analysis. Although the absorbance values in the stationary phase of bacterial suspension were increased in the presence of ethyleneglycol monomers, no significant differences were observed for bacterial numbers throughout the incubation period. Scanning electron microscopy observation revealed the formation of sparse vesicular material surrounding bacterial cells when incubated with ethyleneglycol monomers, and these products were proved to be resin polymers. The results demonstrate that the apparent biomass increase during incubation with ethyleneglycol monomers is due not to promotion of bacterial multiplication, but to the polymerization of resin monomers to form vesicular structures attached to cells.

DOI： 10.1177/154405910408300406

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Language：English   Publisher：INT AMER ASSOC DENTAL RESEARCHI A D R/A A D R  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ACAD PERIODONTOLOGY  

Background: Porphyromonas gingivalls is found in subgingival biofilm and is associated with periodontal disease. Bacteria in biofilms are able to resist higher antimicrobial concentrations than in suspension. Little is known about the susceptibility of P gingivalis in biofilms to antimicrobial agents. The effects of chlorhexidine gluconate, minocycline hydrochloride, and metronidazole on P gingivalis biofilms were examined in vitro.
Methods: P gingivalis strain 381 biofilms were prepared on 32 hydroxyapatite disks. At 0, 24, 72, and 144 hours after perfusion of the three antimicrobial agents, two disks from each device were used to assess the antimicrobial effects by adenosine triphosphate (ATP) bioluminescence, and for morphological investigation by scanning electron microscopy (SEM).
Results: Close relationships were found between the results of the ATP analyses and the SEM observations in all groups examined. A significant decrease (P &lt;0.001) in ATP content was found between the chlorhexidine-treated and control groups. The extracellular matrix structure and P gingivalis cells were altered in the presence of chlorhexidine. Minocycline hydrochloride also caused a decrease (P &lt;0.05) in the ATP content and morphological change on P gingivalls biofilms. Metronidazole showed no significant efficacy against P gingivalis biofilms.
Conclusion: Chlorhexidine gluconate was effective at reducing the viability of P gingivalis 381 cells in biofilms, while minocycline hydrochloride and metronidazole exhibited weaker effects.

DOI： 10.1902/jop.2003.74.11.1647

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Language：English   Publisher：INT AMER ASSOC DENTAL RESEARCHI A D R/A A D R  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

The aim of this study was to examine morphologically the participation of extraradicular biofilm in refractory periapical periodontitis. Six teeth and five extruded root filling gutta-percha points associated with refractory periapical periodontitis were investigated by scanning electron microscope.
In nine of 11 samples examined, bacterial biofilms were seen at the extraradicular area. The gutta-percha surface was covered with glycocalyx-like structures, and filaments, long rods, and spirochete-shaped bacteria were predominant in the extraradicular sites. Planktonic cells, which were filaments and spirochete-shaped bacteria, emigrated from the glycocalyx structures in some spots. In the extracted teeth, biofilm consisting of both bacteria and glycocalyx-like structures were observed on the periapical root surfaces. Next to the residual periodontal ligament, a few filaments, rods, and fusiforms were attached on the healthy cementum surface.
The present findings suggested that bacterial biofilms formed in the extraradicular areas were related to refractory periapical periodontitis.

DOI： 10.1097/00004770-200210000-00001

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MOSHER & LINDER, INC  

Purpose: To evaluate the feasibility of the calcium phosphate precipitation (CPP) method as a treatment for dentin hypersensitivity using vital teeth of beagle dogs. Also, gingival tissue response to two types of CPP methods was examined histologically. Materials and Methods: Dentin tubules of the dogs' vital teeth were exposed by shallow cavity preparation followed by etching with 50% citric acid to simulate the condition of hypersensitive dentin. After CPP treatment was applied to the vital tooth, the dentin surface and longitudinal sections were observed by SEM to evaluate the occluding ability of the CPP method. The precipitate was also analyzed by energy-dispersive X-ray microanalysis to obtain compositional information. The gingival tissue before and after CPP method was examined histologically with light microscopy. Results: Dentin tubules were occluded homogeneously and completely with an apatitic mineral after application of the CPP treatment in vital teeth. However, the depth of the precipitate in dentin tubules from the dentin surface was approximately half that seen in extracted teeth. No histological change was observed in gingival tissues when NaHCO3 was used as a post-treatment solution of the CPP method, whereas another CPP method using NaOH solution resulted in atrophy and degeneration of the epithelium of gingival tissue.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

Objective: Previously, we have reported that incorporation of the antibacterial monomer 12-methacryloyloxydodecylpyridinium bromide (MDPB) was effective in immobilizing bactericide in the resin matrix, and an antibacterial composite without release of the agent could be achieved. In this study, an attempt was made to increase the density of bactericide immobilized in composite, and the inhibitory effects of this modified antibacterial composite on plaque accumulation were determined, focusing on the reliability of the effects and the mechanisms to affect the plaque formation.
Methods: An experimental composite containing immobilized bactericide at 2.83% was prepared by the incorporation of MDPB into a prepolymerized resin filler of control composite, and elution of antibacterial components and inhibition of in vitro plaque accumulation by Streptococcus mutans were determined. The inhibitory effects of the experimental composite on the attachment, glucan synthesis and growth of S. mutans on the surface were also examined in addition to the comparison of surface roughness and hydrophobicity with controls. The results were analyzed using the Student's t-test.
Results: The experimental composite had reproducible inhibitory effects against plaque accumulation compared with control (p &lt; 0.05). although it showed no elution of unpolymerized MDPB. The plaque-inhibitory effect of the experimental composite was found to depend upon the ability to inhibit the attachment, glucan synthesis, and growth of bacteria on its surface as no significant differences in the surface characteristics were obtained between control and experimental composites (p &gt; 0.05).
Significance: It was indicated that the experimental composite containing bactericide-immobilized filler has the possibility to be used clinically with an effective anti-plaque property. (C) 2001 Academy of Dental Materials. Published by Elsevier Science Ltd. All rights reserved.

DOI： 10.1016/S0109-5641(01)00006-9

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INT AMER ASSOC DENTAL RESEARCHI A D R/A A D R  

Some Gram-negative anaerobes are associated with the incidence and progression of periodontal disease. In periodontal pockets, however, the localization of those bacteria is unknown. We investigated the localization of 5 bacterial species in human periodontal pockets. Fifteen teeth with a part of periodontal pockets from 10 adult periodontitis patients were obtained, and the localization of bacteria was examined immunohistochemically. Positive reactions with anti-Prevotella nigrescens antibody were located at the epithelium-associated plaque area in the middle pocket zones. In the middle and deep pocket zones, Fusobacterium nucleatum and Treponema denticola were especially localized in the unattached plaque area, but Eikenella corrodens was observed in the tooth-attached plaque area. Actinobacillus actinomycetemcomitans, detected in 2 of 15 samples examined, was found in the unattached plaque area, in the middle pocket zone. The present findings indicated that the 5 bacterial species examined localized at distinct regions in human periodontal pockets.

DOI： 10.1177/00220345010800101301

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ACAD PERIODONTOLOGY  

Background: Subgingival plaque bacteria live within a biofilm covered with glycocalyx, and little is known of the bacterial species associated with biofilm formation at the bottom of human periodontal pockets, the so-called "plaque-free zone" (PFZ).
Methods: Seventy-seven extracted teeth from 56 patients with severe advanced adult periodontitis were examined. Porphyromonas gingivalis, Campylobacter rectus, Fusobacterium nucleatum, Actinobacillus actinomycetemcomitans, Treponema denticola, Prevotella nigrescens, and Actinomyces viscosus were examined by scanning immunoelectron microscopic techniques, using both secondary and back-scattered imaging, with rabbit antibodies specific for each bacteria.
Results: Secondary electron images showed that rods, filaments, and spirochete-shaped bacteria formed small aggregates in the PFZ. Some of the bacteria were covered with an amorphous film-like structure. By back-scattered electron imaging, positive reactions with anti-P. gingivalis were found in 8 of 13 samples examined, and film-like structures coated several cells of 6 positive samples examined. Labeled cells with anti-C rectus, anti-T. denticola and anti-P. nigrescens were detected in 3 of 11, 5 of 10, and 1 of 8 samples examined. A. viscosus were found in 6 of 11 of the samples. A. viscosus tended to overlay the amorphous capsula and aggregate. F. nucleatum and A. actinomycetemcomitans were not detected in any samples examined.
Conclusions: These findings indicated that P. gingivalis, C. rectus, T. denticola, P. nigrescens, and A. viscosus were present in the PFZ, and that some specified bacteria were possibly related to plaque-biofilm formation of subgingival plaque.

DOI： 10.1902/jop.2000.71.8.1319

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MUNKSGAARD INT PUBL LTD  

The localization and distribution of Porphyromonas gingivalis, Campylobacter rectus and Actinomyces viscosus were studied in human periodontal pockets. After obtaining voluntary consent from 9 patients, 12 teeth and their surrounding periodontal tissue with advanced adult periodontitis were extracted carefully so as not to change the structure of the periodontal pockets. The specimens were processed into serial sections. One of the sections was stained with Brown & Brenn-modified Gram stain to observe the distribution of bacteria. The others were stained immunohistochemically by the Labelled Streptavidin Biotin method (LSAB method) using specific rabbit antibodies against selected bacteria. Some bacteria could be found within epithelial cells. P. gingivalis was found in 9/12 of the samples examined, Small aggregates of P. gingivalis were scattered in all parts of the periodontal pockets, and some of these aggregates could be seen in close contact with the epithelium. Conversely, C. rectus was observed in 5/12 of the samples examined and was predominantly located in the middle and deep pocket zones. C. rectus tended to form large clumps in both the tooth-attached and epithelium-associated plaque area. A. viscosus was observed in 7/12 of the samples examined and was localized predominantly in the tooth-attached plaque area, especially in the shallow and middle pocket zones. Although unexpected spills of unattached plaque from periodontal pockets was possible, immunohistochemical staining with species-specific antibodies was extremely sensitive and revealed the localization and the distribution of periodontal disease-associated bacteria in human periodontal pockets.

DOI： 10.1111/j.1600-0765.1997.tb00937.x

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Language：English   Publisher：AMER ASSOC DENTAL RESEARCH  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society for Oral Pathology  

We immunohistologically examined the cell densities and distribution of T and B cells in periodontitis and analyzed their relationship in terms of periodontal tissue destruction. Specimens were labeled with monoclonal antibodies for T cells (CD3) and B cells (CD20) and developed using the double staining technique. CD3^+ cells appeared in low density infiltrates and increased gradually with the number of total infiltrates; in contrast, CD20^+ cells did not appear in small infiltrates but increased rapidly with the number of infiltrates. The number of fibroblastic cells negatively correlated with that of the total infiltrates and CD20^+ cells, but it did not correlate with the number of CD3^+ cells. These findings suggest that the T cells infiltrated into the inflamed sites at the onset and increased gradually with the development of inflammation and that B cells infiltrated later than T cells and their number increased rapidly with inflammation.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KARGER  

The levels of selected cariogenic and obligately anaerobic bacteria have been compared in carious dentine taken from fissures, smooth surfaces, and root surfaces. The numbers of infected dentinal tubules were determined by immunohistological staining using species-specific antisera against selected bacteria. Selective localization was observed; mutans streptococci were the predominant bacteria in dentine from fissures and smooth surface coronal caries, but not from root surface caries. The proportion of mutans streptococci was higher in the shallow and middle layers of dentine from fissures and smooth surface coronal carious lesions than from the deep layers. In root surface caries, Actinomyces spp. were the predominant bacteria, and were present at higher levels in the deep layer of root lesions than in the shallow and middle layers. The proportion of lactobacillus spp. was relatively low despite its high detection frequency in all layers of the three types of carious lesion. Immunohistological staining with species-specific antisera was able to reveal the distribution and the localization of various bacteria in carious dentine.

DOI： 10.1159/000261635

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

1)Smは小窩裂溝および平滑面齲蝕の全てで検出され,またその細菌侵入率も齲蝕の表層および中層で他の細菌種に比して著明に高かった。2)SSは,Smに次ぐ細菌検出率を示したが,その侵入率はSmよりかなり低かった。3)Av,Lc,Lpおよび偏性嫌気性細菌のPm,Ea,Paは,比較的高い細菌検出率だが,その侵入率はSmに比して有意に低い値を示した。4)Sa,Fn,Peは細菌検出率,細菌侵入率とも著明に低い値を示した

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DOI： 10.1177/00220345900690061401

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Authorship：Corresponding author   Language：Japanese   Publishing type：Research paper, summary (national, other academic conference)  

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Authorship：Lead author,　Last author,　Corresponding author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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Language：Japanese   Publisher：日本バイオフィルム学会  

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口腔において洗口液をはじめとする抗菌成分は、唾液により希釈され時間経過とともに最小発育阻止濃度以下(sub-minimum inhibitory concentration:sub-MIC)となるため、逆にバイオフィルム形成(BF)を促進する可能性がある。そこで本研究では、う蝕病原細菌であるStreptococcus mutans、Streptococcus oralisおよびActinomyces naeslundiiの3菌種から成るin vitro複合バイオフィルムモデルを確立し、sub-MICのグルコン酸クロルヘキシジン(CHG)を作用させた際のS.mutansの局在とBFおよびクオラムセンシング関連遺伝子の転写量の変化について検討した。S.mutansはUA159株と緑色蛍光タンパク発現株(ZsGreen株)の2種類を用いた。SEM像より、コントロール群、CHG作用群ともに球菌を主体とするバイオフィルムが観察された。CLSM像より、CHG作用群はコントロール群と比較してバイオフィルムの厚みが有意に増していた。またCLSMにて構成細菌の局在を観察したところ、S.oralisとA.naesulundiiはバイオフィルム深部に局在し、S.mutans(ZsGreen株)はバイオフィルム中の表層部に局在していた。CHGの有無による局在の差は認めなかった。バイオフィルム中の生菌数は、CHG群とコントロール群で有意差はなかった。バイオフィルム中の各細菌種の割合は、両群とも9割以上Streptococcus属が占有していた。0.24 μg/ml CHG作用群のS.mutansでは、gtfCおよびcomDの転写量がコントロール群と比較し有意に増加していた。このことから今回作製した3菌種による複合バイオフィルムモデルにおいて、sub-MICのCHGは、S.mutansのBF形成関連遺伝子に影響を与えることでBFを促進させることが示唆された。(著者抄録)

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Language：Japanese   Publisher：日本バイオフィルム学会  

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Language：Japanese   Publisher：日本バイオフィルム学会  

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Language：Japanese   Publisher：日本バイオフィルム学会  

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Language：Japanese   Publisher：日本バイオフィルム学会  

J-GLOBAL

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Language：Japanese   Publisher：新潟歯学会  

根尖性歯周組織病変の難治化は、根尖部での複雑な解剖学的問題に加え、残存バイオフィルムによる細菌学的な影響もあるとされている。こうした様々な要因の改善策として外科的歯内療法が選択される。しかし、補綴処置を行う上で考慮する歯冠・歯根長比の問題で複数回に渡り歯根切断が行われることは稀である。今回は、歯根尖切除術を過去3度に渡って施行しても治癒しなかった症例に対して、マイクロスコープの使用下で再歯根尖切除術と根尖部の緊密な封鎖を目的としたmineral trioxide aggregate(以下MTA)による逆根管充填を含めた再外科的歯内療法が奏効した症例を報告する。患者は59歳の女性で、上顎右側中切歯および側切歯に対して打診痛、根尖部圧痛および瘻孔を認める慢性根尖膿瘍と診断し、最初に通常の感染根管治療を行った。その後、病変部の治癒が期待できなかったため歯根尖切除を行い、逆根管形成後にMTAで逆根管充填を行った。手術から1ヵ月後で瘻孔は消失し、さらに6ヵ月後のレントゲン写真では根尖部の透過像が消失しており、治癒傾向を認めた。(著者抄録)

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Language：Japanese   Publisher：新潟歯学会  

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

Evidence Based Dentistry(EBD)について、1)基本的な事象ほどエビデンスとされる文献の発表年数が古い、2)古い文献に基づいたエビデンスほどin vitro研究を根拠としているものが多い等、筆者自身が気になっている疑問がある。これらの疑問を臨床医、特に認定医/専門医の目線で洗い出し、エビデンスのあり方を再考するとともに、今後のEvidence Based Medicine/EBDのあり方、歯科治療、特に保存治療における臨床研究の重要性、認定医/専門医として臨床を通してやるべきことについて述べた。

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Language：Japanese   Publisher：新潟歯学会  

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Language：Japanese   Publisher：新潟歯学会  

根尖性歯周組織病変の難治化は、根尖部での複雑な解剖学的問題に加え、残存バイオフィルムによる細菌学的な影響もあるとされている。こうした様々な要因の改善策として外科的歯内療法が選択される。しかし、補綴処置を行う上で考慮する歯冠・歯根長比の問題で複数回に渡り歯根切断が行われることは稀である。今回は、歯根尖切除術を過去3度に渡って施行しても治癒しなかった症例に対して、マイクロスコープの使用下で再歯根尖切除術と根尖部の緊密な封鎖を目的としたmineral trioxide aggregate(以下MTA)による逆根管充填を含めた再外科的歯内療法が奏効した症例を報告する。患者は59歳の女性で、上顎右側中切歯および側切歯に対して打診痛、根尖部圧痛および瘻孔を認める慢性根尖膿瘍と診断し、最初に通常の感染根管治療を行った。その後、病変部の治癒が期待できなかったため歯根尖切除を行い、逆根管形成後にMTAで逆根管充填を行った。手術から1ヵ月後で瘻孔は消失し、さらに6ヵ月後のレントゲン写真では根尖部の透過像が消失しており、治癒傾向を認めた。(著者抄録)

J-GLOBAL

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Language：Japanese   Publisher：日本バイオフィルム学会  

殺菌に頼ったバイオフィルム(BF)制御は口腔細菌叢の変化の懸念があるだけでなく、付着界面にBF構造が残存し、BF再形成の足場となる可能性が指摘されている。本研究では、結核菌の表層糖脂質をリード化合物として創製した機能性糖脂質(硫酸化ビザンチン:Viz-S)が、ヒト唾液由来細菌のex vivoモデル上でのBF形成に与える影響について検討するとともに、初期定着菌に対する付着抑制効果について検討した。Sub-MIC量のViz-S存在下で24時間培養後のex vivoBF量をクリスタルバイオレット(CV)法で定量したところ、BF量は50μM以上で有意に減少した。蛍光染色を施し、共焦点レーザー顕微鏡(CLSM)観察を行うと、Viz-SはBF中の細菌に殺菌効果を示さず、BF量は濃度依存的に減少した。初期定着菌に各濃度のViz-S溶液を10分間作用させたのち菌体表面性状を評価したところ、100μMにおいて疎水度は有意に低下した。また、50μMもしくは100μMのViz-Sで10分作用後のStreptococcus mutansのガラス表面への細菌付着量をフローセルを用いて解析したところ、ガラス界面への付着量は濃度依存的に減少していた。さらに、Viz-Sを蛍光標識し、S.mutans菌液と混合したのち蛍光顕微鏡観察すると、リング状に蛍光を発する菌体が観察された。これらのことから、Viz-Sはsub-MIC量において細菌壁表層の性状を親水性に変化させることで付着減弱効果を与え、菌種非特異的にBFを易剥離性に変化させることが示唆された。Viz-Sは口腔細菌叢を変動させない新しいBF制御剤として有用である可能性が示された。(著者抄録)

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Language：Japanese   Publisher：(NPO)日本歯周病学会  

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Language：Japanese   Publisher：(株)デンタルダイヤモンド社  

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J-GLOBAL

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J-GLOBAL

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J-GLOBAL

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J-GLOBAL

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Language：Japanese   Publisher：新潟歯学会  

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Language：Japanese   Publisher：(公社)日本矯正歯科学会  

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Language：Japanese   Publisher：(一社)歯科基礎医学会  

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Language：Japanese   Publisher：日本バイオフィルム学会  

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Language：Japanese   Publisher：新潟歯学会  

J-GLOBAL

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Language：Japanese   Publisher：日本バイオフィルム学会  

厚朴由来の抽出物でポリフェノールであるMagnololおよびHonokiolは、生薬として広く用いられ、抗菌作用を有するとされている。本研究では、Streptococcus mutansバイオフィルムに対するMagnololおよびhonokiolの殺菌効果および細胞毒性について検討した。ガラスボトムディッシュ上に形成したS.mutans人工バイオフィルムに対し、Magnolol、Honokiol、およびクロルヘキシジングルコン酸塩を30秒および5分作用させたときの殺菌効果をコロニー数測定法および蛍光プロービング法を用いて評価したところ、Magnolol作用群は、5分作用後において他群と比較して有意に高い殺菌効果を示した。さらに、ヒト歯肉上皮細胞株Ca9-22に対する各抽出物の細胞毒性をMTT試験にて評価したところ、Magnololは、有効殺菌濃度においてヒト歯肉上皮細胞に対して細胞毒性を示さなかった。以上のことから、MagnololはS.mutansバイオフィルムに対する優れた殺菌効果を有し、口腔粘膜への副反応が低いことが示唆された。(著者抄録)

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Language：Japanese   Publisher：新潟歯学会  

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Language：Japanese   Publisher：(公社)日本矯正歯科学会  

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Language：Japanese   Publisher：日本歯科医学教育学会  

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Language：Japanese   Publisher：日本バイオフィルム学会  

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Language：Japanese   Publisher：日本歯内療法学会  

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Language：Japanese   Publisher：日本歯内療法学会  

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Language：Japanese   Publisher：日本バイオフィルム学会  

特定の細菌を用いたin vitroモデルによって口腔環境を再現することは不可能である。そこで、我々は口腔内装置を用いたin situバイオフィルムモデルを新規に開発した。本研究では、我々が開発したモデルを用い実験的にデンタルバイオフィルムを作製し、16S rRNA遺伝子を標的としたシーケンス解析を用いて、その構成細菌を経時的に同定した。その結果、門レベルにおいて、16時間まではFirmicutes門が優勢であり、その後48時間以降にFusobacteria門およびBacteroidetes門の割合が増加した。さらに、属レベルではおよそ16時間までStreptococcus属が20%以上を占め、48時間後以降にFusobacterium属およびPorphyromonas属などの偏性嫌気性菌が優勢となり、96時間後には有意に増加した(p&lt;0.05)。さらに、リアルタイムPCRを用いて構成細菌の経時的定量を行ったところ、全細菌量は二相性の増加傾向を示した。また、Streptococcus属は12時間までで有意に増加し、その後一定となり、Fusobacterium属に関しては経時的に増加傾向を示し96時間後で最も細菌量が多いことが分かった。本研究により、ヒトの口腔で形成した実験的デンタルバイオフィルムは通性嫌気性菌が優位であったのに対し、成熟するにつれてグラム陰性偏性嫌気性菌の割合が増加することにより、全細菌量が二相性に増加することが明らかとなった。(著者抄録)

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Language：Japanese   Publisher：日本バイオフィルム学会  

偏性嫌気性のグラム陰性桿菌であるPorphyromonas gingivalisは、ヒトの歯周ポケット全域に分布し、歯周病原性細菌の1種として重要な役割を果たしている。歯周病が糖尿病や動脈硬化などといったメタボリックシンドローム関連疾患の原因となることが、様々な疫学的調査および動物実験により明らかになっているが、疾患発症のメカニズムについては不明である。近年、新たなメカニズムとして、嚥下されたP.gingivalisが腸管の細菌叢の変化とそれに伴う代謝性内毒素血症を引き起こすことで肝臓・脂肪組織に炎症を誘導し、インスリン抵抗性を惹起することが報告された。しかしながら、宿主の胃を通過する際にP.gingivalisが生存可能であるのかについては未解明である。今回、更なる詳細なメカニズムの解明を目的として、P.gingivalis ATCC 33277株の浮遊細菌およびバイオフィルムに対する人工胃液の影響について解析を行った。浮遊細菌に対して各pHの人工胃液を作用させた結果、pH3、5ならびに7処理群ではP.gingivalisのコロニー形成が認められたが、pH1処理群ではコロニーは形成されなかった。一方、バイオフィルムではpH1の人工胃液処理群においても生菌が認められた。以上の結果より、嚥下されたP.gingivalisが生菌の状態で胃を通過し、下部消化管まで到達することが示唆された。(著者抄録)

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Language：Japanese   Publisher：(一社)日本レーザー歯学会  

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Language：Japanese   Publisher：日本バイオフィルム学会  

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Language：Japanese   Publisher：日本バイオフィルム学会  

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

目的:歯の自家移植は1歯欠損に対する処置法の選択肢の一つであり,天然歯にほぼ一致した組織学的・解剖学的構造の再生が得られ,天然組織の保存を図ることができるという利点を有し,良好な経過を示す臨床症例が多数報告されている.しかしながら,移植歯のサイズや形態に制限があり,症例選択の際には熟考を要する.一般的に,移植歯は移植自体の手技,および移植歯に対する根管治療の難しさから単根歯でかつ歯根の彎曲が小さい歯が用いられることが多いが,今回は歯根の彎曲が著しい上顎右側第三大臼歯を上顎左側第二大臼歯部に移植し良好に経過している症例について報告する.症例の概要:患者は37歳男性.以前より体調不良時に上顎左側第二大臼歯の違和感を覚えていたが,頬側および口蓋側に腫脹を認め来院.来院時,上顎左側第二大臼歯頬側および口蓋側に瘻孔を形成していた.治療経過:補綴物を除去すると髄床底に破折を認め,保存困難と診断した.患者より上顎右側第三大臼歯を用いた移植を希望する申し出があり,上顎右側第三大臼歯には術前のコーンビームCT(CBCT)による診査で歯根の肥大・彎曲を認めたものの,移植可能と判断し移植歯として用いることとした.上顎左側第二大臼歯を抜歯し,同日に上顎右側第三大臼歯を抜歯し口腔外にてNi-Tiロータリーファイルを用いて根管治療を行い,上顎左側第二大臼歯部に移植し固定した.移植歯の固定は1カ月後に除去し,支台築造および暫間被覆冠の作製を行った.移植後6カ月経過時に歯周組織の安定を確認したうえで,最終補綴物を作製した.デンタルエックス線写真において,移植歯周囲歯槽骨および歯槽硬線の回復が認められ,現在術後1年以上経過しているが,経過は良好である.結論:本症例では,CBCTによる術前診査の後に歯根の肥大および彎曲が認められる上顎大臼歯の自家移植を行い,良好な結果が得られた.(著者抄録)

DOI： 10.11471/shikahozon.57.589

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Other Link： http://search.jamas.or.jp/link/ui/2015182155

Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Language：Japanese   Publisher：クインテッセンス出版(株)  

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Language：Japanese   Publisher：(公社)日本生物工学会  

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Other Link： http://search.jamas.or.jp/link/ui/2016045897

Language：Japanese   Publisher：(公社)日本生物工学会  

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Other Link： http://search.jamas.or.jp/link/ui/2016045898

Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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Language：Japanese   Publisher：(株)第一歯科出版  

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Language：Japanese   Publisher：日本歯内療法学会  

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Language：Japanese   Publisher：日本バイオフィルム学会  

1990年代に入り歯科領域にバイオフィルム感染症の概念が導入され、我々は、初めてバイオフィルムという概念の下でデンタルプラークを微細形態学的に観察した。そして、菌体外マトリックスに被覆された種々の形態型からなる細菌種がバイオフィルムを形成していることを明らかにした。また、免疫組織学的手法を用いて、歯周病原性が高いと推察されているPorphyromons gingivalisが、歯肉縁下のデンタルバイオフィルム全域に分布していることを明らかにした。一方で、P.gingivalisはバイオフィルム形成過程で遺伝子の発現を巧みに調整していることや、遺伝子の1つ(PGN_0088)がバイオフィルムの糖合成を抑制し、バイオフィルムの3次元的構造や物理的強度に関わっていることを遺伝子工学的および分子生物学的手法により示唆した。う蝕、歯周病、感染根管といった歯科領域が対象とする主な感染症の原因は、いずれもデンタルバイオフィルムである。従ってこのバイオフィルムの実態をよく理解し、適切に対応することが歯科臨床の王道である。う蝕や歯周病の予防・治療法の第1選択としてバイオフィルムの機械的除去を行っていることは合理的ではあるが、機械的除去のみしか対応手段を持たないのは残念なことである。近い将来、遺伝子工学や分子生物学などの知見に基づいた、デンタルバイオフィルムのコントロール法が開発されることを期待している。(著者抄録)

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Language：Japanese   Publisher：日本バイオフィルム学会  

Eikenella corrodensは、対数増殖期の中後期にAI-2を産生し、バイオフィルム形成に影響を及ぼす。しかし、定常期に入るとAI-2活性は劇的に減少することから、AI-2の分解または不活化酵素の存在が示唆された。本研究では、本菌の培養上清からAI-2不活化酵素を精製し、その同定を試みた。E.corrodensの定常期の培養上清を硫安分画することにより、AI-2不活化活性を有する画分(30%画分)を得た。この画分は、本菌の培養上清から精製したAI-2のみならず、センサー株に対してAI-2活性を示すメチルヒドロキシフラノン(MHF)も不活化した。30%画分とインキュベートしたMHFをTLCおよび逆相HPLCで解析したところ、スポットの消失やピークのシフトが見られた。これらの結果から、この画分にはAI-2を別の形に変換する酵素が含まれることが示唆された。次に、30%画分をイオン交換クロマトグラフィーで精製を行った。精製タンパク質をSDS-PAGEで解析すると、約40kDaの単一なバンドが確認された。そのN末端アミノ酸配列は外膜ポーリン(PorA)のものと一致した。porA欠損株の培養上清からはAI-2不活化活性は見られなかった。これらの結果から、E.corrodensにおいて可溶性ポーリンがAI-2の不活化に関与することが示唆された。(著者抄録)

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

J-GLOBAL

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Language：Japanese   Publisher：(公社)日本生物工学会  

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Other Link： http://search.jamas.or.jp/link/ui/2014247450

Language：Japanese   Publisher：日本バイオフィルム学会  

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Language：Japanese   Publisher：日本バイオフィルム学会  

Porphyromoas gingivalisはグラム陰性の偏性嫌気性桿菌で、辺縁性歯周炎や根尖性歯周炎などの歯科疾患に密接に関与している。マクロライド系抗生物質のアジスロマイシン(AZM)はP.gingivalisに対して最小発育阻止濃度以下の濃度(sub-MIC)でバイオフィルム形成を阻害することが報告されている。本研究では、P.gingivalis ATCC 33277株のバイオフィルム形成において中・後期間に顕著な遺伝子発現の減少を認めたPGN_0088の役割を検討し、AZMおよび同じくマクロライド系抗生物質のエリスロマイシン(ERY)がPGN_0088欠損株に及ぼす影響を検索した。微細形態学的および3次元的観察において、PGN_0088欠損株のバイオフィルムは網目様の構造を示した。さらに、バイオフィルム中の菌体当りの糖成分の量は欠損株において、野生株および相補株と比べて有意に増加し、PGN_0088は、バイオフィルム中の糖成分の合成を抑制し、バイオフィルムの立体構造に関与していると示唆された。欠損株および相補株はAZMおよびERYに対する耐性を有しているにも関わらず、全菌株においてsub-MICのAZMおよびERYによりバイオフィルム形成が有意に抑制され、AZMおよびERYはP.gingivalisに対し、抗菌作用とは異なる機序でバイオフィルム形成を抑制していると推察された。(著者抄録)

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Language：Japanese   Publisher：(株)第一歯科出版  

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

目的:Minimal Interventionの概念が普及して,歯髄保存の重要性がより注目される一方で,う蝕象牙質の除去範囲についてはいまだに明確な基準が存在しない.本研究では,う蝕除去後の残存象牙質に対するEr:YAGレーザーの照射が,残存細菌に与える影響について多面的評価を行った.材料と方法:う蝕治療が必要な患歯57歯を対象とした.う蝕検知液にて象牙質が淡いピンク色に染色されるまでう蝕象牙質を除去した後,レーザー照射群(28歯)ではEr:YAGレーザーをう蝕除去後の残存象牙質に照射し,同部の象牙質採取を行い,定性的かつ定量的な細菌学的検索に供した.非照射群(29歯)では,レーザー照射を行わずに象牙質を採取し,レーザー照射群と同様の手順で実験を行った.また,一部の試料では採取した象牙質を共焦点レーザー顕微鏡にて観察した.成績:レーザー照射群・非照射群とも,術後に臨床症状が出現した症例は認めなかった.また,非照射群では69%の試料から細菌が検出されたのに対し,レーザー照射群では43%の試料から細菌が検出され,照射群では有意に残存細菌の検出率が低下していた(p&lt;0.05).一方,細菌が検出された試料で細菌数について評価したところ,非照射群と比較してレーザー照射群では検出された細菌数が有意に減少しており(p&lt;0.05),このことは共焦点レーザー顕微鏡による観察でも確認された.検出された細菌種については,レーザー照射群と非照射群の間で明確な違いは認められなかった.結論:う蝕象牙質除去後の残存細菌に対する殺菌効果について,低出力Er:YAGレーザーの照射は有用であることが明らかとなった.(著者抄録)

DOI： 10.11471/shikahozon.56.1

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：(公社)日本生物工学会  

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Other Link： http://search.jamas.or.jp/link/ui/2013125531

Language：Japanese   Publisher：(公社)日本生物工学会  

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Other Link： http://search.jamas.or.jp/link/ui/2013125532

Language：Japanese   Publisher：日本バイオフィルム学会  

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Porphyromonas gingivalisは辺縁性歯周炎および根尖性歯周炎といった歯科疾患に密接に関与している。P.gingivalisバイオフィルム形成過程での遺伝子発現の変化については解明されたが、菌体構成成分やバイオフィルムの微細構造を経時的に解析した報告は無く、遺伝子・菌体構成成分・性状の経時的な変化について、未だ不明である。本研究では、P.gingivalis ATCC 33277株のバイオフィルム中における糖成分及びタンパク質成分の量、また、バイオフィルムの微細形態学的構造を、各形成過程において解析した。バイオフィルム形成を微細形態学的に観察した結果、形成3日後から菌体がマトリックス様構造物に覆われていたことから、バイオフィルム中の菌体外マトリックスは、初期付着の時点で既に生成されている事が明らかとなった。一方、形成14日後のバイオフィルムにおいて、菌体当りの糖成分の量は他の時点と比べて有意に減少しており、さらに、バイオフィルム中において菌体やベシクル様構造物の密度が減少し、菌体がゴーストセル化していたことから、バイオフィルム形成の後期では、菌体間の接着力が低下し、デタッチメントを引き起こしている可能性が示唆された。(著者抄録)

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Language：Japanese   Publisher：日本バイオフィルム学会  

バイオフィルムに起因する感染症である辺縁性歯周炎や根尖性歯周炎の予防・治療法はバイオフィルムの機械的除去が第一選択であり、補助的に化学療法を使用している。しかしながら、バイオフィルムを完全に排除することは困難であり、バイオフィルムに起因する疾患は再発を繰り返す。本研究では辺縁性歯周炎や根尖性歯周炎の病原性細菌の1つであるPorphyromonas gingivalisのバイオフィルムに対して有効であるグルコン酸クロルヘキシジン(CHX)を作用させた後のバイオフィルムの構造および成分について検討した。微細形態学的観察の結果、CHXを作用させると、バイオフィルムを形成している菌体細胞は破壊されるが、バイオフィルムの表層構造に影響はなく、菌体死滅後もバイオフィルムは残存することが明らかとなった。続いて、残存したバイオフィルムの成分を定量的および定性的に解析した結果、破壊された菌体から溢出した液体成分はバイオフィルム内に留まることが明らかとなった。さらに、CHX作用後のバイオフィルムではベシクル様構造物が多数観察された。これらの結果より、CHXはバイオフィルム中の菌体細胞を破壊するが、バイオフィルムの構造を破壊することはできず、残存したバイオフィルムが疾患の難治化や再燃に関与する可能性が示唆された。(著者抄録)

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Language：Japanese   Publisher：日本バイオフィルム学会  

【目的】歯周病関連細菌Eikenella corrodensは菌体表層にNアセチルガラクトサミン(GalNAc)特異的なレクチンを有し、これが本菌の歯周病原性に大きく関与する。最近、プラスミド上にコードされたリコンビナーゼがゲノム上の線毛遺伝子領域に組み換えを起こし、GalNAc特異的レクチン活性を上昇させること、バイオフィルム形成能が増加することなどを明らかにしてきた。今回は上皮細胞への付着と侵入に及ぼす影響を調べた。【結果】23834株にリコンビナーゼを導入すると付着能力が高まった。この付着能力の上昇は、GalNAcの添加により抑制されることから、リコンビナーゼ導入によるレクチン活性の上昇が寄与することが示唆された。一方、23834株へのリコンビナーゼ導入によって口腔上皮細胞への侵入は抑制された。以前の研究で組換えによりタイプ4線毛の消失が見られたことから、この侵入能力の抑制は線毛の消失によることが考えられた。しかし、相変異株では侵入能力の抑制は見られなかったことから、ゲノム再編によって線毛以外の変化によって侵入能力が抑制されることが示唆された。さらに、リコンビナーゼ導入株を感染させたときには上皮細胞からのIL-8の誘導産生が減少した。このことから、ゲノム再編株は好中球の走化性を抑え、口腔内での生存を有利にしていることが示唆された。これまでの報告と本研究の結果を考え合わせると、E.corrodensはゲノム再編により口腔内で高病原化することが示唆された。(著者抄録)

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Language：Japanese   Publisher：(株)ヒョーロン・パブリッシャーズ  

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Other Link： http://search.jamas.or.jp/link/ui/2012181259

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Language：Japanese   Publisher：日本嫌気性菌感染症学会  

59歳女。死亡18日前から発熱および右側腹部痛を自覚した。自宅にて心肺機能停止状態で発見され、救急救命部に搬入された。CT検査を行なうことによって、肝右葉の大型mass、および右心房内の血栓様病変の存在を確認した。さらに、肝穿刺によって茶褐色混濁かつ悪臭を伴う膿汁を採取し、肝右葉のmassは肝膿瘍である可能性が高いことが臨床的に診断した。搬入後間もなく死亡の確認が行われ、死因究明を目的に病理解剖を実施した。肝右葉にグラム陰性菌感染による多房性の肝膿瘍の形成を認めた。重度の歯周および歯内疾患を罹患し、複数回のそれら疾患の急性発作を経験していた。死因は、肝膿瘍内の肝静脈が侵襲され、歯周病細菌が血管内に流れ込んだ結果、一気に全身に散布されたことによる感染性血栓形成性のショックおよびLPSショックと考えられた。

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Language：Japanese   Publisher：日本歯内療法学会  

根尖切除を行う際、切除する歯が単根歯であれば根尖の切断面の形態はおおよそ予測可能であるが、2根管以上を有する歯の場合には、根管の分岐や合流、イスムスなど存在により、切断部位に現れる根管の形態は様々であるため、解剖学的形態を熟知しておかなければ感染源を見逃してしまうおそれがある。そこで今回、上顎小臼歯の根尖切除を行った2例の術中に認められた根管形態を示しながら、手術を成功に導くために必要な解剖学的知識を整理した。上顎小臼歯の根尖切除においては、解剖学的形態を理解したうえで、術前にX線写真などで確実な診断を行い、かつ実際に切断面を観察してから適切な処置を行うことが求められる。

DOI： 10.20817/jeajournal.32.3_217

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Language：Japanese   Publisher：日本歯内療法学会  

最近のシステマティックレビューでは、初回根管治療と再根管治療の成功率はそれぞれ74.6%、76.7%である。成功率低下の一因として、根尖孔外バイオフィルムの形成があげられる。根尖孔外バイオフィルムの検出や根管治療で排除・抑制することは極めて困難である。根管治療をよりエビデンスに基づいた治療形態へと近づける機器として、コーンビームCTやマイクロスコープの有用性が報告されている。また、抗バイオフィルム薬が開発途中にある。

DOI： 10.20817/jeajournal.32.2_87

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Porphyromonas gingivalisは辺縁性歯周炎および根尖性歯周炎といった歯科疾患に密接に関与している。バイオフィルムに関与する遺伝子は多数報告されているが、それら遺伝子のバイオフィルム形成過程での発現の変化についての報告はない。本研究では、P.gingivalisのバイオフィルム形成における遺伝子発現の経時的変化を、フローセル系および静置系の2種類のバイオフィルム形成モデルを用いて検索した。フローセル系モデルを用いて作成したP.gingivalisバイオフィルム細菌においては、形成過程のいずれの時点間においても5倍以上の変化が認められた遺伝子は存在しなかったが、浮遊細菌及び静置系バイオフィルム細菌ではそれぞれ、50個及び1個の遺伝子において5倍以上の変化が認められた。これは、フローセル系内のバイオフィルム細菌は代謝活性が低下していることが一因と推察された。また、2倍以上発現量の変化する遺伝子数が最多となる期間は、浮遊細菌は誘導期から対数増殖期前期の間で、細胞分裂に関わる遺伝子であると推察された。一方、フローセル系バイオフィルム細菌ではバイオフィルム成熟期付近で2倍以上発現量の変化する遺伝子数が最多となり、菌体外マトリックス形成やクオラムセンシングに関与する遺伝子が含まれていると推察された。(著者抄録)

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Language：Japanese   Publisher：日本微生物生態学会  

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Language：Japanese   Publisher：(株)第一歯科出版  

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DOI： 10.14833/amjsp.2010f.0.36.0

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Language：Japanese   Publisher：日本バイオフィルム学会  

Porphyromonas gingivalisは辺縁性歯周炎および根尖性歯周炎といった歯科疾患に密接に関与している。マクロライド系抗生物質であるアジスロマイシン(AZM)は、緑膿菌に対し最小発育阻止濃度(MIC)以下の濃度(sub-MIC)でバイオフィルム形成を阻害する。本研究では、種々の抗生物質がP.gingivalisバイオフィルムに及ぼす影響をsub-MICを含む各種濃度において生物学的および共焦点レーザー顕微鏡(CLSM)を用い3次元的に検索した。静置系モデルにおいて、P.gingivalisバイオフィルムはAZM、エリスロマイシン(EM)添加群ではsub-MICにおいて有意に減少した(p&lt;0.01)。アンピシリン(ABPC)、オフロキサシン(OFX)添加群ではMIC以上で有意にバイオフィルムを抑制したが、ゲンタマイシン(GM)は抑制効果を示さなかった。また、フローセルモデルにおいてsub-MICでP.gingivalisバイオフィルムを抑制したものはAZMのみで、CLSM観察においてもバイオフィルムの3次元的抑制像が観察された。静置およびフローセルの両系においてsub-MICで有意にP.gingivalisバイオフィルムを抑制したものはAZMのみであり、P.gingivalisを含む病原性デンタルバイオフィルムをAZMの全身投与により臨床的にコントロールできる可能性が示唆された。(著者抄録)

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Language：Japanese   Publisher：(一社)日本病理学会  

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DOI： 10.11471/shikahozon.50.648

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Language：Japanese   Publisher：(株)医薬ジャーナル社  

歯周病を含め、多くの口腔疾患はデンタルバイオフィルム(デンタルプラーク)を代表とするオーラルバイオフィルム細菌に起因する感染症である。オーラルバイオフィルムは、メタボリック症候群、骨粗鬆症、誤嚥性肺炎、糖尿病、感染性心内膜炎といった全身疾患のリスクファクターとしても注目されている。オーラルバイオフィルム中には500種類の細菌が共生・拮抗しながら棲息しており、その形成と成長には体細胞間の情報伝達機構が関与していることなどがわかってきた。多彩な様相を呈するヒトのオーラルバイオフィルムの全貌を解明することは困難であるが、本稿では歯槽骨吸収を惹起するデンタルバイオフィルムの特徴と実態を概説するとともに、バイオフィルムと骨量減少との関係による歯槽骨吸収のメカニズムについて考察した。(著者抄録)

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過去3ヵ月以内に感染根管治療の既往がなく、抗生物質投与を受けていない全身疾患がない同意の得られた35名(男17名、女18名、26〜77歳)を対象に、根管拡大後のEr:YAGレーザー照射が、根尖孔部付近の根管内に残存した細菌に及ぼす影響について細菌学的・組織学的に検討した。レーザー照射前に細菌が残存していた15根管中9試料からは照射後も細菌は検出されなかった。6試料からは細菌が検出されたがうち5試料は検出限界以下の細菌数で、1試料も10CFUの細菌数で殺菌作用としての効果は認められた。蛍光顕微鏡によるレーザー照射後の生死の観察で生菌が認められた1試料からは死菌が検出されず、ターゲットとした根尖孔部の根管内象牙質にレーザー光が当たっていない可能性が示唆され、感染根管治療時における根尖部の残存根管内細菌に対するEr:YAGレーザー照射の有効性が示された。

DOI： 10.11471/shikahozon.49.537

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

生後18〜20ヵ月、体重10〜15kgのビーグル犬を用い、抗菌性モノマーMDPBを5%濃度で配合したセルフエッチングプライマーを組み込んだ接着システムが、歯髄に対して為害作用を及ぼさずin vivoにおいて良好な接着を示すという仮説を立てて検証した。5%MDPB配合のセルフエッチングプライマーを用いコンポジットレジン修復を行ったところ、7日、30日、75日のいずれの期間経過後も歯髄の炎症反応は全くみられなかった。5%MDPB配合セルフエッチングプライマーと試作フッ素徐放性ボンディングレジンを組み合わせたシステムによってin vivoで形成された象牙質接着界面を透過型電子顕微鏡で観察したところ微細構造はin vivoと同様で、象牙質との良好な接着状態を表すものであった。このことから抗菌性モノマーMDPBを配合したセルフエッチングシステムは生体に使用した場合も良好な親和性と接着界面を示すことが示唆された。

DOI： 10.11471/shikahozon.49.523

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Language：Japanese  

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Language：Japanese   Publisher：(株)医薬ジャーナル社  

歯科の2大疾患であるう蝕と歯周病だけでなく,口腔疾患の多くはデンタルプラークを代表とする口腔バイオフィルム細菌に起因する感染症である.口腔のバイオフィルム感染症は,誤嚥性肺炎,糖尿病,細菌性心内膜炎といった全身疾患のリスクファクターとしても注目されている.口腔バイオフィルム中には500種以上の細菌が棲息していること,その成長にはクオラムセンシングという菌体細胞間の密度調整機構が関与していることなどが明らかにされた.しかしながら,多彩なヒトの口腔バイオフィルムの全貌は解明されておらず,バイオフィルムに有効な化学的コントロール法は確立されていない.将来的には,既存の機械的なコントロール法に加え,作用機序が明確なバイオフィルムの新しいコントロール法が確立されるものと期待される(著者抄録)

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Language：Japanese  

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

マウス由来線維芽細胞様細胞(L929細胞)に対しEr:YAGレーザーをパルスエネルギー30mJ・60mJ・90mJ・120mJでそれぞれ3秒間照射した.照射条件はいずれのパルスエネルギーにおいても,パルスレート10pps,レーザーチップ先端から計測面までの距離10cmとした.照射後の生存細胞率はパルスエネルギーが大きいほど有意に低かったが,60mJと90mJの間,90mJと120mJの間には有意差を認めなかった.実際の照射面積から計算すると60mJ以上で細胞への障害作用がかなり強くなると推測された.走査型電顕による細胞形態の観察でも60mJ以上で明らかな変化が確認された.これらの結果からEr:YAGレーザーはパルスエネルギー60mJ以上で線維芽細胞様細胞への障害が強くなることが示唆された

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Language：Japanese  

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

開発した三次元レーザー計測システムによる定量分析と走査電子顕微鏡による形態観察から明らかにしたin vivoでのセラミックインレーの辺縁劣化を,in vitroにおいて可及的に再現する劣化試験の確立を試みた.さらに,確立した劣化試験にて,レジンセメント幅が辺縁劣化量に及ぼす影響について検討を加えた.セメント幅の拡大に伴って辺縁劣化量が増加し,可及的に小さなセメント幅を達成することが,辺縁劣化の防止に有効であることが示唆された

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Language：English  

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Language：Japanese   Publisher：金原出版(株)