Effect of periodontitis induced by Fusobacterium nucleatum on the microbiota of the gut and surrounding organs.

Detection of the oral bacterium Fusobacterium nucleatum in colorectal cancer tissues suggests that periodontitis may alter gut microbiota. The purpose of this study was to analyze the influence and infection route of periodontal inflammation caused by F. nucleatum, and microbiota of the gut and surrounding organs (heart, liver, kidney). Wistar female rats were orally inoculated with F. nucleatum to establish an experimental periodontitis model that was confirmed by X-ray imaging and histopathological analysis. The mandibles, gut, liver, heart, and kidneys were collected from the experimental group at 2, 4, and 8 weeks, and from the uninfected control group at 0 weeks, for DNA extraction for PCR amplification and comprehensive microbiota analysis using the Illumina MiSeq platform. Imaging confirmed the onset of periodontitis at 2 weeks post-inoculation, and histopathology showed inflammatory cell infiltration from 2 to 8 weeks. PCR and comprehensive microbiota analysis showed the presence of F. nucleatum in the heart and liver at 2 weeks, and in the liver at 4 and 8 weeks. There were changes of microbiota of the gut, heart, liver, and kidneys at 4 weeks: namely, decreased Verrucomicrobia and Bacteroidetes, and increased Firmicutes. F. nucleatum induced the onset of periodontitis and infected the heart and liver in rats. As the periodontic lesion progressed, the microbiota of the gut, liver, heart, and kidneys were altered.

Antibacterial effects of surface pre-reacted glass-ionomer (S-PRG) filler eluate on polymicrobial biofilms.

PURPOSE: To analyze the effects of surface pre-reacted glass-ionomer (S-PRG) filler eluate on polymicrobial biofilm metabolism and live bacterial count. METHODS: Biofilm was formed using glass disks 12 mm in diameter and 150 µm in thickness. Stimulated saliva was diluted 50-fold with buffered McBain 2005 and cultured in anaerobic conditions at 37°C for 24 hours in anaerobic conditions (10% CO2, 10% H2, 80% N2) to form the biofilm on the glass disks. Following this, biofilms were treated with (1) sterilized deionized water (control), (2) 0.2% chlorhexidine digluconate (0.2CX), (3) S-PRG eluate diluted to 10% (10% S-PRG)，(4) 20% S-PRG，(5) 40% S-PRG，(6) 80% S-PRG，and (7) S-PRG for 15 minutes (n= 10 per group), and samples were subdivided into two groups for measuring live bacterial count immediately after treatment and after 48 hours of culturing after treatment. The pH of the spent medium collected at the time of culture medium exchange was tested. RESULTS: Immediately after treatment, the live bacterial count of samples treated with drug solutions was significantly lower than the control (8.2 × 108), and the counts of samples treated with 0.2CX (1.3 × 107) and S-PRG (1.4 × 107) were significantly lower than those treated with diluted S-PRG (4.4 × 107-1.4 x 108). When the medium was measured again after culturing for 48 hours, growth was continually inhibited in all treatment groups and the bacterial count of samples treated with S-PRG (9.2 x 107) was significantly lower than that of samples treated with 0.2CX (1.8 × 108). The pH of spent medium immediately after treatment was significantly higher in groups treated with drug solutions (5.5-6.8) than the controls (4.2), and it was highest in the S-PRG-treated group (6.8). Thereafter, when culturing was continued for 48 hours, the pH of all treated groups decreased; however, the pH of the S-PRG-treated group was significantly higher than groups treated with other drug solutions. CLINICAL SIGNIFICANCE: Surface pre-reacted glass-ionomer (S-PRG) filler eluate not only reduced the live bacterial count of polymicrobial biofilm, but also continuously inhibited the lowering of pH.

Effect of toothpaste containing multiple ions-releasing filler on polymicrobial biofilm regrowth and dentin demineralization.

PURPOSE: To compare the efficacy of toothpaste containing surface pre-reacted glass-ionomer (S-PRG) filler particles to that of conventional sodium fluoride (NaF) toothpaste for the prevention of dentin demineralization and biofilm regrowth. METHODS: Bovine root dentin specimens and glass coverslips were used as biofilm growth substrates. To establish biofilms, glass and dentin specimens were incubated for 72 hours in 0.2% sucrose McBain medium inoculated with stimulated saliva from a single donor. Specimens then received a single 5-minute treatment with S-PRG toothpaste, fluoride toothpaste, or sterilized deionized water and were incubated in McBain medium for 120 hours to allow biofilm regrowth. Output parameters during regrowth (72-192 hours) were pH of spent medium, colony-forming unit (CFU) counts of biofilms, and dentin mineral profiles, integrated mineral loss (IML: vol% × µm), and lesion depth (Ld). Treatment group differences were tested by one-way ANOVA followed by Tukey's multiple range test (P< 0.05). RESULTS: At 144 hours, medium pH was significantly higher in the S-PRG-treated dentin group than in the NaF-treated dentin group. In addition, at 192 hours, the CFU count, IML, and Ld were lower in the S-PRG-treated dentin group than in the NaF-treated dentin group. There were significant differences of pH among dentin groups at 72 hours. Treatment with S-PRG toothpaste markedly inhibited dentin demineralization compared to that with NaF toothpaste. CLINICAL SIGNIFICANCE: Toothpaste containing multiple ions-releasing filler suppressed bacterial viability and inhibited dentin demineralization.

Protective effects of (6R)-5,6,7,8-tetrahydro-l-biopterin on local ischemia/reperfusion-induced suppression of reactive hyperemia in rat gingiva.

We herein investigated the regulatory mechanism in the circulation responsible for rat gingival reactive hyperemia (RH) associated with ischemia/reperfusion (I/R). RH was analyzed using a laser Doppler flowmeter. RH and I/R were elicited by gingival compression and release with a laser Doppler probe. RH increased in a time-dependent manner when the duration of compression was between 30 s and 20 min. This increase was significantly suppressed by N (ω)-nitro-l-arginine-methyl-ester (l-NAME), 7-nitroindazole (7-NI), and 2,4-diamino-6-hydroxypyrimidine (DAHP). However, RH was markedly inhibited following 60 min of compression. This inhibition was significantly decreased by treatments with superoxide dismutase (SOD), (6R)-5,6,7,8-tetrahydro-l-biopterin (BH4), and sepiapterin. The luminescent intensity of superoxide anion (O2 (•-))-induced 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo-[1,2-a] pyrazine-3-one (MCLA) was markedly decreased by SOD and BH4, but only slightly by sepiapterin. BH4 significantly decreased O2 (•-) scavenging activity in a time-dependent manner. These results suggested that nitric oxide (NO) secreted by the nitrergic nerve played a role in regulating local circulation in rat gingiva. This NO-related regulation of local circulation was temporarily inhibited in the gingiva by the I/R treatment. The decrease observed in the production of NO, which was caused by suppression of NO synthase (NOS) activity subsequent to depletion of the NOS co-factor BH4 by O2 (•-), played a partial role in this inhibition.

Inhibitory effects of French pine bark extract, Pycnogenol®, on alveolar bone resorption and on the osteoclast differentiation.

Pycnogenol(®) (PYC) is a standardized bark extract from French maritime pine (Pinus pinaster Aiton). We examined the inhibitory effects of PYC on alveolar bone resorption, which is a characteristic feature of periodontitis, induced by Porphyromonas gingivalis (P. gingivalis) and osteoclast differentiation. In rat periodontitis model, rats were divided into four groups: group A served as the non-infected control, group B was infected orally with P. gingivalis ATCC 33277, group C was administered PYC in the diet (0.025%: w/w), and group D was infected with P. gingivalis and administered PYC. Administration of PYC along with P. gingivalis infection significantly reduced alveolar bone resorption. Treatment of P. gingivalis with 1 µg/ml PYC reduced the number of viable bacterial cells. Addition of PYC to epithelial cells inhibited adhesion and invasion by P. gingivalis. The effect of PYC on osteoclast formation was confirmed by tartrate-resistant acid phosphatase staining. PYC treatment significantly inhibited osteoclast formation. Addition of PYC (1-100 µg/ml) to purified osteoclasts culture induced cell apoptosis. These results suggest that PYC may prevent alveolar bone resorption through its antibacterial activity against P. gingivalis and by suppressing osteoclastogenesis. Therefore, PYC may be useful as a therapeutic and preventative agent for bone diseases such as periodontitis.

Formation of subsurface dentin lesions using a polymicrobial biofilm model.

PURPOSE: To simulate an oral demineralization environment by multiple species of bacteria by inducing subsurface dentin lesions with a polymicrobial biofilm model. METHODS: Polymicrobial biofilms consisting of multiple species of bacteria were generated from stimulated saliva using a high-throughput active attachment model. Biofilms were grown on dentin specimens in McBain medium containing 0, 0.2 or 2.5 ppm F and on glass without fluoride for 192 hours. The medium was refreshed twice daily, after 10 and 14 hours, until 72 hours, followed by treatment for 5 minutes with 400 ppm fluoride. Specimens were recovered after 192 hours. The number of colony forming units (CFU) was measured, and integrated mineral loss (IML) was determined by transversal microradiography. RESULTS: Mineral profiles in specimens grown with 0.2F and 2.5F revealed surface layers and initial lesions distinct from those grown with 0F. IML was significantly lower with 0.2F and 2.5F than with 0F (P < 0.05), although CFUs were similar. CFUs of biofilms grown on dentin in medium containing 0F were 10-fold higher than on glass (P < 0.05). Subsurface lesions on dentin formed consistently, with their growth progression inhibited by application of fluoride. To our knowledge, this is the first report describing the induction of subsurface dentin lesions by a polymicrobial biofilm model, and this model may be useful for studies of demineralization supporting in situ and in vivo models.

Relationship between aging and periodontal disease severity in gauge-raised cynomolgus monkeys (Macaca fascicularis).

The study aimed to evaluate the periodontal disease status in different age groups and clarify the relationship between aging and the severity of periodontal disease. The test animals were cynomolgus monkeys that were born and raised at the Tsukuba Primate Research Center of the National Institutes of Biomedical Innovation, Health, and Nutrition. The participants were divided into three groups: young (5-10 years old), middle (10-19 years old), and old (≥20 years old). The plaque Index (PLI), Gingival Index (GI), probing pocket depth (PPD), and Bleeding on probing (BOP) were used for the periodontal examination. Representative teeth were also examined. Polymerase chain reaction (PCR) was used to identify Porphyromonas macacae in dental plaque. Multiple comparisons and regression analyses were used to analyze the relationship between each age group and each oral examination index. Statistically significant differences were found between the age groups and periodontal examination index. Multiple regression analysis revealed that age was strongly correlated with each oral examination index. Based on these results, oral examinations of cynomolgus monkeys kept in the same environment confirmed an association between aging and periodontal disease severity. Monkeys at this facility are expected to serve as new experimental models for elucidating the mechanisms underlying the progression of age-related periodontal disease.

Effect of the Progression of Fusobacterium nucleatum-induced Apical Periodontitis on the Gut Microbiota.

INTRODUCTION: Fusobacterium nucleatum, which is involved in the development of periodontal disease and apical lesions, can be transmitted to the colon and metastasize to colorectal cancer, suggesting a link between oral and systemic diseases. We analyzed the effects of F. nucleatum on bacterial flora in the gut and surrounding organs in a rat model of apical periodontitis and analyzed the infection route to the gut and distant organs. METHODS: We induced apical periodontitis in rat molars by infecting the dental pulp with F. nucleatum and then took X-ray images and performed histopathologic analyses. Next, we removed the maxilla, gut, heart, liver, and kidney from the rats at 0, 2, 4, and 8 weeks postsurgery and then extracted DNA samples and performed polymerase chain reaction and microbiome analyses using the Illumina MiSeq (Illumina Co, Tokyo, Japan). RESULTS: The presence of inflammatory cell infiltration confirmed apical periodontitis from 2-8 weeks. Polymerase chain reaction and microbiome analyses revealed F. nucleatum in the rat gut from 2 weeks and in the kidney from 8 weeks. The rat gut, heart, liver, and kidney exhibited altered bacterial flora, including a marked decrease in Verrucomicrobia and an increase in Proteobacteria after 2 weeks and increases in Bacteroidetes and Firmicutes after 4 weeks. CONCLUSIONS: The onset of F. nucleatum-induced apical periodontitis changed the bacterial flora in the rat gut, heart, liver, and kidney, with a confirmed progressing infection in the large intestines.

Evaluation of Antimicrobial Effects on Dental Impression Materials and Biofilm Removal by Sodium Dichloroisocyanurate.

Dental materials are inevitably contaminated with oral microorganisms. To prevent transmission of infectious diseases, impressions need to be disinfected. In the present study, we examined the disinfection effects on impression materials and biofilm removal by sodium dichloroisocyanurate (SDIC). Exponentially growing Streptococcus mutans, Escherichia coli, Staphylococcus aureus and Candida albicans, and dental plaque bacteria were suspended in phosphate buffered saline (PBS) and exposed for 1, 5 and 10 min to 1 mL of the 10 ppm, 100 ppm, 1,000 ppm, and 10,000 ppm SDIC solutions. The bactericidal effect was evaluated by colony forming units of each microorganisms. Moreover, the effect of SDIC solution on S. mutans biofilm was examined. Bactericidal effects of SDIC solutions on oral bacteria on dental impression surfaces were assessed and the surface quality of dental casts after immersion in SDIC solution for 30 min was observed under a scanning electron microscope. The number of all bacterial strains, including plaque bacteria, were significantly decreased by SDIC solution treatment in a dose-dependent manner. Significant S. mutans biofilm removing activity of SDIC was observed in 1,000 and 10,000 ppm solution. The number of oral bacteria adhering to the surfaces of impressions markedly decreased following 10-min immersion in the 1,000 ppm SDIC solution. The 30-min immersion of dental impression in the 1,000 ppm SDIC solution did not adversely affect the surface roughness of dental casts. The results indicate that SDIC Solution is useful to deactivate oral bacteria on dental impression.

Inhibitory effect of omega-3 fatty acids on alveolar bone resorption and osteoclast differentiation.

In this study, a Porphyromonas gingivalis (P.g.)-infected mouse periodontitis model was used to investigate the effect of omega-3 fatty acid intake on differentiation and maturation of cultured osteoclast. Four-week-old C57BL/6JJcl mice were divided into four groups according to the diets they were fed from the beginning of the experiment (i.e., food containing omega-3 or omega-6 fatty acids) and whether they were orally administered P.g. Thirty-three days after beginning the experiment, bone marrow cells were sampled from the femoral bone of mice from each group and differentiated into osteoclasts; the effects of the ingestion of different fatty acids were subsequently investigated. There was no statistical interaction between the different fatty acids and P.g. infection on the number of osteoclasts (P = 0.6). However, the fatty acid type affected the number of osteoclasts in mice (P = 0.0013), with the omega-3 groups demonstrating lower osteoclast numbers than the omega-6 groups. Furthermore, the addition of resolvin E1 (RvE1), which is an omega-3 fatty acid-derived lipid mediator, suppressed the differentiation of mouse cultured osteoclasts (P < 0.0001). Therefore, the ingestion of omega-3 fatty acids may suppress osteoclast differentiation while inhibiting bone resorption and tissue destruction due to periodontitis.

Purification and characterization of a fimbrial protein from Porphyromonas salivosa ATCC 49407.

Periodontal disease is a significant problem in companion animals such as dogs and cats. However, there is little information available about fimbriae association of periodontal disease in companion animals. In this study, we have purified and characterized a fimbriae from Porphyromonas salivosa ATCC 49407. The molecular mass of this protein was approximately 60-kDa, as estimated by SDS-PAGE. Immunogold electron microscopy revealed that anti-60-kDa fimbrial serum bound to fimbria on the cell surface of P. salivosa ATCC 49407. However, fimbriae of P. gingivalis and P. gulae were not labeled with the same antibody. Immunoelectron-microscopic studies and immunoblot analysis revealed that antigenicity and molecular weight were distinct from previously reported Porphyromonas fimbrial proteins. The amino acid sequence of the N-terminal 15 residues of the 60-kDa fimbrillin protein revealed only 3 of 15 residues identical to other Porphyromonas species fimbrillin proteins. Thus, the N-terminal amino acid sequence of the 60-kDa fimbrillin protein of P. salivosa clearly differed from previously reported fimbrillin proteins. The level of adherence of the P. salivosa was 1.81%. It was confirmed that P. salivosa can adheres to human cells. These results suggest that the 60-kDa fimbriae of P. salivosa ATCC 49407 is a new type of fimbria and may have an important factor in the adherence host cells. We suggest that the surface structure of P. salivosa may have a role in the colonization of this organism in periodontal pockets in companion animals.

Inhibitory effects of azithromycin on the adherence ability of Porphyromonas gingivalis.

BACKGROUND: Porphyromonas gingivalis is a major pathogen and has a high detection rate in periodontal disease. Fimbriae and hemagglutinin are expressed by P. gingivalis, and these play an important role in the adherence of the bacteria to periodontal tissue and biofilm formation. The aim of this study was to investigate the effects of sub-minimal inhibitory concentrations (sub-MICs) of azithromycin on the adherence of P. gingivalis, focusing on the inhibition of fimbriae expression and hemagglutinin activity. METHODS: P. gingivalis ATCC 33277 were incubated anaerobically with sub-MICs of azithromycin at 37°C by gentle shaking for 18 hours. The bacterial cells were harvested, washed twice with phosphate-buffered saline (PBS), and the proteins analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. Adherence assay and hemagglutinin activity tests were done with the same culture. RESULTS: The results of SDS-PAGE indicated that the sub-MICs of azithromycin inhibited 41-kDa fimbrial protein expression and hemagglutinin activities. The disappearance of 41-kDa fimbrial protein expression and long fimbriae in 0.4 µg/mL, 0.2 µg/mL, and 0.1 µg/mL of azithromycin was confirmed by western blotting and transmission electron microscopy. The adherence of P. gingivalis to human gingival epithelial cells was reduced by sub-MICs of azithromycin compared with the adherence levels without antibiotic. CONCLUSIONS: These results suggest that sub-MICs of azithromycin may reduce the adherence of P. gingivalis to host cells, by inhibiting production of fimbriae and hemagglutinin activities. Therefore, azithromycin can be used as a biofilm treatment of periodontal disease caused by P. gingivalis.

Molecular and antigenic similarities of the fimbrial major components between Porphyromonas gulae and P. gingivalis.

Porphyromonas gulae is black-pigmented anaerobic bacteria associated with canine periodontitis. There is little information available about the specific identify and relative occurrence of pigmented anaerobes in companion animals. Our aim was to clarify the factor involved in the adherence and colonization of the organism in the oral cavity. Fimbrial protein was purified from P. gulae ATCC 51700. The molecular mass of this protein was approximately 41kDa as estimated by SDS-PAGE. An antibody against 41-kDa fimbrial protein from P. gingivalis ATCC 33277 reacted with fimbrillin of P. gulae ATCC 51700. Immunogold electron microscopy revealed that the anti-41kDa fimbrial serum bound to fimbria on the cell surface of P. gulae ATCC 51700. Thus, fimbrial protein of P. gulae ATCC 51700 had the same size and antigenicity as 41-kDa fimbriae of P. gingivalis ATCC 33277. The nucleotide sequence of the fimA gene from P. gulae ATCC 51700 showed 94% homology with that of P. gingivalis ATCC 33277. Moreover, the deduced amino acid sequences have 96.8% identity. P. gulae has adherent ability to gingival epithelial cells. The properties of P. gulae fimbriae are similar to those of P. gingivalis fimbriae. We suggest that the surface structure of P. gulae may play a role in the colonization of this organism in periodontal pockets in companion animals.

Effects of French Pine Bark Extract Chewing Gum on Oral Malodor and Salivary Bacteria.

Frequent or persistent malodor (halitosis) represents a considerable embarrassment to those affected. French pine bark extract, Pycnogenol® (PYC), has displayed antibacterial activity against a broad range of bacterial species. In the present study, anticipated benefits of PYC on diminishing halitosis were investigated. Ten healthy males and 11 females, aged 40.1±12.3 y, were recruited based on threshold breath sulfur compounds presence, diagnosed by portable gas chromatography. Subjects were randomly assigned to either sugar-free gums, or gums bearing an additional 2.5 mg PYC per piece. The subjects were required to consume two pieces of PYC or placebo gum six times daily for 15 min. The levels of volatile sulfur compounds (VSCs), measured by OralChromaTM, and tongue-coating score were recorded at baseline, 2, and 4 wk. Hydrogen sulfide-producing bacteria in saliva were cultured on Brucella blood agar plates containing 0.05% cysteine, 0.12% glutathione, and 0.02% lead acetate. The group consuming PYC chewing gum reduced exhaled hydrogen sulfide, methyl mercaptan and dimethyl sulfide significantly (p<0.01) after 2 wk versus baseline. Continuation of daily PYC-gum consumption for 4 wk remarkably lowered the tongue-coating score and exhaled hydrogen sulfide was significantly decreased compared to the placebo group. PYC chewing gum significantly reduced hydrogen sulfide-producing bacteria in saliva after 4 wk (p<0.01), with no effects observed in the placebo control. The results suggest that PYC chewing gum is effective in reducing oral malodor by decreasing the accumulation of tongue coating and the number of hydrogen sulfide-producing bacteria in saliva.

The r40-kDa outer membrane protein human monoclonal antibody protects against Porphyromonas gingivalis-induced bone loss in rats.

BACKGROUND: Porphyromonas gingivalis has been implicated as an important pathogen in the development of adult periodontitis, and its colonization of subgingival sites is critical in the pathogenic process. We recently reported the construction and characterization of human immunoglobulin G isotype clones, which were specifically reactive with recombinant (r) 40-kDa outer membrane protein (OMP) of P. gingivalis. The aim of this study was to investigate the efficacy of human monoclonal antibody (hMAb) against r40-kDa OMP of P. gingivalis to the protection alveolar bone loss by P. gingivalis in rats. METHODS: The role of 40-kDa OMP in the adherence of P. gingivalis to human gingival epithelial cells (HGECs) was examined by preincubating with r40-kDa OMP hMAb before adding the HGECs. Moreover, we used a rat model to examine the effect of the anti-r40-kDa OMP hMAb in alveolar bone loss by oral infection. Forty-six days after the last infection, the periodontal bone level was assessed morphometrically on defleshed rat jaws. RESULTS: The adherence to HGECs was reduced by 84% compared to adherence levels without the antibody. P. gingivalis could not be detected from rats in a P. gingivalis-non-infected group and a group that was administered the anti-r40-kDa OMP hMAb. The bone loss in P. gingivalis-infected animals that were administered the anti-r40-kDa OMP hMAb was significantly lower than that of P. gingivalis-infected rats. CONCLUSIONS: Our results suggest that transchromosomic mouse-derived hMAb against r40-kDa OMP of P. gingivalis protects against periodontal bone loss. This newly constructed anti-r40-kDa OMP hMAb was used to protect against periodontal diseases caused by P. gingivalis infection.

Expression of toll-like receptor 2 and 4 in dental pulp.

Toll-like receptors (TLRs) are important factors in innate immune responses because they mediate signals from bacterial cell wall components during inflammatory reactions. However, the role of TLR in dental pulp, which is bounded by hard tissues, is little understood. The present study investigated the expression of TLR-2 and TLR-4 in experimentally inflamed pulp by quantitative real-time polymerase chain reaction and immunohistochemistry. Total RNA isolated from pulp tissue from 0 to 72 hours after bacterial dentinal infection. The TLR-2 messenger RNA (mRNA) level was 30-fold higher than the TLR-4 mRNA level at 9 hours. The TLR-2 mRNA level in pulp began to increase by 3 hours after bacterial infection, reaching a maximum level after 9 hours and gradually decreasing from 9 to 72 hours. Numerous TLR-2- and CD64-positive cells detected on macrophage and dendritic-like cells, TLR-4-positive cells detected a little in the pulp at 9 hours. These results suggest that TLR-2 may be mainly regulated during the early stage of pulp inflammation triggered by bacterial infection.

Expression of bone extracellular matrix proteins on osteoblast cells in the presence of mineral trioxide.

The biocompatibility of periapical tissue with mineral trioxide aggregate (MTA) affects its ability to repair and regenerate itself. Here we report the cytotoxicity of MTA and how it affects the expression of bone extracellular matrix protein in MC3T3-E1 osteoblast cells. We quantified the cytotoxicity of MTA, amalgam, and Dycal (Dentsply/Caulk, Milford, DE) on MC3T3-E1 cells by measuring the ability of cells to cleave a tetrazolium salt to produce formazan dye during a period of 24, 48, or 96 hours. We used reverse-transcriptase polymerase chain reaction with primer sets for type I collagen, osteocalcin, and bone sialoprotein to measure the gene-expression response of MC3T3-E1 cells treated with MTA. MTA, amalgam, and Dycal were less toxic after 48 hours. MC3T3-E1 cell growth with MTA and Dycal was greater than nonstimulated controls. MTA caused an upregulation of type I collagen and osteocalcin messenger RNA expression after 24 hours. These results showed that, in the presence of MTA, cells grow faster and produce more mineralized matrix gene expression in osteoblasts.

Antibacterial Action of a Condensed Tannin Extracted from Astringent Persimmon as a Component of Food Addictive Pancil PS-M on Oral Polymicrobial Biofilms.

The purpose of this study was to evaluate the antibacterial activity against polymicrobial (PM) biofilms of a condensed tannin extracted from astringent persimmon (PS-M), which is contained in refreshing beverages commercially available in Japan. Salivary PM biofilms were formed anaerobically on glass coverslips for 24 and 72 h and were treated for 5 min with sterilized deionized water (DW), 0.05 and 0.2 wt% chlorhexidine digluconate (CHX), and 0.5-4.0 wt% PS-M solution. The colony forming units (CFU/mL) were determined and morphological changes of the biofilms were observed by scanning electron microscopy (SEM). The CFUs were lower in all PS-M and CHX groups compared to the DW group. PS-M exerted a dose-dependent effect. PS-M (1.53 × 10(7)) at a dose of 4.0 wt% had the same effect as 0.2 wt% CHX (2.03 × 10(7)), regardless of the culture period. SEM revealed the biofilm structures were considerably destroyed in the 4.0 wt% PS-M and 0.2 wt% CHX. These findings indicate that the antibacterial effects of PS-M, a naturally derived substance, are comparable to those of CHX. PS-M may keep the oral cavity clean and prevent dental caries and periodontal disease related to dental plaque, as well as systemic disease such as aspiration pneumonitis.

Porphyromonas gulae 41-kDa fimbriae induced osteoclast differentiation and cytokine production.

Porphyromonas gulae is considered to be associated with canine periodontitis. We have previously reported that the P. gulae American Type Culture Collection (ATCC) 51700 comprised 41-kDa fimbriae. The purpose of the present study was to demonstrate the roles of 41-kDa fimbrial protein in periodontal disease. In this study, we examined the involvement of the 41-kDa fimbrial protein in osteoclast differentiation and cytokine production in murine macrophages. Furthermore, alveolar bone resorption induced by P. gulae infection in rats was evaluated. To estimate osteoclast differentiation, bone marrow cells and MC3T3-G2/PA6 cells were cultured with or without the 41-kDa fimbrial protein for 7 days. BALB/c mouse peritoneal macrophages were stimulated with the 41-kDa fimbrial protein, and the levels of interleukin (IL)-1ß and tumor necrosis factor (TNF)-α production were determined by enzyme-linked immunosorbent assay. Osteoclast differentiation was significantly enhanced by treatment with the 41-kDa fimbrial protein in a dose-dependent manner. The total area of pits formed on the dentine slices with osteoclasts incubated with the 41-kDa fimbrial protein was significantly greater than that of the control. The purified 41-kDa fimbrial protein induced IL-1ß and TNF-α production in BALB/c mouse peritoneal macrophages after 6 hr of incubation in a dose-dependent manner. The bone loss level in rats infected with P. gulae was significantly higher than that of the sham-infected rats. These results suggest that P. gulae 41-kDa fimbriae play important roles in the pathogenesis of periodontal disease.

Porphyromonas gingivalis 67-kDa fimbriae induced cytokine production and osteoclast differentiation utilizing TLR2.

Porphyromonas gingivalis, a major etiological agent of adult periodontitis, has two distinctly different types of fimbriae on the cell surface. The major fimbriae, which consist of a 41-kDa fimbrillin of P. gingivalis ATCC 33277, have been known to induce inflammatory cytokine production in murine peritoneal macrophages. In this study, we examined the effects of the minor fimbriae of P. gingivalis, composed of a 67-kDa fimbrillin, on cytokine production in murine peritoneal macrophages and the ability to induce osteoclast differentiation. Murine peritoneal macrophages were stimulated with P. gingivalis 67-kDa minor fimbriae for 24 h, then the levels of interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha and IL-6 production were determined by enzyme-linked immunosorbent assay (ELISA). To estimate osteoclast differentiation, mouse osteoclast precursors were placed on dentine slices, and cultured with or without P. gingivalis 67-kDa minor fimbriae for 7 days. P. gingivalis 67-kDa minor fimbriae clearly induced IL-1beta, TNF-alpha and IL-6 production in mouse macrophages. Furthermore, pit formations on the dentine slices were significantly extended when the osteoclast precursors were incubated with P. gingivalis 67-kDa minor fimbriae. Pretreatment with anti-Toll-like receptor 2 (TLR2) antibody significantly inhibited IL-1beta, TNF-alpha and IL-6 induction (P<0.05) in mouse macrophages and pit-forming activity of osteoclast precursor cells stimulated with P. gingivalis 67-kDa minor fimbriae. These results suggest that P. gingivalis 67-kDa minor fimbriae may provoke host inflammatory response and be involved in periodontal tissue breakdown.