Yoshiaki Nomura

Salivary biomarkers for predicting the progression of chronic periodontitis

OBJECTIVE Predicting the progression of periodontitis would allow for targeted supportive periodontal therapy. The purpose of this study was to determine the usefulness of salivary biomarkers for predicting the progression of periodontitis. DESIGN Eighty-five chronic periodontitis patients were enrolled in an 18-month longitudinal study. Amongst them, 57 experienced progression of periodontitis, indicated at the end of the 18 months by at least one site with >3mm loss of attachment compared with baseline. We determined the levels of aspartate aminotransferase, alanine aminotransferase (ALT), lactate dehydrogenase, alkaline phosphatase and free haemoglobin as biomarkers, as well as the counts of Porphyromonas gingivalis, Prevotella intermedia and Tannerella forsythia, which represented the periodontal bacteria, in the stimulated saliva. The Mann-Whitney U test was used to compare patients with and without progression. After categorising the diagnostic values, the chi-square test was applied. RESULTS Counts and ratios (ratio to total bacteria) of P. gingivalis and P. intermedia were found to be significant predictors of the progression of periodontitis. To increase prediction accuracy, combination analyses were performed. The combination of ALT level and the P. gingivalis ratio showed the highest likelihood (p<0.001, sensitivity 0.40, specificity 0.96, likelihood 11.30). CONCLUSION Our findings suggest that salivary ALT level and the P. gingivalis ratio may be potential indicators for the progression of periodontitis. Such a salivary test could be a useful diagnostic tool for predicting periodontal disease progression.

Assessment of the Plasma/Serum IgG Test to Screen for Periodontitis

Chronic periodontitis is a silent infectious disease prevalent worldwide and affects lifestyle-related diseases. Therefore, efficient screening of patients is essential for general health. This study was performed to evaluate prospectively the diagnostic utility of a blood IgG antibody titer test against periodontal pathogens. Oral examination was performed, and IgG titers against periodontal pathogens were measured by ELISA in 1,387 individuals. The cut-off value of the IgG titer was determined in receiver operating characteristic curve analysis, and changes in periodontal clinical parameters and IgG titers by periodontal treatment were evaluated. The relationships between IgG titers and severity of periodontitis were analyzed. The best cut-off value of IgG titer against Porphyromonas gingivalis for screening periodontitis was 1.682. Both clinical parameters and IgG titers decreased significantly under periodontal treatment. IgG titers of periodontitis patients were significantly higher than those of healthy controls, especially in those with sites of probing pocket depth over 4 mm. Multiplied cut-off values were useful to select patients with severe periodontitis. A blood IgG antibody titer test for Porphyromonas gingivalis is useful to screen hitherto chronic periodontitis patients (ClinicalTrials.gov number NCT01658475).

Human periodontal ligament fibroblasts are the optimal cell source for induced pluripotent stem cells.

Among the various kinds of fibroblasts existing in the human body, the periodontal ligament (PDL) fibroblasts have been suggested as multipotent cells. Periodontal ligament fibroblasts are characterized by rapid turnover, a high remodeling capacity and remarkable capacity for renewal and repair. They also differentiate into osteoblasts and cementoblasts. We established iPS cells from human PDL fibroblasts by introducing the ES cell markers Oct3/4, Sox2, Nanog, Klf4 and Lin28 by retrovirus transduction, even without the oncogene c-Myc. The iPS cells established in this study expressed the ES cell markers and formed teratomas in SCID mice. The c-Myc expression level in the PDL fibroblasts was higher than that in the iPS cells by quantitative RT-PCR. Therefore, we have concluded that PDL fibroblasts could be an optimal cell source for iPS cells.

Morphological changes in the rat periodontal ligament and its vascularity after experimental tooth movement using superelastic forces.

The aim of this study was to statistically assess the morphological changes of the rat periodontal ligament (PDL) and its vascularity in relation to varied magnitudes of superelastic force in experimental tooth movement using nickel-titanium (NiTi) alloy wire. Forces of 0.8, 1.6, 4, 8, and 18 g were applied to the upper first molars of five groups of 10-week-old male Wistar rats (300-320 g) for 1, 7, 14, 21, and 28 days. A control group with no orthodontic appliance application was assessed in accordance with the five experimental periods. The specimens were observed under light microscopy, processed by computer imaging, and analysed statistically with Tukeys HSD non-parametric test. One day after the start of the experiment, a few blood vessels could be seen in the compressed PDL with forces of 0.8 and 1.6 g. The cross-sectional areas of blood vessels (CAV) and periodontal ligament (CAPL) in the experimental groups where a force of over 4 g was applied were significantly smaller than those where 0.8 and 1.6 g forces were used, and in the control group. On day 7, large CAV were seen in the 1.6, 4, and 8 g groups. On day 28, the 8 and 18 g groups showed significantly larger CAPL than the 0.8, 4 g, or control groups. The findings suggest that a light continuous force, under 1.6 g, maintains the vascular structure during experimental tooth movement. In contrast, a heavy continuous force over 4 g causes the vascular structure to be absent in the early stages of tooth movement, but a dynamic regeneration of the PDL with vascularity and expansion follows.

Time-lapse observation of rat periodontal ligament during function and tooth movement, using microcomputed tomography

The aim of this study was to observe the time-lapse changes in the rat periodontal ligament (PDL) during function and tooth movement. Under Nembutal anaesthesia, time-lapse changes in the thickness of the PDL of the first molars were investigated in five 12-week-old adolescent rats with microcomputed tomography. Three-dimensional (3D) images were reconstructed from the data. Histological observation was also performed, using undecalcified frozen sections of the maxillary first molar area. The PDL appeared as a radiolucent furrow on the 3D images. A slight change in the thickness of the PDL was observed 1 hour after initiation of orthodontic force loading, which became significant after 6 hours, with the appearance of pressure-tension zones during the tooth movement. These changes were more significant 3 days after orthodontic loading. Histological observation of the lingual cervical PDL (pressure zone) in nine 12- to 13-week-old rats demonstrated that the periodontal space had become narrow and the cellular elements appeared to be densely packed in the narrowed PDL 6 hours after orthodontic loading. Degeneration of tissues appeared 3 days after loading. Observation of the buccal cervical PDL (tension zone) demonstrated that the PDL was extended 6 hours after orthodontic force loading, and the extension continued for up to 3 days. Alkaline phosphatase activity was distributed in the PDL, except for the degenerating tissues in the pressure zone 3 days after loading. The results suggest that the periodontal reaction was initiated within 6 hours after orthodontic force loading, which was related to the structural changes of the PDL. The changes probably induced an early response in individual cells of the PDL.

Screening of Probiotic Candidates in Human Oral Bacteria for the Prevention of Dental Disease

The oral cavity in healthy subjects has a well-balanced microbiota that consists of more than 700 species. However, a disturbance of this balance, with an increase of harmful microbes and a decrease of beneficial microbes, causes oral disorders such as periodontal disease or dental caries. Nowadays, probiotics are expected to confer oral health benefits by modulating the oral microbiota. This study screened new probiotic candidates with potential oral health benefits and no harmful effects on the oral cavity. We screened 14 lactobacillus strains and 36 streptococcus strains out of 896 oral isolates derived from healthy subjects. These bacteria did not produce volatile sulfur compounds or water-insoluble glucan, had higher antibacterial activity against periodontal bacteria, and had higher adherence activity to oral epithelial cells or salivary-coated hydroxyapatite in vitro. We then evaluated the risk of primary cariogenicity and infective endocarditis of the selected oral isolates. As a result, Lactobacillus crispatus YIT 12319, Lactobacillus fermentum YIT 12320, Lactobacillus gasseri YIT 12321, and Streptococcus mitis YIT 12322 were selected because they showed no cariogenic potential in an artificial mouth system and a lower risk of experimental infective endocarditis in a rat model. These candidates are expected as new probiotics with potential oral health benefits and no adverse effects on general health.

Application of chimeric glucanase comprising mutanase and dextranase for prevention of dental biofilm formation

Water‐insoluble glucan (WIG) produced by mutans streptococci, an important cariogenic pathogen, plays an important role in the formation of dental biofilm and adhesion of biofilm to tooth surfaces. Glucanohydrolases, such as mutanase (α‐1,3‐glucanase) and dextranase (α‐1,6‐glucanase), are able to hydrolyze WIG. The purposes of this study were to construct bi‐functional chimeric glucanase, composed of mutanase and dextranase, and to examine the effects of this chimeric glucanase on the formation and decomposition of biofilm. The mutanase gene from Paenibacillus humicus NA1123 and the dextranase gene from Streptococcus mutans ATCC 25175 were cloned and ligated into a pE‐SUMOstar Amp plasmid vector. The resultant his‐tagged fusion chimeric glucanase was expressed in Escherichia coli BL21 (DE3) and partially purified. The effects of chimeric glucanase on the formation and decomposition of biofilm formed on a glass surface by Streptococcus sobrinus 6715 glucosyltransferases were then examined. This biofilm was fractionated into firmly adherent, loosely adherent, and non‐adherent WIG fractions. Amounts of WIG in each fraction were determined by a phenol‐sulfuric acid method, and reducing sugars were quantified by the Somogyi–Nelson method. Chimeric glucanase reduced the formation of the total amount of WIG in a dose‐dependent manner, and significant reductions of WIG in the adherent fraction were observed. Moreover, the chimeric glucanase was able to decompose biofilm, being 4.1 times more effective at glucan inhibition of biofilm formation than a mixture of dextranase and mutanase. These results suggest that the chimeric glucanase is useful for prevention of dental biofilm formation.

Salivary pathogen and serum antibody to assess the progression of chronic periodontitis: a 24-mo prospective multicenter cohort study

BACKGROUND AND OBJECTIVE A diagnosis of periodontitis progression is presently limited to clinical parameters such as attachment loss and radiographic imaging. The aim of this multicenter study was to monitor disease progression in patients with chronic periodontitis during a 24-mo follow-up program and to evaluate the amount of bacteria in saliva and corresponding IgG titers in serum for determining the diagnostic usefulness of each in indicating disease progression and stability. MATERIAL AND METHODS A total of 163 patients with chronic periodontitis who received trimonthly follow-up care were observed for 24 mo. The clinical parameters and salivary content of Porphyromonas gingivalis, Prevotella intermedia and Aggregatibacter actinomycetemcomitans were assessed using the modified Invader PLUS assay, and the corresponding serum IgG titers were measured using ELISA. The changes through 24 mo were analyzed using cut-off values calculated for each factor. One-way ANOVA or Fishers exact test was used to perform between-group comparison for the data collected. Diagnostic values were calculated using Fishers exact test. RESULTS Of the 124 individuals who completed the 24-mo monitoring phase, 62 exhibited periodontitis progression, whereas 62 demonstrated stable disease. Seven patients withdrew because of acute periodontal abscess. The ratio of P. gingivalis to total bacteria and the combination of P. gingivalis counts and IgG titers against P. gingivalis were significantly related to the progression of periodontitis. The combination of P. gingivalis ratio and P. gingivalis IgG titers was significantly associated with the progression of periodontitis (p = 0.001, sensitivity = 0.339, specificity = 0.790). CONCLUSIONS It is suggested that the combination of P. gingivalis ratio in saliva and serum IgG titers against P. gingivalis may be associated with the progression of periodontitis.

Streptococcus troglodytae sp. nov., from the chimpanzee oral cavity.

Six strains, TKU 25, TKU 28, TKU 30, TKU 31(T), TKU 33 and TKU 34, were isolated from the oral cavity of a chimpanzee (Pan troglodytes). Colonies of strains grown on Mitis-Salivarius agar were similar in morphology to that of Streptococcus mutans. The novel strains were Gram-stain-positive, facultatively anaerobic cocci that lacked catalase activity. Analysis of the partial 16S rRNA gene sequences of these isolates showed that the most closely related strain was the type strain of S. mutans (96.4 %). The next closely related strains to the isolates were the type strains of Streptococcus devriesei (94.5 %) and Streptococcus downei (93.9 %). These isolates could be distinguished from S. mutans by inulin fermentation and alkaline phosphatase activity (API ZYM system). The peptidoglycan type of the novel isolates was Glu-Lys-Ala(3). Strains were not susceptible to bacitracin. On the basis of phenotypic characterization, partial 16S rRNA gene and two housekeeping gene (groEL and sodA) sequence data, we propose a novel taxon, Streptococcus troglodytae sp. nov.; the type strain is TKU 31(T) ( = JCM 18038(T) = DSM 25324(T)).

Laser capture microdissection of rat periodontal ligament for gene analysis

The periodontal ligament (PDL) is a connective tissue interposed between two hard tissues, viz., the root of a tooth and the alveolar bone, which makes it difficult to obtain directly. In the study reported here, PDL, subgingival connective tissue and pulp of rat molars were extracted directly by laser capture microdissection and the gene expression of TGF-β1 on the microdissected PDL was examined. The maxillae of rats were dissected and rapidly immersed in isopentane cooled with liquid nitrogen. Serial frontal sections of the rat first molar area were used for immunohistochemistry and for laser capture microdissection to localize the TGF-β1 gene. Gene expression and immunohistochemical localization of TGF-β1 also were examined in the pulp and subgingival connective tissues. TGF-β1 was located immunohistochemically in the fibroblasts in the PDL. A considerable amount of RNA was obtained by laser capture microdissection of these three tissues for analysis of gene expression. The reverse transcription- polymerase chain reaction demonstrated that glyceraldehyde 3-phosphate dehydrogenase was amplified in all three tissues, and that TGF-β1 was detected in the PDL. Laser capture microdissection makes it possible to analyze the gene expression of PDL and expression of TGF-β1in the PDL suggests that this gene could function in maintaining PDL.