Periodontal Regenerative Therapy Using rhFGF-2 and Deproteinized Bovine Bone Mineral versus rhFGF-2 Alone: 4-Year Extended Follow-Up of a Randomized Controlled Trial.

The aim of this study was to evaluate longitudinal outcomes of recombinant human fibroblast growth factor (rhFGF)-2 plus deproteinized bovine bone mineral (DBBM) therapy in comparison with rhFGF-2 alone for treating periodontal intrabony defects. This study describes 4-year follow-up outcomes of the original randomized controlled trial. Intrabony defects in periodontitis patients were treated with rhFGF-2 (control) or rhFGF-2 plus DBBM (test). Clinical, radiographic, and patient-reported outcome (PRO) measures were used to evaluate the outcomes. Thirty-two sites were able to be followed up. At 4 years postoperatively, clinical attachment level (CAL) gains in the test and control groups were 3.5 ± 1.4 mm and 2.7 ± 1.4 mm, respectively, showing significant improvement from preoperative values but no difference between groups. Both groups showed an increase in radiographic bone fill (RBF) over time. At 4 years, the mean value for RBF in the test group (62%) was significantly greater than that in the control group (42%). In 1-2-wall defects, the test treatment yielded significantly greater RBF than the control treatment. No significant difference in PRO scores was noted between the groups. Although no significant difference in CAL gain was found between the groups at the 4-year follow-up, the combination treatment significantly enhanced RBF. Favorable clinical, radiographic outcomes, and PRO in both groups can be maintained for at least 4 years.

Taxonomic and Gene Category Analyses of Subgingival Plaques from a Group of Japanese Individuals with and without Periodontitis.

Periodontitis is an inflammation of tooth-supporting tissues, which is caused by bacteria in the subgingival plaque (biofilm) and the host immune response. Traditionally, subgingival pathogens have been investigated using methods such as culturing, DNA probes, or PCR. The development of next-generation sequencing made it possible to investigate the whole microbiome in the subgingival plaque. Previous studies have implicated dysbiosis of the subgingival microbiome in the etiology of periodontitis. However, details are still lacking. In this study, we conducted a metagenomic analysis of subgingival plaque samples from a group of Japanese individuals with and without periodontitis. In the taxonomic composition analysis, genus Bacteroides and Mycobacterium demonstrated significantly different compositions between healthy sites and sites with periodontal pockets. The results from the relative abundance of functional gene categories, carbohydrate metabolism, glycan biosynthesis and metabolism, amino acid metabolism, replication and repair showed significant differences between healthy sites and sites with periodontal pockets. These results provide important insights into the shift in the taxonomic and functional gene category abundance caused by dysbiosis, which occurs during the progression of periodontal disease.

Role of Hyalin-like Protein in Gliding and Biofilm Formation by Capnocytophaga Ochracea.

Capnocytophaga ochracea possesses a type-IX secretion system that exports proteins which have a gliding motility-associated C-terminal (CTD) domain. This system is found in several species of the Bacteroidetes phylum. Hyalin, a large protein encoded by Coch_0033 in C. ochracea ATCC 27872, has a CTD domain and is posited to be involved in quorum sensing according to the database of the Kyoto Encyclopedia of Genes and Genomes. This suggests that it plays a role in biofilm formation via interbacterial communication. The aim of this study was to investigate the potential role of the hyalin-like protein coded by the Coch_0033 gene in gliding and biofilm formation of C. ochracea. A hyalin-like protein-deficient mutant strain of C. ochracea, designated mutant WR-1, was constructed through insertion of the ermF-ermAM cassette into the target gene. The spreading feature at the edge of the colony was lost in the mutant strain. Crystal violet and confocal laser scanning microscopy revealed no difference between the quantity of biofilm organized by the mutant and that organized by the wild-type strain. These data suggest that the hyalin-like protein encoded by the Coch_0033 gene is indeed involved in C. ochracea gliding activity.

Periodontal surgery using rhFGF-2 with deproteinized bovine bone mineral or rhFGF-2 alone: 2-year follow-up of a randomized controlled trial.

AIM: To compare outcomes of rhFGF-2 + DBBM therapy with rhFGF-2 alone in the treatment of intrabony defects. This study provides 2-year follow-up results from the previous randomized controlled trial. MATERIALS AND METHODS: Defects were randomly allocated to receive rhFGF-2 + DBBM (test) or rhFGF-2 (control). Treated sites were re-evaluated at 2 years postoperatively, using original clinical and patient-centred measures. RESULTS: Thirty-eight sites were available for re-evaluation. At 2 years, both groups showed a significant improvement in clinical attachment level (CAL) from baseline. A gain in CAL of 3.4 ± 1.3 mm in the test group and 3.1 ± 1.5 mm in the control group was found. No significant inter-group difference was noted. Both groups showed a progressive increase in radiographic bone fill (RBF). The test treatment yielded greater RBF (56%) compared with the control group (41%). The control treatment performed better in contained defects in terms of CAL and RBF. There was no significant difference in patient-reported outcomes between groups. CONCLUSIONS: At 2-year follow-up, the test and cotrol treatments were similarly effective in improving CAL, whereas the test treatment achieved a significantly greater RBF. In both treatments, favourable clinical, radiographic, and patient-reported outcomes can be sustained for at least 2 years. TRIAL REGISTRATION: The University Hospital Medical Information Network-Clinical Trials Registry (UMIN-CTR) 000025257.

Treatment of intrabony periodontal defects using rhFGF-2 in combination with deproteinized bovine bone mineral or rhFGF-2 alone: A 6-month randomized controlled trial.

AIM: To evaluate the use of recombinant human fibroblast growth factor (rhFGF)-2 in combination with deproteinized bovine bone mineral (DBBM) compared with rhFGF-2 alone, in the treatment of intrabony periodontal defects. MATERIALS AND METHODS: Patients with periodontitis who had received initial periodontal therapy and had intrabony defects of ≥ 3 mm in depth were enrolled. Sites were randomly assigned to receive a commercial formulation of 0.3% rhFGF-2 + DBBM (test) or rhFGF-2 alone (control). Clinical parameters and a patient-reported outcome measure (PROM) were evaluated at baseline and at 3 and 6 months postoperatively. RESULTS: Twenty-two sites in each group were evaluated. A significant improvement in clinical attachment level (CAL) from baseline was observed in both groups at 6 months postoperatively. CAL gain was 3.16 ± 1.45 mm in the test group and 2.77 ± 1.15 mm in the control group, showing no significant difference between groups. Radiographic bone fill was significantly greater in the test group (47.2%) than in the control group (29.3%). No significant difference in PROM between groups was observed. CONCLUSIONS: At 6 months, no significant difference in CAL gain or PROM between the two treatments was observed, although combination therapy yielded an enhanced radiographic outcome.

Treatment of Gingival Fenestration with Connective Tissue Graft: A Case Report.

Here, we report a case of gingival fenestration requiring periodontal plastic surgery. The patient was a 32-year-old man presenting with the chief complaint of esthetic impairment and gingival twitching due to gingival fenestration. Baseline examination revealed localized periodontal breakdown, including gingival fenestration in the lower right central incisor (#41). Periodontal examination revealed 3% of sites with a probing depth of ≥4 mm and 8.9% with bleeding on probing. Radiographic examination revealed vertical bone loss in #15 and 36, together with buccal fenestration in #41. Based on a clinical diagnosis of chronic periodontitis with gingival fenestration, initial periodontal therapy comprised plaque control and scaling and root planing. Following suppression of inflammation, occlusal adjustment was performed in the anterior teeth. As plastic surgery, combined use of an elevated flap and a connective tissue graft was applied at #41. Following reevaluation, the patient was placed on maintenance care. The patient's periodontal condition has remained stable over a 6-month period.

Clinical effect of a dentifrice containing three kinds of bactericidal ingredients on periodontal disease: a pilot study in patients undergoing supportive periodontal therapy.

OBJECTIVE: This study aimed to evaluate clinically the effect of a novel dentifrice containing three kinds of bactericidal ingredients on periodontal disease. RESULTS: This was a single-arm, prospective clinical study that enrolled patients with periodontitis undergoing supportive periodontal therapy. Periodontal examination, microbiological testing of saliva samples, and evaluation of inflammatory markers (IL-1ß, IL-6, IL-8, TNF-α) in gingival crevicular fluid were performed. After 4 weeks of the use of test dentifrice, these parameters were re-evaluated. The use of dentifrice was also subjectively evaluated by clinicians and participants. Among 30 participants, there were significant improvements in the periodontal and microbiological parameters, and the level of interleukin-1ß in the gingival crevicular fluid, following the use of the test dentifrice. In clinicians' subjective evaluation of the overall usefulness of the dentifrice, 'mild' and 'moderate' improvement accounted for 83% of the total responses. In the participants' subjective evaluation, the majority indicated their experience of the use as favorable. Within the limitations of this study, it is suggested that the progression of periodontal disease during the supportive periodontal therapy can be prevented by the use of the test dentifrice. Trial registration UMIN Clinical Trials Registry (UMIN-CTR) 000023175. Date of formal registration: July 14, 2016 ( https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000026716 ).

SigCH, an extracytoplasmic function sigma factor of Porphyromonas gingivalis regulates the expression of cdhR and hmuYR.

Extracytoplasmic function (ECF) sigma factors play an important role in the bacterial response to various environmental stresses. Porphyromonas gingivalis, a prominent etiological agent in human periodontitis, possesses six putative ECF sigma factors. So far, information is limited on the ECF sigma factor, PGN_0319. The aim of this study was to investigate the role of PGN_0319 (SigCH) of P. gingivalis, focusing on the regulation of hmuY and hmuR, which encode outer-membrane proteins involved in hemin utilization, and cdhR, a transcriptional regulator of hmuYR. First, we evaluated the gene expression profile of the sigCH mutant by DNA microarray. Among the genes with altered expression levels, those involved in hemin utilization were downregulated in the sigCH mutant. To verify the microarray data, quantitative reverse transcription PCR analysis was performed. The RNA samples used were obtained from bacterial cells grown to early-log phase, in which sigCH expression in the wild type was significantly higher than that in mid-log and late-log phases. The expression levels of hmuY, hmuR, and cdhR were significantly decreased in the sigCH mutant compared to wild type. Transcription of these genes was restored in a sigCH complemented strain. Compared to the wild type, the sigCH mutant showed reduced growth in log phase under hemin-limiting conditions. Electrophoretic mobility shift assays showed that recombinant SigCH protein bound to the promoter region of hmuY and cdhR. These results suggest that SigCH plays an important role in the early growth of P. gingivalis, and directly regulates cdhR and hmuYR, thereby playing a potential role in the mechanisms of hemin utilization by P. gingivalis.

In vitro Effect of Mouthrinse Containing Essential Oils on Proliferation and Migration of Gingival Epithelial Cells.

We aimed to investigate in vitro the effects of mouthrinses containing essential oils (EOs) on proliferation and migration of gingival epithelial cells. Human gingival epithelial cells were treated with predetermined dilutions of commercially available EO mouthrinses with or without ethanol and a mouthrinse containing cetyl pyridinium chloride (CPC) for 60 s. Cell proliferation was evaluated using WST-1 assay. Cell migration was assessed using a wound closure model. Within 10 s of exposure to EO mouthrinse without ethanol, the epithelial cells became aberrant and shrank. No statistically significant difference in cell migration or proliferation was observed among cells pretreated by the EO mouthrinse with ethanol, CPC mouthrinse and control (phosphate buffered saline). In contrast, the EO mouthrinse without ethanol significantly reduced cell proliferation (p < 0.001) to approximately 20% relative to control. As for the EO mouthrinse without ethanol, it was not possible to assess its effect on cell migration using this model, because treated cells could be easily detached from the culture plate upon scratch, possibly because of the surfactant ingredient in the formulation. Within the limitations of the study, the EO mouthrinse with ethanol exerted no inhibitory effect on proliferation and migration of the gingival epithelial cells. Copyright © 2016 John Wiley & Sons, Ltd.

Involvement of the Type IX Secretion System in Capnocytophaga ochracea Gliding Motility and Biofilm Formation.

Capnocytophaga ochracea is a Gram-negative, rod-shaped bacterium that demonstrates gliding motility when cultured on solid agar surfaces. C. ochracea possesses the ability to form biofilms; however, factors involved in biofilm formation by this bacterium are unclear. A type IX secretion system (T9SS) in Flavobacterium johnsoniae was shown to be involved in the transport of proteins (e.g., several adhesins) to the cell surface. Genes orthologous to those encoding T9SS proteins in F. johnsoniae have been identified in the genome of C. ochracea; therefore, the T9SS may be involved in biofilm formation by C. ochracea. Here we constructed three ortholog-deficient C. ochracea mutants lacking sprB (which encodes a gliding motility adhesin) or gldK or sprT (which encode T9SS proteins in F. johnsoniae). Gliding motility was lost in each mutant, suggesting that, in C. ochracea, the proteins encoded by sprB, gldK, and sprT are necessary for gliding motility, and SprB is transported to the cell surface by the T9SS. For the ΔgldK, ΔsprT, and ΔsprB strains, the amounts of crystal violet-associated biofilm, relative to wild-type values, were 49%, 34%, and 65%, respectively, at 48 h. Confocal laser scanning and scanning electron microscopy revealed that the biofilms formed by wild-type C. ochracea were denser and bacterial cells were closer together than in those formed by the mutant strains. Together, these results indicate that proteins exported by the T9SS are key elements of the gliding motility and biofilm formation of C. ochracea.

Involvement of luxS in Biofilm Formation by Capnocytophaga ochracea.

Capnocytophaga ochracea is present in the dental plaque biofilm of patients with periodontitis. Biofilm cells change their phenotype through quorum sensing in response to fluctuations in cell-population density. Quorum sensing is mediated by auto-inducers (AIs). AI-2 is involved in intercellular signaling, and production of its distant precursor is catalyzed by LuxS, an enzyme involved in the activated methyl cycle. Our aim was to clarify the role of LuxS in biofilm formation by C. ochracea. Two luxS-deficient mutants, TmAI2 and LKT7, were constructed from C. ochracea ATCC 27872 by homologous recombination. The mutants produced significantly less AI-2 than the wild type. The growth rates of these mutants were similar to that of the wild-type in both undiluted Tryptic soy broth and 0.5 × Tryptic soy broth. However, according to crystal violet staining, they produced significantly less biofilm than the wild type. Confocal laser scanning microscopy and scanning electron microscopy showed that the biofilm of the TmAI2 strain had a rougher structure than that of the wild type. Complementation of TmAI-2 with extrinsic AI-2 from the culture supernatant of wild-type strain did not restore biofilm formation by the TmAI2 strain, but complementation of LKT7 strain with luxS partially restored biofilm formation. These results indicate that LuxS is involved in biofilm formation by C. ochracea, and that the attenuation of biofilm formation by the mutants is likely caused by a defect in the activated methyl cycle rather than by a loss of AI-2.

Effect of a mouthrinse containing rice peptide CL(14-25) on early dental plaque regrowth: a randomized crossover pilot study.

BACKGROUND: We aimed to evaluate clinically the effect of mouthrinse containing a rice peptide on early dental plaque regrowth. METHODS: The study was designed as a double-masked, two-group crossover randomized pilot trial, involving 10 periodontally healthy volunteers. After receiving a professional tooth cleaning at baseline, over the next 3 days each participant refrained from all oral hygiene measures and had two daily rinses with 20 ml of the test mouthrinse containing 0.4 % rice peptide CL(14-25) or placebo rinse. At the end of each experimental period, plaque score was assessed using the modified Volpe's method, and the participants filled out a questionnaire. Each participant underwent a 7-day washout period followed by a second allocation. The plaque score was the primary outcome of the study and participant perception was the secondary outcome. RESULTS: No adverse effects were observed in the participants during the study. Clinically, the mean plaque score of the examined teeth was significantly lower in the test group (2.44 ± 0.74, CI: 1.91-2.96) than the placebo group (2.65 ± 0.63, CI: 2.20-3.10) (P < 0.05). When analyzed according to the type of teeth, a significantly lower score of the premolars/molars was observed in the test group (2.39 ± 0.68, CI: 2.08-2.71) than that in the placebo group (2.66 ± 0.58, CI: 2.39-2.93) (P < 0.05). CONCLUSIONS: The mouthrinse containing 0.4 % rice peptide CL(14-25) was effective in reducing the early regrowth of dental plaque. However, clinical relevance of this efficacy needs to be validated in a future large-scale study. TRIAL REGISTRATION: UMIN Clinical Trials Registry (UMIN-CTR) R000014000. Date of formal registration: November 1, 2013.

Changes in antimicrobial susceptibility profile and prevalence of quinolone low-sensitive strains in subgingival plaque from acute periodontal lesions after systemic administration of sitafloxacin.

This study aimed to assess changes in antimicrobial susceptibilities of subgingival bacteria in acute periodontal lesions following systemic administration of a new-generation fluoroquinolone, sitafloxacin and to monitor the occurrence and fate of quinolone low-sensitive strains. Patients with acute phase of chronic periodontitis were subjected to microbiological assessment of their subgingival plaque samples at baseline (A1). Sitafloxacin was then administered systemically (100 mg/day for 5 days). The microbiological examinations were repeated one week after administration (A2). Susceptibilities of clinical isolates from acute sites to various antimicrobials were determined using broth and agar dilution methods. At A2, subgingival bacteria with low sensitivity to levofloxacin were identified in four patients, and they were subjected to a follow-up microbiological examination at on the average 12 months after sitafloxacin administration (A3). The patients received initial and supportive periodontal therapy during the period A2 to A3. From the examined subgingival sites, 8 and 19 clinical isolates were obtained at A2 and A3, respectively. Some Streptococcus strains isolated at A2 were found to be resistant to levofloxacin (MIC 16-64 µg/ml), azithromycin (MIC 2->128 µg/ml) or clarithromycin (MIC 1->32 µg/ml). At A3, isolated streptococci were highly susceptible to levofloxacin (MIC 0.5-2 µg/ml), while those resistant to azithromycin or clarithromycin were still isolated. It is suggested that the presence of the quinolone low-sensitive strains in initially acute lesions after sitafloxacin administration was transient, and they do not persist in the subgingival milieu during the periodontal therapy.

Effects of systemic administration of sitafloxacin on subgingival microflora and antimicrobial susceptibility profile in acute periodontal lesions.

The aim of this study was to assess the effect(s) of systemic administration of sitafloxacin on subgingival microbial profiles of acute periodontal lesions. Antimicrobial susceptibility of clinical isolates was also investigated. Patients with acute phases of chronic periodontitis were subjected to clinical examination and microbiological assessment of their subgingival plaque samples by culture technique. Sitafloxacin was then administered (100 mg/day for 5 days) systemically. The clinical and microbiological examinations were repeated 6-8 days after administration. Susceptibilities of clinical isolates to various antimicrobials were determined using the broth and agar dilution methods. From the sampled sites in 30 participants, a total of 355 clinical isolates (34 different bacterial species) were isolated and identified. Parvimonas micra, Prevotella intermedia and Streptococcus mitis were the most prevalent cultivable bacteria in acute sites. Systemic administration of sitafloxacin yielded a significant improvement in clinical and microbiological parameters. Among the antimicrobials tested, sitafloxacin was the most potent against the clinical isolates with an MIC90 of 0.12 µg/ml at baseline. After administration, most clinical isolates were still highly susceptible to sitafloxacin although some increase in MICs was observed. The results suggest that systemic administration of sitafloxacin is effective against subgingival bacteria isolated from acute periodontal lesions.

Prevalence of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythia in Japanese patients with generalized chronic and aggressive periodontitis.

This study aimed to investigate the prevalence and levels of major periodontal pathogens, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythia in subgingival plaque samples of a group of Japanese patients with aggressive periodontitis (AgP) and chronic periodontitis (CP). A total of 40 patients with clinical diagnosis of AgP or CP and 10 periodontally healthy volunteers were subjected to clinical and microbiological analysis. Subgingival plaque samples were analyzed for A. actinomycetemcomitans, P. gingivalis and T. forsythia with a real-time polymerase chain reaction (PCR) technique. The prevalence of P. gingivalis and T. forsythia was relatively high in patients with periodontitis: over 60% of AgP or CP patients harbored these pathogens whereas they were not detected in the subgingival plaque samples from periodontally healthy individuals. P. gingivalis and T. forsythia were relatively frequently detected together in AgP and CP patients. No significant differences in the prevalence or level of the 3 pathogens were found between periodontitis groups. The proportion of T. forsythia was approximately 4-fold higher in CP group than in AgP group (P = 0.02). In periodontitis patients, a significant positive correlation was found between periodontal parameters (probing depth and clinical attachment level) and the numbers of total bacteria, P. gingivalis and T. forsythia. No distinct pattern of the subgingival profile of these pathogens was discerned between the two disease entities, except for the difference in the proportion of T. forsythia. The red complex bacteria, P. gingivalis and T. forsythia were highly prevalent in this population of Japanese AgP and CP patients, collaborating their roles in periodontitis.

Porphyromonas gingivalis entry into gingival epithelial cells modulated by Fusobacterium nucleatum is dependent on lipid rafts.

Host cell invasion by a major periodontal pathogen, Porphyromonas gingivalis, has been proposed as an important mechanism involved in host-pathogen interactions in periodontal and cardiovascular diseases. The present study sought to gain insight into the underlying mechanism(s) involved in previously demonstrated fusobacterial modulation of host cell invasion by P. gingivalis. An immortalized human gingival cell line Ca9-22 was dually infected with P. gingivalis ATCC 33277 and Fusobacterium nucleatum TDC 100, and intracellular invasion was assessed by scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM). SEM observation showed that P. gingivalis and F. nucleatum formed consortia and were in the process of penetrating into Ca9-22 by 30-60 min after infection. In CSLM, Ca9-22 cells that contained both P. gingivalis and F. nucleatum were frequently observed after 2 h, although cells that contained exclusively P. gingivalis were also found. Infection by P. gingivalis and/or F. nucleatum revealed evident colocalization with a lipid raft marker, GM1-containing membrane microdomains. In an antibiotic protection assay, depletion of epithelial plasma membrane cholesterol resulted in a significant reduction of recovered P. gingivalis or F. nucleatum (∼33% of untreated control; p < 0.001). This inhibition was also confirmed by CSLM. Sequential infection experiments showed that timing of infection by each species could critically influence the invasion profile. Co-infection with F. nucleatum significantly enhanced host cell invasion by P. gingivalis 33277, its serine phophatase SerB mutant and complemented strains, suggesting that the SerB does not play a major role in this fusobacterial enhancement of P. gingivalis invasion. Thus, the interaction between F. nucleatum and host cells may be important in the fusobacterial enhancement of P. gingivalis invasion. Collectively, these results suggest that lipid raft-mediated process is at least one of the potential mechanisms involved in fusobacterium-modulated host cell invasion by P. gingivalis.