An exploratory clinical trial to evaluate the safety and efficacy of combination therapy of REGROTH® and Cytrans® granules for severe periodontitis with intrabony defects.

Introduction: Currently, flap operation (FOP) using REGROTH® (0.3% basic fibroblast growth factor [FGF-2]) is the standard treatment for periodontal regenerative therapy in Japan. However, the periodontal tissue regenerative effect with REGROTH® monotherapy is inadequate for severe alveolar bone defects. Therefore, in this study, we evaluated the safety and effectiveness of periodontal regenerative therapy for patients with severe periodontitis using REGROTH® (test medicine) combined with Cytrans® Granules (test device: carbonated apatite granules), which is a new artificial bone. Methods: The study participants included 10 patients with severe periodontitis (mean age: 47.4 years). All participants provided written informed consents. In each patient, the intrabony defect site (mean bone defect depth: 5.7 mm) was defined as the test site. FOP was performed for the test site after the baseline investigation; moreover, the test medicine and test device were administered simultaneously. Furthermore, the observation of subjects' general condition and test sites was conducted and the blood, urine, and periodontal tissue tests were performed up to 36 weeks after FOP. The rate of bone increase (%), clinical attachment level (CAL), probing pocket depth (PPD), bleeding on probing (BOP), tooth mobility (Mo), width of keratinized gingiva (KG), gingival recession (REC), gingival index (GI), and plaque index (PlI) were evaluated during the periodontal tissue investigation. Results: As the primary endpoint, no adverse events related to the test medicine and test device occurred during the entire observation period of this study. Regarding the secondary endpoints, there was a significant increase in new alveolar bone (p = 0.003) and CAL acquisition (p = 0.001) as well as decrease in PPD (p = 0.002) and BOP (p = 0.016) at 36 weeks after administration of the test medicine and test device compared with the preoperative values. Furthermore, at 36 weeks after surgery, the Mo, GI, and PlI decreased to preoperative levels at 40%, 60%, and 30% of sites, respectively. However, at 36 weeks after surgery, there was no difference in KG and REC compared with their preoperative values. Conclusions: The safety of periodontal regenerative therapy using the test medicine in combination with the abovementioned test device was confirmed. In addition, it was suggested that this periodontal regenerative therapy is effective for tissue regeneration in severe alveolar bone defects.This clinical trial was conducted after registering and publicizing as a specified clinical trial in the Japan registry of clinical trials (jRCTs051190045).

Porphyromonas gingivalis induces entero-hepatic metabolic derangements with alteration of gut microbiota in a type 2 diabetes mouse model.

Periodontal infection induces systemic inflammation; therefore, aggravating diabetes. Orally administered periodontal pathogens may directly alter the gut microbiota. We orally treated obese db/db diabetes mice using Porphyromonas gingivalis (Pg). We screened for Pg-specific peptides in the intestinal fecal specimens and examined whether Pg localization influenced the intestinal microbiota profile, in turn altering the levels of the gut metabolites. We evaluated whether the deterioration in fasting hyperglycemia was related to the changes in the intrahepatic glucose metabolism, using proteome and metabolome analyses. Oral Pg treatment aggravated both fasting and postprandial hyperglycemia (P < 0.05), with a significant (P < 0.01) increase in dental alveolar bone resorption. Pg-specific peptides were identified in fecal specimens following oral Pg treatment. The intestinal Pg profoundly altered the gut microbiome profiles at the phylum, family, and genus levels; Prevotella exhibited the largest increase in abundance. In addition, Pg-treatment significantly altered intestinal metabolite levels. Fasting hyperglycemia was associated with the increase in the levels of gluconeogenesis-related enzymes and metabolites without changes in the expression of proinflammatory cytokines and insulin resistance. Oral Pg administration induced gut microbiota changes, leading to entero-hepatic metabolic derangements, thus aggravating hyperglycemia in an obese type 2 diabetes mouse model.

Periodontal inflamed surface area is associated with hs-CRP in septuagenarian Japanese adults in cross-sectional findings from the SONIC study.

Periodontal disease is a chronic inflammatory condition that affects various peripheral organs. The periodontal inflamed surface area (PISA) quantifies periodontitis severity and the spread of inflammatory wounds. This study aimed to investigate the association between PISA and high-sensitivity C-reactive protein (hs-CRP), a systemic inflammation marker. This study included 250 community-dwelling septuagenarians (69-71 years). We collected information on their medical (e.g., diabetes and dyslipidemia) and dental examinations (e.g., measurement of the probing pocket depth). Generalized linear model analysis was used to explore the association between PISA and hs-CRP levels. There was a significant difference in hs-CRP levels between groups with PISA ≥ 500 and < 500 (p = 0.017). Moreover, the generalized linear model analysis revealed a significant association between PISA and hs-CRP levels (risk ratio = 1.77; p = 0.033) even after adjusting other factors. Further, we found a correlation between PISA and hs-CRP (Spearman's rank correlation coefficient, rs = 0.181; p = 0.023). Our findings suggest that PISA is an effective index for estimating the effect of periodontitis on the whole body, enabling medical-dental cooperation.

Association of periodontal disease with atherosclerosis in 70-year-old Japanese older adults.

Periodontal disease and arteriosclerotic disease are greatly affected by aging. In this study, the association of conventional risk factors and periodontal disease with atherosclerosis was longitudinally examined in Japanese older adults. Subjects in this study were 490 community-dwelling septuagenarians (69-71 years) randomly recruited from the Basic Resident Registry of urban or rural areas in Japan. At the baseline examination, all subjects underwent socioeconomic and medical interviews; medical examinations, including examinations for carotid atherosclerosis, hypertension, diabetes mellitus, and dyslipidemia; and conventional dental examinations, including a tooth count and measurement of probing pocket depth (PPD). After 3 years, 182 septuagenarians who had no atherosclerosis at the baseline examination were registered and received the same examination as at the baseline. In the re-examination conducted 3 years after the baseline survey, 131 (72.0%) of the 182 participants who had no atherosclerosis at the baseline examination were diagnosed with carotid atherosclerosis. Adjusting and analyzing the mutual relationships of the conventional risk factors for atherosclerosis by multiple logistic regression analysis for the 171 septuagenarians with a full set of data, the proportion of teeth with PPD ≥ 4 mm was independently related to the prevalence of atherosclerosis (odds ratio: 1.029, P < 0.022). This longitudinal study of Japanese older adults suggests that periodontal disease is associated with the onset/progression of atherosclerosis. Maintaining a healthy periodontal condition may be an important factor in preventing the development and progression of atherosclerosis.

Development of Oral Care Chip, a novel device for quantitative detection of the oral microbiota associated with periodontal disease.

Periodontal disease, the most prevalent infectious disease in the world, is caused by biofilms formed in periodontal pockets. No specific bacterial species that can cause periodontitis alone has been found in any study to date. Several periodontopathic bacteria are associated with the progress of periodontal disease. Consequently, it is hypothesized that dysbiosis of subgingival microbiota may be a cause of periodontal disease. This study aimed to investigate the relationship between the subgingival microbiota and the clinical status of periodontal pockets in a quantitative and clinically applicable way with the newly developed Oral Care Chip. The Oral Care Chip is a DNA microarray tool with improved quantitative performance, that can be used in combination with competitive PCR to quantitatively detect 17 species of subgingival bacteria. Cluster analysis based on the similarity of each bacterial quantity was performed on 204 subgingival plaque samples collected from periodontitis patients and healthy volunteers. A significant difference in the number of total bacteria, Treponema denticola, Campylobacter rectus, Fusobacterium nucleatum, and Streptococcus intermedia bacteria in any combination of the three clusters indicated that these bacteria gradually increased in number from the stage before the pocket depth deepened. Conversely, Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, and Streptococcus constellatus, which had significant differences only in limited clusters, were thought to increase in number as the pocket depth deepened, after periodontal pocket formation. Furthermore, in clusters where healthy or mild periodontal disease sites were classified, there was no statistically significant difference in pocket depth, but the number of bacteria gradually increased from the stage before the pocket depth increased. This means that quantitative changes in these bacteria can be a predictor of the progress of periodontal tissue destruction, and this novel microbiological test using the Oral Care Chip could be effective at detecting dysbiosis.

FGF-2 promotes initial osseointegration and enhances stability of implants with low primary stability.

OBJECTIVES: The aim of this study was to examine the effect of basic fibroblast growth factor (FGF-2) on osseointegration of dental implants with low primary stability in a beagle dog model. MATERIALS AND METHODS: Customized titanium implants that were designed to have low contact with the existing bone were installed into the edentulous mandible of beagle dogs. To degrade the primary stability of the implants, the diameters of the bone sockets exceeded the implant diameters. FGF-2 (0.3%) plus vehicle (hydroxypropyl cellulose) or vehicle alone was topically applied to the sockets in the FGF-2 and control groups, respectively. In Study 1, the new bone area and length of new bone-to-implant contact (BIC) were evaluated at 4, 8, and 12 weeks after installation using histomorphometry and scanning electron microscopy. In Study 2, the implant stability quotient (ISQ) values were sequentially measured for 16 weeks using an Osstell system. RESULTS: The histomorphometric analysis revealed that the new bone area and length of BIC in the FGF-2 group were significantly larger than those in the control group at 4 weeks. Electron microscopic observation showed intimate contact between the mature lamellar bone and the implant surfaces, osseointegration, in both groups. The ISQ values in the FGF-2 group were significantly increased from 6 to 16 weeks compared with those in the control group. CONCLUSIONS: Taken together, our study demonstrates that FGF-2 promoted new bone formation around the dental implants and subsequent osseointegration, resulting in promotion of stability of implants with low primary stability.

Prevalence and risk factors for peri-implant diseases in Japanese adult dental patients.

We investigated the prevalences and risk factors for peri-implant diseases in Japanese adult dental patients attending a follow-up visit at dental hospitals or clinics as part of their maintenance program. This cross-sectional multicenter study enrolled patients with dental implants who attended regular check-ups as part of a periodontal maintenance program during the period from October 2012 through September 2013. Patients with implants with at least 3 years of loading time were included in the study. The condition of peri-implant tissue was examined and classified into the following categories: healthy, peri-implant mucositis, and peri-implantitis. Patients were also evaluated for implant risk factors. A total of 267 patients (110 men, 157 women; mean age: 62.5 ± 10.7 years) were analyzed. The prevalence of patient-based peri-implant mucositis was 33.3% (n = 89), and the prevalence of peri-implantitis was 9.7% (n = 26). Poor oral hygiene and a history of periodontitis were strong risk factors for peri-implant disease. The present prevalences were lower than those previously reported. The quality of periodontal therapy before and after implant installation and patient compliance and motivation, as indicated by plaque control level, appear to be important in maintaining peri-implant tissue health.

Metabolomic Analysis of Gingival Crevicular Fluid Using Gas Chromatography/Mass Spectrometry.

Periodontitis is one of the most prevalent threats to oral health as the most common cause of tooth loss. In order to perform effective treatment, a clinical test that detect sites where disease activity is high and predicts periodontal tissue destruction is strongly desired, however, it is still difficult to prognose the periodontal tissue breakdown on the basis of conventional methods. The aim of this study is to examine the usefulness of gas chromatography/mass spectrometry (GC/MS), which could eventually be used for on-site analysis of metabolites in gingival crevicular fluid (GCF) in order to objectively diagnose periodontitis at a molecular level. GCF samples were collected from two diseased sites (one site with a moderate pocket and another site with a deep pocket) from each patient and from clinically healthy sites of volunteers. Nineteen metabolites were identified using GC/MS. Total ion current chromatograms showed broad differences in metabolite peak patterns between GCF samples obtained from healthy sites, moderate-pocket sites, and deep-pocket sites. The intensity difference of some metabolites was significant at sites with deep pockets compared to healthy sites. Additionally, metabolite intensities at moderate-pocket sites showed an intermediate profile between the severely diseased sites and healthy sites, which suggested that periodontitis progression could be observed with a changing metabolite profile. Principal component analysis confirmed these observations by clearly delineating healthy sites and sites with deep pockets. These results suggest that metabolomic analysis of GCF could be useful for prediction and diagnosis of periodontal disease in a single visit from a patient and provides the groundwork for establishing a new, on-site diagnostic method for periodontitis.

Long-term Observation of Regenerated Periodontium Induced by FGF-2 in the Beagle Dog 2-Wall Periodontal Defect Model.

The long-term stability and qualitative characteristics of periodontium regenerated by FGF-2 treatment were compared with normal physiological healing tissue controls in a Beagle dog 2-wall periodontal defect model 13 months after treatment by assessing tissue histology and three-dimensional microstructure using micro-computed tomography (µCT). After FGF-2 (0.3%) or vehicle treatment at the defect sites, serial changes in the bone mineral content (BMC) were observed using periodic X-ray imaging. Tissues were harvested at 13 months, evaluated histomorphometrically, and the cortical bone volume and trabecular bone structure of the newly formed bone were analyzed using µCT. FGF-2 significantly increased the BMC of the defect area at 2 months compared with that of the control group, and this difference was unchanged through 13 months. The cortical bone volume was significantly increased by FGF-2, but there was no difference between the groups in trabecular bone structure. Bone maturation was occurring in both groups because of the lower cortical volume and denser trabecular bone than what is found in intact bone. FGF-2 also increased the area of newly formed bone as assessed histomorphometrically, but the ratios of trabecular bone in the defect area were similar between the control and FGF-2 groups. These results suggest that FGF-2 stimulates neogenesis of alveolar bone that is of similar quality to that of the control group. The lengths of the regenerated periodontal ligament and cementum, measured as the distance from the defect bottom to the apical end of the gingival epithelium, and height and area of the newly formed bone in the FGF-2 group were larger than those in the control group. The present study demonstrated that, within the limitation of artificial periodontal defect model, the periodontal tissue regenerated by FGF-2 was maintained for 13 months after treatment and was qualitatively equivalent to that generated through the physiological healing process.

Action Mechanism of Fibroblast Growth Factor-2 (FGF-2) in the Promotion of Periodontal Regeneration in Beagle Dogs.

Fibroblast growth factor-2 (FGF-2) enhances the formation of new alveolar bone, cementum, and periodontal ligament (PDL) in periodontal defect models. However, the mechanism through which FGF-2 acts in periodontal regeneration in vivo has not been fully clarified yet. To reveal the action mechanism, the formation of regenerated tissue and gene expression at the early phase were analyzed in a beagle dog 3-wall periodontal defect model. FGF-2 (0.3%) or the vehicle (hydroxypropyl cellulose) only were topically applied to the defect in FGF-2 and control groups, respectively. Then, the amount of regenerated tissues and the number of proliferating cells at 3, 7, 14, and 28 days and the number of blood vessels at 7 days were quantitated histologically. Additionally, the expression of osteogenic genes in the regenerated tissue was evaluated by real-time PCR at 7 and 14 days. Compared with the control, cell proliferation around the existing bone and PDL, connective tissue formation on the root surface, and new bone formation in the defect at 7 days were significantly promoted by FGF-2. Additionally, the number of blood vessels at 7 days was increased by FGF-2 treatment. At 28 days, new cementum and PDL were extended by FGF-2. Moreover, FGF-2 increased the expression of bone morphogenetic protein 2 (BMP-2) and osteoblast differentiation markers (osterix, alkaline phosphatase, and osteocalcin) in the regenerated tissue. We revealed the facilitatory mechanisms of FGF-2 in periodontal regeneration in vivo. First, the proliferation of fibroblastic cells derived from bone marrow and PDL was accelerated and enhanced by FGF-2. Second, angiogenesis was enhanced by FGF-2 treatment. Finally, osteoblastic differentiation and bone formation, at least in part due to BMP-2 production, were rapidly induced by FGF-2. Therefore, these multifaceted effects of FGF-2 promote new tissue formation at the early regeneration phase, leading to enhanced formation of new bone, cementum, and PDL.

Periodontal disease in a patient with Prader-Willi syndrome: a case report.

INTRODUCTION: Prader-Willi syndrome is a complex genetic disease caused by lack of expression of paternally inherited genes on chromosome 15q11-q13. The prevalence of Prader-Willi syndrome is estimated to be one in 10,000 to 25,000. However, descriptions of the oral and dental phenotype are rare. CASE PRESENTATION: We describe the clinical presentation and periodontal findings in a 20-year-old Japanese man with previously diagnosed Prader-Willi syndrome. Clinical and radiographic findings confirmed the diagnosis of periodontitis. The most striking oral findings were anterior open bite, and crowding and attrition of the lower first molars. Periodontal treatment consisted of tooth-brushing instruction and scaling. Home care involved recommended use of adjunctive chlorhexidine gel for tooth brushing twice a week and chlorhexidine mouthwash twice daily. Gingival swelling improved, but further treatment will be required and our patient's oral hygiene remains poor. The present treatment of tooth-brushing instruction and scaling every three weeks therefore only represents a temporary solution. CONCLUSIONS: Rather than being a direct result of genetic defects, periodontal diseases in Prader-Willi syndrome may largely result from a loss of cuspid guidance leading to traumatic occlusion, which in turn leads to the development of periodontal diseases and dental plaque because of poor oral hygiene. These could be avoided by early interventions to improve occlusion and regular follow-up to monitor oral hygiene. This report emphasizes the importance of long-term follow-up of oral health care by dental practitioners, especially pediatric dentists, to prevent periodontal disease and dental caries in patients with Prader-Willi syndrome, who appear to have problems maintaining their own oral health.

Effects of concomitant use of fibroblast growth factor (FGF)-2 with beta-tricalcium phosphate (ß-TCP) on the beagle dog 1-wall periodontal defect model.

The effects of concomitant use of fibroblast growth factor-2 (FGF-2) and beta-tricalcium phosphate (ß-TCP) on periodontal regeneration were investigated in the beagle dog 1-wall periodontal defect model. One-wall periodontal defects were created in the mesial portion of both sides of the mandibular first molars, and 0.3% FGF-2 plus ß-TCP or ß-TCP alone was administered. Radiographic evaluation was performed at 0, 3, and 6 weeks. At 6 weeks, the periodontium with the defect site was removed and histologically analyzed. Radiographic findings showed that co-administration of FGF-2 significantly increased bone mineral contents of the defect sites compared with ß-TCP alone. Histologic analysis revealed that the length of the regenerated periodontal ligament, the cementum, distance to the junctional epithelium, new bone height, and area of newly formed bone were significantly increased in the FGF-2 group. No abnormal inflammatory response or ankylosis was observed in either group. These findings indicate the efficacy of concomitant use of FGF-2 and ß-TCP as an osteoconductive material for periodontal regeneration following severe destruction by progressive periodontitis.

Human gingival epithelial cells produce chemotactic factors interleukin-8 and monocyte chemoattractant protein-1 after stimulation with Porphyromonas gingivalis via toll-like receptor 2.

BACKGROUND: The mechanism of stimulation of human gingival epithelial cells (HGEC) by Porphyromonas gingivalis (Pg) has not been fully clarified yet. In order to investigate the possible activation of HGEC by Pg through Toll-like receptors (TLRs), we analyzed the production of chemotactic factors and the activated nuclear factor-kappa B (NF-kappaB). METHODS: The mRNA expression of TLRs and the protein expression of TLR2 and TLR4 in HGEC and gingival tissue were assessed using reverse transcription-polymerase chain reaction (RT-PCR) assay and immunohistochemical staining. Primary cultured HGEC (nHGEC) and HGEC transformed by simian virus 40 T antigen (OBA-9) were activated by a sonic extract (SE) of Pg to examine cytokine production and NF-kappaB activation using enzyme-linked immunosorbant assay (ELISA). In addition, Pg mediated activation of NF-kappaB in a TLR2-transfectant was also investigated. RESULTS: RT-PCR results revealed that HGEC expressed mRNA of TLR2, TLR4, TLR5, and TLR9, although the expression profiles of each cell line were slightly different. In addition, immunostaining revealed the prominent expression of TLR2 not only in nHGEC, but also in the gingival epithelium of the tissue specimen. Interestingly, nHGEC and OBA-9 secreted IL-8 and monocyte chemoattractant protein (MCP)-1 upon stimulation with Pg SE more efficiently than LPS and fimbriae of Pg. Furthermore, Pg SE increased the activated NF-kappaB not only in OBA-9, but also in 293T cells transfected with the human TLR2 gene. CONCLUSION: TLR2 participates, at least partly, in the signaling pathway to induce chemokine production in gingival epithelium as a reaction against Pg component(s), probably other than lipopolysaccharide and fimbriae.

IL-15 up-regulates iNOS expression and NO production by gingival epithelial cells.

To investigate the biological activity of epithelial cells in view of host defense, we analyzed the mRNA expression of inducible NOS (iNOS) as well as NO production by human gingival epithelial cells (HGEC) stimulated with IL-15. RT-PCR analysis revealed that HGEC expressed IL-15 receptor alpha-chain mRNA. In addition, stimulation with IL-15 enhanced iNOS expression by HGEC through an increase of both mRNA and protein levels. Moreover, IL-15 up-regulated the production of NO(2)(-)/NO(3)(-), a NO-derived stable end product, from HGEC. The enhanced NO production by IL-15 was inhibited by AMT, an iNOS-specific inhibitor. These results suggest that IL-15 is a potent regulator of iNOS expression by HGEC and involved in innate immunity in the mucosal epithelium.