FGF-2 stimulates periodontal regeneration: results of a multi-center randomized clinical trial.

The efficacy of the local application of recombinant human fibroblast growth factor-2 (FGF-2) in periodontal regeneration has been investigated. In this study, a randomized, double-blind, placebo-controlled clinical trial was conducted in 253 adult patients with periodontitis. Modified Widman periodontal surgery was performed, during which 200 µL of the investigational formulation containing 0% (vehicle alone), 0.2%, 0.3%, or 0.4% FGF-2 was administered to 2- or 3-walled vertical bone defects. Each dose of FGF-2 showed significant superiority over vehicle alone (p < 0.01) for the percentage of bone fill at 36 wks after administration, and the percentage peaked in the 0.3% FGF-2 group. No significant differences among groups were observed in clinical attachment regained, scoring approximately 2 mm. No clinical safety problems, including an abnormal increase in alveolar bone or ankylosis, were identified. These results strongly suggest that topical application of FGF-2 can be efficacious in the regeneration of human periodontal tissue that has been destroyed by periodontitis.

The association of periodontal disease with oral malodour in a Japanese population.

AIM: The purpose of this study was to evaluate the association between oral malodour and periodontal disease, and to determine the effect of periodontal therapy on oral malodour. MATERIALS AND METHODS: Oral malodour parameters, including volatile sulphur compound (VCS) measurement, methyl mercaptan/hydrogen sulphide ratio by gas chromatography, organoleptic testing, tongue coating score, and periodontal parameters were evaluated in 823 patients complaining of oral malodour. Amongst these patients, 89 with oral pathogenic halitosis received tongue cleaning and periodontal therapy. Oral malodour and periodontal parameters were measured at baseline and after treatment. RESULTS: Amongst 823 patients, 102 were diagnosed with gingivitis and 721 with periodontitis. VCS levels and periodontal parameters increased according to the severity of oral malodour. Organoleptic testing significantly correlated with periodontal probing depth and a percentage of periodontal pocket depth ≥4mm (r=0.40 and 0.39 respectively). There were significant correlations between methyl mercaptan/hydrogen sulphide ratio and periodontal parameters. Significant decrease in oral malodour and periodontal parameters in 89 patients with oral pathogenic halitosis was also observed after periodontal treatment. CONCLUSIONS: Oral malodour is associated with periodontal disease, and periodontal therapy combined with tongue cleaning is beneficial for oral pathogenic halitosis.

Human gingival fibroblasts release high-mobility group box-1 protein through active and passive pathways.

INTRODUCTION: The nuclear protein high-mobility group box-1 (HMGB1) acts as a late mediator of inflammation when secreted in the extracellular milieu. In this study, we examined the effect of lipopolysaccharides from periodontal pathogens and apoptotic and necrotic cell death on HMGB1 production in human gingival fibroblasts (HGF). METHODS: HGF from healthy periodontal tissue were cultured and stimulated with lipopolysaccharides (LPS) from Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Escherichia coli. We also initiated apoptotic and necrotic cell deaths in HGF. The HMGB1 released in the supernatants from stimulated or dying cells was measured. Immunocytochemical staining against HMGB1 was performed in LPS-stimulated HGF. RESULTS: A significantly higher amount of HMGB1 was detected from necrotic and apoptotic HGF. LPS from A. actinomycetemcomitans, P. gingivalis, and E. coli significantly induced the production of HMGB1 in a time-dependent manner. After 6 h of LPS stimulation, HMGB1 was present in the cytoplasm of cells whereas its location was mainly nuclear after 24 h. CONCLUSIONS: LPS from two major periodontal pathogens, A. actinomycetemcomitans and P. gingivalis, induced HMGB1 secretion from HGF. Apoptotic and necrotic cell deaths resulted in the enhancement of HMGB1. Our results suggest that HGF can be a source of HMGB1 by both active secretion and passive release, and that HMGB1 from HGF may contribute to periodontal tissue destruction.

Oxidative stress causes alveolar bone loss in metabolic syndrome model mice with type 2 diabetes.

BACKGROUND AND OBJECTIVE: Alveolar bone loss is caused by a host response to periodontal pathogens, and its progression is often enhanced by systemic conditions such as insulin resistance. Alveolar bone dehiscence has been observed in KK-A(y) mice, which are metabolic syndrome model mice with type 2 diabetes. The aim of this study was to investigate inducements responsible for alveolar bone dehiscence in the KK-A(y) mice. MATERIAL AND METHODS: The expression of endothelial nitric oxide synthase in the mandibles of mice was detected using immunohistochemical staining and the reverse transcription-polymerase chain reaction. After administration of N-acetylcysteine, an antioxidant, to KK-A(y) mice, alveolar bone loss and the expression of endothelial nitric oxide synthase protein in gingival keratinocytes and of hydrogen peroxide concentrations in plasma, were analyzed. The effect of hydrogen peroxide on endothelial nitric oxide synthase expression in keratinocytes was examined using cultured keratinocytes. RESULTS: The expression of endothelial nitric oxide synthase was decreased in gingival keratinocytes from KK-A(y) mice compared with gingival keratinocytes from control mice. Administration of N-acetylcysteine to the mice restored endothelial nitric oxide synthase expression in the gingival keratinocytes, suppressed the alveolar bone loss and decreased the hydrogen peroxide concentrations in plasma without the improvement of obesity or diabetes. In vitro, stimulation with hydrogen peroxide decreased the expression level of endothelial nitric oxide synthase in cultured keratinocytes, which was restored by the addition of N-acetylcysteine. CONCLUSION: Reactive oxygen species, such as hydrogen peroxide, are responsible for the alveolar bone loss accompanied by decreased endothelial nitric oxide synthase expression in KK-A(y) mice. Therefore, we propose a working hypothesis that the generation of oxidative stress is an underlying systemic condition that enhances alveolar bone loss in periodontitis occurring as a complication of diabetes.

Hydrogen sulfide inhibits cell proliferation and induces cell cycle arrest via an elevated p21 Cip1 level in Ca9-22 cells.

BACKGROUND AND OBJECTIVE: Volatile sulfur compounds such as hydrogen sulfide (H(2)S) and methyl mercaptan (CH(3)SH) are the main causes of oral mal odor. However, the physiological functions of H(2)S have not been investigated in oral tissues. The aim of this study was to evaluate the effect of H(2)S on cell proliferation and the cell cycle in oral epithelial-like cells. MATERIAL AND METHODS: Ca9-22 cells were used in this study. Cells were cultured in 5% CO(2)/95% air with (5 or 10 ng/mL) or without H(2)S. DNA synthesis was measured using a 5-bromo-2-deoxyuridine enzyme-linked immunosorbent assay. The cell cycle was analyzed using a flow cytometer. The expressions of phosphorylated retinoblastoma protein (Rb), p21(Cip1) and p27(Kip1) were evaluated by western blotting. RESULTS: Exposure to 5 and 10 ng/mL of H(2)S significantly decreased DNA synthesis (p < 0.05). Cell cycle analysis also showed that exposure to both concentrations of H(2)S significantly increased the proportion of cells in G(1) phase (p < 0.001) and significantly decreased the proportion of cells in S phase (p < 0.01). Western blotting showed that Rb phosphorylation was reduced and p21(Cip1) was enhanced by exposure to H(2)S. CONCLUSION: The results indicated that H(2)S inhibits cell proliferation and induces cell cycle arrest via the expression of p21(Cip1) in Ca9-22 cells.

Arginine-specific gingipains from Porphyromonas gingivalis deprive protective functions of secretory leucocyte protease inhibitor in periodontal tissue.

Chronic periodontitis is correlated with Porphyromonas gingivalis infection. In this study, we found that the expression of secretory leucocyte protease inhibitor (SLPI), an endogenous inhibitor for neutrophil-derived proteases, was reduced in gingival tissues with chronic periodontitis associated with P. gingivalis infection. The addition of vesicles of P. gingivalis decreased the amount of SLPI in the media of primary human gingival keratinocytes compared to untreated cultures. We therefore investigated how arginine-specific gingipains (Rgps) affect the functions of SLPI, because Rgps are the major virulence factors in the vesicles and cleave a wide range of in-host proteins. We found that Rgps digest SLPI in vitro, suppressing the release of SLPI. Rgps proteolysis of SLPI disrupted SLPI functions, which normally suppresses neutrophil elastase and neutralizes pro-inflammatory effects of bacterial cell wall compounds in cultured human gingival fibroblasts. The protease inhibitory action of SLPI was not exerted towards Rgps. These results suggest that Rgps reduce the protective effects of SLPI on neutrophil proteases and bacterial proinflammatory compounds, by which disease in gingival tissue may be accelerated at the sites with P. gingivalis infection.

Hepatocyte growth factor in gingival crevicular fluid and the distribution of hepatocyte growth factor-activator in gingival tissue from adult periodontitis.

Hepatocyte growth factor (HGF), also known as scatter factor, is a broad-spectrum and multifunctional cytokine required for the development, growth and regeneration of various organs and tissues. The expression of HGF in human gingival fibroblasts is induced by inflammatory cytokines such as interleukin 1. Thus, although it is possible that content of HGF in gingival crevicular fluid (GCF) in periodontitis is increased, this has not so far been reported because the volume of GCF is too small to determine HGF by the available enzyme-linked immunosorbent assay (ELISA). A recently developed, highly sensitive ELISA for HGF, with a detection limit of 1 pg/ml sample, has now enabled HGF to be measured in GCF.The mean HGF content in GCF from sites with clinically healthy gingiva, defined by the absence of overt signs of gingival inflammation and a probing depth (PD) <3 mm, was 1.7 ng/ml, and that of periodontitis, defined by obvious alveolar bone loss detected by radiographic examination and a PD> or =3 mm, was 3.23 ng/ml. Although treating the periodontitis did not significantly decrease the HGF concentration despite significantly improved clinical scores such as PD and Gingival Index, the total amount of HGF in GCF did decrease significantly after treatment. HGF was expressed by gingival fibroblasts and inflammatory cells as determined by in situ hybridization. HGF-activator (HGFA), which converts inactive pro-HGF to active mature HGF, was detected in gingival epithelial cells by immunostaining. The expression of HGFA was also confirmed in gingival tissue by reverse transcription-polymerase chain reaction (RT-PCR). These findings indicate that HGF is synthesized and activated in gingiva that is clinically healthy or associated with periodontitis.

The effects of methyl mercaptan on epithelial cell growth and proliferation.

AIM: Previous studies have demonstrated that methyl mercaptan (CH3SH), one of the main causes of oral malodour, might contribute to the initiation and progression of periodontal disease. These studies suggested that CH3SH may affect the epithelial cells of the gingival crevice, which form a barrier to the penetration of microbial substances. In this study, the effects of CH3SH on the epithelial cells and gingival fibroblasts were investigated. METHOD: Human oral epithelial carcinoma cell line (KB), human oral squamous cell carcinoma cell line (HSC-2), and human gingival fibroblasts (HGF) derived from healthy gingiva were used in this study. These cells were cultured in conditions of 5% CO2/95% air with or without CH3SH (10 ng/ml or 50 ng/ml) for 5 days. Cell numbers, proliferation and cytotoxicity were evaluated. RESULTS: CH3SH inhibited epithelial cell growth and proliferation at the concentration of 50 ng/ml, and a cytotoxic effect of CH3SH was also noted. On the other hand, HGF cells were not affected by 50 ng/ml CH3SH. CONCLUSION: High concentrations of CH3SH such as 50 ng/ml have an inhibitory effect on the growth and proliferation of epithelial cells, but not on those of fibroblasts.

Preparation and characterization of an Actinomyces naeslundii aggregation factor that mediates coaggregation with Porphyromonas gingivalis.

Intergeneric coaggregation is responsible for the complexity of the microbiota in human dental plaque and is believed to be important in the initial bacterial colonization of the human oral cavity. Actinomyces naeslundii, an early colonizer of the tooth surface, may enhance subsequent colonization by Porphyromonas gingivalis which is associated with adult periodontitis. The purpose of this study was to isolate and characterize the A. naeslundii aggregation factor (AnAF) that mediates coaggregation with P. gingivalis. AnAF was isolated from A. naeslundii sonic extract (SE) by gel filtration on a Sephacryl S-400HR, by hydrophobic interaction chromatography on a HiTrap Octyl Sepharose 4FF, and by ion exchange chromatography on a HiTrap Q. The specific activity increased 12-fold with a yield of 2.5%. SDS-PAGE analysis of AnAF revealed a protein band of high molecular weight in excess of 200 kDa. Carbohydrate was detected as the only material coinciding with the protein band, indicating that the AnAF was a glycoprotein. Immunoblotting analysis indicated that AnAF directly bound to P. gingivalis cells. AnAF was sensitive to sodium metaperiodate treatment but not to heat or protease treatments. These results suggest that the AnAF carbohydrate component mediated coaggregation with P. gingivalis cells. AnAF also inhibited coaggregation with other periodontal disease-associated bacteria such as Prevotella intermedia, Fusobacterium nucleatum, Capnocytophaga ochracea, but not streptococci.

[Influence of residual plaque score during initial treatment on pocket reduction rate at individual tooth surfaces].

The purpose of this study is to examine the relation ship between residual plaque score at an individual tooth surface during initial treatment and the pocket reduction rate at the evaluation time. Thirty-nine adult periodontal patients (mean age 30.2 years) who O'Leary plaque control record (PCR) reached a level of 10% PCR, were selected for this study. A total of 5638 surfaces were monitored by recording probing pocket depths at the disto-buccal (a), mid-buccal (b), mesio-buccal (c), disto-lingual (d), mid-lingual and (e), mesio-lingual (f) regions, and by examining O'Leary plaque control records. The results were as follows: 1) The residual plaque score at all teeth surfaces was 14.74 +/- 19.21%. 2) The lowest plaque score were seen at 54/45, and 21/12. 3) The highest plaque score were seen at 7/7, and 76/67. 4) Well-responding sites that showed a high residual plaque score and a good pocket reduction rate, were 5/5 (initial probing pocket depth 3.0-3.5mm, a), and 4/4 (initial probing pocket depth 3.0-3.5 mm,f; 5.0-5.5 mm, f). 5) 7/7 (initial probing pocket depth 3.0 mm a, b, d), and 7/7 (initial probing pocket depth 3.0-3.5 mm, f) showed a high residual plaque score and a low pocket reduction rate. 6) The normal pocket response occurred at a level of residual plaque score and a low pocket reduction rate. 6) The normal pocket response occurred at a level of residual plaque score of less than 25%. In order to gain good pocket response, it is important to keep the level of residual plaque score at less than 25% rather than to keep the level of PCR at 10% or 20% (as several authors have previously stated). 7) A low residual plaque score during treatment is therefore more important than a low PCR.