Miho Machigashira

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.

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.

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.

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.

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.

Cleavage of Host Cytokeratin-6 by Lysine-Specific Gingipain Induces Gingival Inflammation in Periodontitis Patients

Background/Purpose Lysine-specific gingipain (Kgp) is a virulence factor secreted from Porphyromonas gingivalis (P. gingivalis), a major etiological bacterium of periodontal disease. Keratin intermediate filaments maintain the structural integrity of gingival epithelial cells, but are targeted by Kgp to produce a novel cytokeratin 6 fragment (K6F). We investigated the release of K6F and its induction of cytokine secretion. Methods K6F present in the gingival crevicular fluid of periodontal disease patients and in gingipain-treated rat gingival epithelial cell culture supernatants was measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometer-based rapid quantitative peptide analysis using BLOTCHIP. K6F in gingival tissues was immunostained, and cytokeratin 6 protein was analyzed by immunofluorescence staining and flow cytometry. Activation of MAPK in gingival epithelial cells was evaluated by immunoblotting. ELISA was used to measure K6F and the cytokines release induced by K6F. Human gingival fibroblast migration was assessed using a Matrigel invasion chamber assay. Results We identified K6F, corresponding to the C-terminus region of human cytokeratin 6 (amino acids 359–378), in the gingival crevicular fluid of periodontal disease patients and in the supernatant from gingival epithelial cells cultured with Kgp. K6F antigen was distributed from the basal to the spinous epithelial layers in gingivae from periodontal disease patients. Cytokeratin 6 on gingival epithelial cells was degraded by Kgp, but not by Arg-gingipain, P. gingivalis lipopolysaccharide or Actinobacillus actinomycetemcomitans lipopolysaccharide. K6F, but not a scrambled K6F peptide, induced human gingival fibroblast migration and secretion of interleukin (IL)-6, IL-8 and monocyte chemoattractant protein-1. These effects of K6F were mediated by activation of p38 MAPK and Jun N-terminal kinase, but not p42/44 MAPK or p-Akt. Conclusion Kgp degrades gingival epithelial cell cytokeratin 6 to K6F that, on release, induces invasion and cytokine secretion by human gingival fibroblasts. Thus, Kgp may contribute to the development of periodontal disease.

Proliferation of Osteoblast-Like Cells on Zirconia/Alumina Nanocomposite

The aim of this study was to evaluate the biocompatibility with the proliferation of osteoblast-like cell (MC3T3-E1) on zirconia/alumina nanocomposite (NANOZR) in comparison to yttria stabilized zirconia (3Y-TZP) and titanium (Ti). Cellular proliferations after 1-, 3-, 6-, and 9-day incubation were calculated from the measurement of the MTT activities of the proliferated cell and were analyzed by two-way ANOVA. Time-dependent proliferation of MC3T3-E1 in all the sample was observed in all three materials with culture days. However, these were no significant differences in the proliferation between three kinds of material, indicating all the materials have a similar-good biocompatibility.

Early Cellular Responses of Osteoblast-Like Cells on Acid-Etching and Alkaline Treated Titanium

The effects of four surface modification (acid-etching, alkaline treatment, acid-etching /alkaline treatment, and sandblasting) of commercially pure titanium (cpTi) on the early cellular responses of osteoblast-like MC3T3-E1 cells were investigated. MTT assay was used to measure the levels of cell attachment to the different surface specimens after 1-, 2-, and 3-hr cell incubation. All data were submitted to two-way analysis of variance. Cell morphology was observed by scanning electron microscopy (SEM). Results showed that initial adhesion of osteoblast-like cells was independent on the surface of cpTi modified with different method.

In Vitro Evaluation of Hydroxyapatite-Containing Glass Coating on Zirconia

The aim of this study was to investigate the response of osteoblast-like cells (MC3T3-E1) to the surface of hydroxyapatite-containing glass coating on zirconia (HA-G-Zr) in comparison to yttria stabilized zirconia (Y-TZP). The MC3T3-E1 cells were cultured on HA-G-Zr and Y-TZP specimens in 24-well tissue culture plates. Surface properties were evaluated by X-ray diffractometry, Fourier tranfer infrared microscopy (FT-IR) and scanning electron microscopy (SEM). Cell proliferation in each well was measured by MTT assay. Cell morphology was observed by SEM. Cell differentiation was evaluated by alkaline phosphatase (ALP) activity and osteocalcin levels. After the glass coating on Y-TZP, X-ray diffraction peaks due to hydroxyapatite (HA) were observed clearly. HA-G-Zr appeared on surface of uneven and roughened state, wherein microcracks on the microns in width and many voids of several microns in size were present. Time-dependent proliferation and differentiation of MC3T3-E1 cells were found in all the specimens. MC3T3-E1 cells on HA-G-Zr plates showed higher differentiation than Y-TZP. These results demonstrated that HA-G-Zr showed better cellular biocompatibility than Y-TZP.

Inhibitory Effect of Acid Electrolyzed Water on Plaque Formation.

本研究の目的は, 超電解イオン水の洗口によるプラーク形成抑制効果を検討することである。臨床的に健康な歯周組織を有する7名 (男性4名, 女性3名) を被験者とし, 実験開始にあたって全顎のGingival Index (GI) および上顎両側犬歯の歯肉溝滲出液 (GingivalCrevicularFluid: GCF) 量の診査を行いエアーポリッシャーおよびハンドスケーラーにより全歯面上のプラークを除去した。口腔清掃を停止し, 洗口 (1日4回, 約150mlにて1分間) のみを4日間行った。2日および4日後に上顎両側小臼歯の頬側の歯肉縁上プラークを採取し, 歯面上の生菌数をColony forming unit (CFU) により調べた。また, 4日後のGI, GCF unitsおよび全顎のPlaque Index (PlI) の変化も検討した。コントロールとして, ポビドンヨード, 生理食塩水を用いた。4日目のGIにおいては, 超電解イオン水では生理食塩水に比べ有意に低い値を示した。一方, 4日目のPlIは, 超電解イオン水では, 生理食塩水と同程度であり, ポビドンヨードに比べ有意に高いプラーク付着を示した。GCF量に関しては, 各被験液間で有意な差はみられなかった。超電解イオン水における2日目のCFUは, ポビドンヨードと同様, 生理食塩水に比べ有意に低い値を示した。4日目におけるCFUは, 各被験液間で有意な差はみられなかった。以上のことから洗口剤として超電解イオン水を使用した際, 2日目においてはポビドンヨードと同様の殺菌作用を示すが, 4日目においてはいずれも殺菌作用は低く, また超電解イオン水のプラーク形成抑制作用はポビドンヨードよりもやや劣ると思われる。