Kazushi Kunimatsu

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.

Randomized Placebo-Controlled and Controlled Non-Inferiority Phase III Trials Comparing Trafermin, a Recombinant Human Fibroblast Growth Factor 2, and Enamel Matrix Derivative in Periodontal Regeneration in Intrabony Defects

We investigated the efficacy, safety, and clinical significance of trafermin, a recombinant human fibroblast growth factor (rhFGF)‐2, for periodontal regeneration in intrabony defects in Phase III trials. Study A, a multicenter, randomized, double‐blind, placebo‐controlled study, was conducted at 24 centers. Patients with periodontitis with 4‐mm and 3‐mm or deeper probing pocket depth and intrabony defects, respectively, were included. A total of 328 patients were randomly assigned (2:1) to receive 0.3% rhFGF‐2 or placebo, and 323 patients received the assigned investigational drug during flap surgery. One of the co‐primary endpoints, the percentage of bone fill at 36 weeks after drug administration, was significantly greater in the rhFGF‐2 group at 37.131% (95% confidence interval [CI], 32.7502 to 41.5123; n = 208) than it was in the placebo group at 21.579% (95% CI, 16.3571 to 26.8011; n = 100; p < 0.001). The other endpoint, the clinical attachment level regained at 36 weeks, was not significantly different between groups. Study B, a multicenter, randomized, blinded (patients and evaluators of radiographs), and active‐controlled study was conducted at 15 centers to clarify the clinical significance of rhFGF‐2. Patients with 6‐mm and 4‐mm or deeper probing pocket depth and intrabony defects, respectively, were included. A total of 274 patients were randomly assigned (5:5:2) to receive rhFGF‐2, enamel matrix derivative (EMD), or flap surgery alone. A total of 267 patients received the assigned treatment during flap surgery. The primary endpoint, the linear alveolar bone growth at 36 weeks, was 1.927 mm (95% CI, 1.6615 to 2.1920; n = 108) in the rhFGF‐2 group and 1.359 mm (95% CI, 1.0683 to 1.6495; n = 109) in the EMD group, showing non‐inferiority (a prespecified margin of 0.3 mm) and superiority of rhFGF‐2 to EMD. Safety problems were not identified in either study. Therefore, trafermin is an effective and safe treatment for periodontal regeneration in intrabony defect, and its efficacy was superior in rhFGF‐2 compared to EMD treatments.

Production of hydrogen sulfide by two enzymes associated with biosynthesis of homocysteine and lanthionine in Fusobacterium nucleatum subsp. nucleatum ATCC 25586.

Fusobacterium nucleatum produces a large amount of the toxic metabolite hydrogen sulfide in the oral cavity. Here, we report the molecular basis of F. nucleatum H(2)S production, which is associated with two different enzymes: the previously reported Cdl (Fn1220) and the newly identified Lcd (Fn0625). SDS-PAGE analysis with activity staining revealed that crude enzyme extracts from F. nucleatum ATCC 25586 contained three major H(2)S-producing proteins. Two of the proteins with low molecular masses migrated similarly to purified Fn0625 and Fn1220. Their kinetic values suggested that Fn0625 had a lower enzymic capacity to produce H(2)S from L-cysteine (approximately 30%) than Fn1220. The Fn0625 protein degraded a variety of substrates containing betaC-S linkages to produce ammonia, pyruvate and sulfur-containing products. Unlike Fn0625, Fn1220 produced neither pyruvate nor ammonia from L-cysteine. Reversed-phase HPLC separation and mass spectrometry showed that incubation of L-cysteine with Fn1220 produced H(2)S and an uncommon amino acid, lanthionine, which is a natural constituent of the peptidoglycans of F. nucleatum ATCC 25586. In contrast, most of the sulfur-containing substrates tested, except L-cysteine, were not used by Fn1220. Real-time PCR analysis demonstrated that the fn1220 gene showed several-fold higher expression than fn0625 and housekeeping genes in exponential-phase cultures of F. nucleatum. Thus, we conclude that Fn0625 and Fn1220 produce H(2)S in distinct manners: Fn0625 carries out beta-elimination of L-cysteine to produce H(2)S, pyruvate and ammonia, whereas Fn1220 catalyses the beta-replacement of L-cysteine to produce H(2)S and lanthionine, the latter of which may be used for peptidoglycan formation in F. nucleatum.

Evaluation of bleeding on probing and gingival crevicular fluid enzyme activity for detection of periodontally active sites during supportive periodontal therapy

This study aimed to analyze the enzyme activity in gingival crevicular fluid (GCF) and its association with clinical parameters, especially bleeding on probing (BOP), and thus reconsider the significance and accuracy of recording BOP. A total of 184 patients who had entered supportive periodontal therapy were selected and GCF was collected from 401 sites before recording the clinical parameters, probing pocket depth (PPD), BOP, clinical attachment level, gingival index and plaque index. The enzyme activity of neutrophil elastase and aspartate aminotransferase and amount of protein in GCF were also analyzed. In the clinical parameters for biochemical data, amount of GCF showed the most correlation. A cut-off value for BOP and PPD were determined by the ROC curve and Youden index. Analysis was performed with all clinical parameters and biochemical data. Of the 401 sites, 51 were less than the cut-off value and were BOP-negative. On the other hand, 29 sites had values more than the cut-off value, with 14 BOP-negative sites and 15 BOP-positive sites. A conclusion is as follows: twenty-nine sites with values more than the cut-off value were diagnosed as sites requiring periodontal management, however, 14 of these were BOP-negative. These results suggest that combining other biochemical tests with examination of BOP and PPD may improve the validity of periodontal disease diagnosis. In future studies, it will be essential to find a marker that can precisely detect periodontal disease activity, and to develop a diagnostic tool for chair-side use.

Use of a novel assay to evaluate enzymes that produce hydrogen sulfide in Fusobacterium nucleatum

To evaluate enzymes that produce hydrogen sulfide (H(2)S) in species of Fusobacterium nucleatum, we developed an assay based on SDS-polyacrylamide gel electrophoresis with renaturation followed by active staining. This assay provided precise insight into the enzymes that produce H(2)S in terms of their number and molecular weights.

Possible roles of medullasin in nifedipine-induced human gingival overgrowth

In order to clarify a possible pathophysiological role of medullasin, a neutrophil elastase-like proteinase, in nifedipine (NF)-induced gingival overgrowth, the distributions of medullasin-positive cells immunostained in specimens from patients with NF-induced gingival overgrowth and chronic marginal gingivitis were compared in three different biopsy areas. Twenty gingival biopsies were obtained from five patients with gingival overgrowth and 20 biopsies from another five patients with chronic marginal gingivitis. In the marginal gingivitis group, the mean percentage of positive cells in the vicinity of pocket epithelium (zone I) was significantly higher than in the areas of connective tissue of the mid-portion (zone II) and adjacent to oral epithelium ( zone III) (p < 0.05). In the gingival overgrowth group, on the contrary, the positive cells significantly increased in zone II as compared with zones I and III (p < 0.05). Further, medullasin-positive cells of zones II and III in the overgrowth group had infiltrated more extensively than those in the gingivitis group (p < 0.001), indicating the participation of this enzyme in the mechanism of NF-induced gingival overgrowth. These observations suggest that medullasin may play a part in NF-induced overgrowth both in host defence and in immunoregulation, possibly cytotoxically.

Role of medullasin in nifedipine-induced gingival overgrowth in rats

To clarify the possible pathophysiological role of medullasin, a neutrophil elastase-like proteinase, in nifedipine (NF)-induced gingival overgrowth, a rat model of gingival overgrowth was first established using a diet containing NF. The relation between histopathological changes and the distribution of the proteinase was then investigated. Thirty-two, specific pathogen-free 20 day-old, male, Fisher 344 rats were fed a diet containing NF and killed at 2, 8, 16 and 32 weeks. Control rats (n = 32) were fed the same diet but without the drug. The mandible of each rat was resected and sectioned at 4-microm thickness buccolingually between the first and second molars. Computer image analysis was used to evaluate the extent of overgrowth in the approximal gingiva of each sample. To examine medullasin activity, the mean percentage of medullasin-positive cells per total cells counted in the pocket epithelium and the connective tissue adjacent to the epithelium of approximal gingiva was determined immunohistochemically. The height of the mid-portion and the area in NF-treated group increased significantly with time (with the exception of area at 2 weeks) compared with the corresponding regions in the control group. A marked inflammatory-cell infiltration and elongated rete pegs, especially in the mid-portion of approximal gingiva, were seen in the NF-treated group. The mean percentages of medullasin-positive cells in the NF-treated group at 8, 16 and 32 weeks were significantly higher than those of the control. Although medullasin-positive cells were mainly neutrophils, in several samples of the NF-treated group they were recognized as macrophage-like. These findings suggest that medullasin may be involved in host defence and immunoregulation in a NF-induced rat model of gingival overgrowth.

Immunohistochemical Study on Intraepithelial Distribution and Density of Langerhans Cells Identified Using Anti-S-100 Protein Antibody in Phenytoin-induced Overgrown Gingival Tissues.

宿主免疫機能に関与するランゲルハンス細胞の歯肉増殖症における役割を調べる目的で, フェニトイン誘発性ヒト増殖歯肉組織中における動態を抗S-100蛋白抗体を用いて検索し, 全身疾患を持たない成人性歯周炎罹患歯肉組織における動態と比較検討した。フェニトイン歯肉増殖症患者ならびに成人性歯周炎患者それぞれ5名ずつを被検者とし, 歯周外科時に試料を採取してパラフィン包理連続切片を作製した。これらの切片にウサギ抗S-100蛋白ポリクローナル抗体を用いて免疫染色を施し, 歯肉上皮内におけるS-100蛋白陽性細胞の組織学的検索を行った。その結果, S-100蛋白陽性細胞は, 増殖症ならびに成人性歯周炎ともに歯肉上皮の基底層から有棘層にかけて散在性に存在していた。また, 歯肉上皮の単位面積あたりに存在するS-100蛋白陽性細胞数および総歯肉上皮細胞数に対するS-100蛋白陽性細胞数の存在比率を計測すると, いずれも増殖症歯肉において有意に高い傾向 (p<0.01) を認めた。さらに, 多くのS-100蛋白陽性細胞が存在する歯肉上皮直下結合組織中にCD3陽性細胞が浸潤しており, 特に増殖歯肉において顕著であった。以上の結果より, フェニトイン服用による増殖歯肉においては, 成人性歯周炎と比べ口腔上皮内にS-100蛋白陽性細胞が有意に増加することから, 本疾患においてこの細胞の果たす役割が増強されている可能性が示唆された。

Total Protein Concentration of Gingival Crevicular Fluid as an Indicator of the Severity of Periodontal Disease.

歯周疾患における歯肉溝滲出液 (GCF) のタンパク濃度の臨床パラメーターとしての有用性を検討した。対象は成人型歯周炎患者 (23名) で, 平均年齢は52.7歳であった。被検部位はレントゲン写真上で明らかな歯槽骨破壊の認められる歯周ポケット93ヵ所を任意に選択した。本研究に用いたパラメーターは, Plaque Index (PlI), GCF量, probing depth (PD), attachment level (AL), Gingival Index (GI) および動揺度 (Mo) の6種類であった。GCFは, ペリオペーパー ®を 歯肉縁下約1mmに3分間保持, 採取し, ペリオトロン (®6) 0 00にて液量を測定した後, Lowry法を用いてタンパク濃度を算出した。その結果, タンパク濃度と6種のパラメーターとの相関係数は, 上記の記載順に0.41, 0.70, 0.31, 0.29, 0.60および0.39であり, Spearmanの順位相関ですべて有意であった (p<0.01) 。この中で, タンパク濃度は特にGCF量と高い相関を示した。以上より, タンパク濃度もまた歯周疾患の有効なパラメーターとなりうる可能性が示唆された。

A role of human neutrophil lysosomal proteinases in periodontal tissue breakdown.

To clarify a role of neutrophil lysosomal proteinases in periodontal tissue breakdown, their nature in neutrophils and their levels in gingival crevicular fluid (GCF) of periodontitis patients were investigated. When the distribution of these proteinases in the neutrophil granular subfractions was analyzed, cathepsins B and H were made soluble by hypotonic shock. Cathepsins L and G were made soluble after washing the membrane with 1M NaCI. On the other hand, about 30% of hemoglobin (Hb) -hydrolase (s) was tightly associated with the membrane after both treatments. Immunochemical analyses revealed that about 35% of the total Hb-hydrolase activity was attributed to cathepsin D, which was found in the content fraction, and the rest were due to pepstatin-insensitive serine proteinase (s) . Differing from rat neutrophils, human neutrophils do not contain cathepsin E. The ratio of cathepsin D to the serine proteinase (s) in neutrophils was not changed in GCF from periodontitis patients and these enzymes were associated with development of the disease.The quantity of cathepsin B in GCF of periodontitis patients determined by enzyme immunoassay was closely correlated with that by activity measurement, indicating that cathepsin B increases with the increase of severity of the disease and that its level in GCF can be inferred by activity measurement.