Junichi Tatsumi

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.

Modification of forsythia detaching factor by gingival crevicular fluid in periodontitis.

OBJECTIVES Forsythia detaching factor (FDF) is a virulence factor of Tannerella forsythia detected as a mixture of the 60-kDa form of FDF and the 28-kDa C-terminal fragment (FDFc). The objective of the present study was to clarify the proteolytic activity of gingival crevicular fluid (GCF) from patients with periodontitis and healthy subjects using recombinant FDF (rFDF) as substrate. DESIGN Eleven patients with periodontitis and 6 healthy subjects were recruited. Modification of rFDF and subsequent production of rFDFc by proteolytic activity of GCF was determined by Western blotting. Proteolytic activity of GCF was evaluated using an Ac-Arg-Ala-Lys-p-nitroaniline substrate. Correlation analysis between two different sets of variables was performed. Variables used in this analysis were proteolytic activity, clinical parameters, relative band density of rFDFc and those of rFDF. RESULTS Proteolytic activity in GCF was significantly higher in patients with periodontitis than in healthy subjects. Production of rFDFc was determined by treatment of rFDF with GCF from patients with periodontitis and with GCF from healthy subjects. Correlations between clinical parameters and proteolytic activity in GCF were significantly positive. On the other hand, correlations between relative band density of rFDFc or rFDF on Western blot and cleaving activity or clinical parameters were significantly negative. CONCLUSION The detected extend of GCF-activity generating rFDFc from rFDF and/or even further degrading rFDF correlates with severity of periodontitis.

23(S)25(R)-1,25-Dihydroxyvitamin D3-lactone, a naturally occurring metabolite of 1,25-dihydroxyvitamin D3, inhibits osteoclast-like cell formation in human bone marrow cultures

Abstract: We have used a human bone marrow culture system that forms multinucleated cells (MNCs), 50% of which express the osteoclast phenotype, to examine the 23(S)25(R)-1,25-dihydroxyvitamin D3-26,23-lactone (1,25-D3-lactone) on osteoclast-like cell formation. The 1,25-D3-lactone is a vitamin D3 metabolite that has recently been detected in human serum under physiological conditions at concentrations of approximately 131 pg/ml (3 × 10−10 M) and can inhibit bone resorption induced by 1,25-dihydroxyvitamin D3 (1,25-D3) in vivo and in vitro. We examined the effects of the 1,25-D3-lactone on the formation of MNC that cross-reacted with 23C6 monoclonal antibody (23C6-positive MNC), which preferentially binds to osteoclasts. All metabolites of 1,25-D3 except the 1,25-D3-lactone increased both total and 23C6-positive MNC formation in a dose-dependent manner. In contrast, the 1,25-D3-lactone inhibited both total and 23C6-positive MNC formation, whether the cultures were treated with 1,25-D3, parathyroid hormone, or interleukin-1β, all potent stimulators of MNC formation. This inhibitory action of 1,25-D3-lactone on MNC formation was very similar to the inhibitory effects of calcitonin. These data suggest that (1) 1,25-D3-lactone is a potent natural inhibitor of formation of cells with the osteoclast phenotype at physiological concentrations and (2) the inhibition of these cells by 1,25-D3-lactone may not result solely from its competitive binding to the 1,25-D3 receptor.

The N-terminal fragment of osteocalcin is released during osteoclastic bone resorption in vitro

Abstract: As a novel method for measuring bone resorption, an immunoassay system for human N-terminal osteocalcin (N-OC) was developed to determine if osteocalcin molecules are released from bone degraded by osteoclasts in vitro. The assay system employed a monoclonal antibody to the first 20 N-terminal residues of osteocalcin as the solid phase and polyclonal antibodies against these same residues as an enzyme conjugate. This assay system could detect the N-terminal portion of osteocalcin formed during the degradation of the human osteocalcin molecule by trypsin or cathepsin D. Osteoclasts were isolated from human alveolar bone and cultured on human bone slices; the osteocalcin content in the media from these cultures was measured by this N-OC assay method. The N-terminal fragment of osteocalcin was the major form of osteocalcin released during osteoclastic bone resorption along with small amounts of intact osteocalcin. Interleukin-1 (IL-1β) and interleukin-6 (IL-6), stimulators of osteoclastic bone resorption, increased the N-OC level by 1.5 fold compared with the level of N-OC in osteoclast cultures in the absence of stimulators. In contrast, treatment of the cultures with calcitonin or an inhibitor of cathepsin D (E-64), both of which inhibit osteoclastic bone resorption, decreased the amount of N-OC in the culture supernatants. These results suggest that the N-terminal fragment of osteocalcin is released during osteoclastic bone resorption and that its level may serve as an index of bone resorption in vitro.

Lysine-specific proteolytic activity responsible for forsythia detaching factor modification

OBJECTIVES The objective of the present study was to clarify the lysine-specific proteolytic activity derived from periodontal pathogens responsible for Forsythia detaching factor (FDF) modification. DESIGN The activity responsible for FDF modification in Tannerella forsythia and Porphyromonas gingivalis were evaluated by colorimetric assay using Ac-Arg-Ala-Lys-p-nitroaniline as a substrate. FDF modification in T. forsythia and P. gingivalis were evaluated by Western blotting using recombinant FDF (rFDF) as a substrate. Furthermore, the activity in GCF of 20 patients with periodontitis and 10 healthy subjects was also evaluated by colorimetric assay. Bacteria in subgingival plaque were detected using polymerase chain reaction. RESULTS The activity of both bacteria in colorimetric assay were 21.35 unit (P. gingivalis) and 3.61 unit (T. forsythia), respectively. Western blot analysis revealed that P. gingivalis was found to efficiently degrade rFDF and T. forsythia partially cleaved rFDF. The activity in GCF from patients with periodontitis (clinically healthy sites: CH, deep bleeding sites: DB and deep non-bleeding sites: DNB) was significantly higher than those from healthy subjects (healthy sites: H). Among the patients with periodontitis, the activity from CH was significantly lower than those from DB and DNB. T. forsythia was detected in 68.4% of DNB, in 78.4% of DB and in none of CH. P. gingivalis was detected in 63.2% of DNB, in 84.0% of DB and in 10.5% of CH. No bacterium was detected in healthy subjects. CONCLUSION The lysine-specific proteolytic activity responsible for FDF modification correlates with the presence of major periodontal pathogens.

Participation of Dexamethasone(Dex) and LPS to Alveolar Bone Resorption.

近年, 精神的ストレスが, 歯周疾患発症のリスクファクターの一つであるという幾つかの研究が報告された。それらの研究では, 精神的ストレスが加わると, 生体内では血清中のglucocorticoidの上昇, あるいはNK細胞活性の低下が認められることを示唆している。そこで本研究ではストレス負荷下におけるglucocorticoidに注目した。そこでin vitroにおけるglucocorticoidおよび歯周組織の組織破壊因子の1つであるLPSの骨吸収への関与を検討した。造血幹細胞からの破骨細胞形成系は, マウス骨髄細胞を用い, glucocorticoid様物質のDexamethasone (Dex) および歯周病原性細菌由来のリポ多糖体 (Lipopolysaccharide: 以下LPS) の刺激により出現したTRAP陽性多核細胞を測定した。さらに, DexおよびLPSの培養液中のInterleukin-1 beta (IL-1β) 量をEnzyme-linked immunosorbent assay (ELISA) kitにより測定した。DexおよびLPSによるTRAP陽性多核細胞の形成を比較した結果, Dex (10-7M) とLPS (100ng/ml) を共に添加するとTRAP陽性多核細胞数の増加が得られた。IL-1β 量は, LPSにDexを添加することによりIL -1β 量を抑制した。以上の結果から, Dexが直接破骨細胞形成を促進し, 歯槽骨吸収機構に関与する可能性が示唆され, 精神的ストレスが歯周疾患の誘発あるいは進行に影響を及ぼすことが示唆された。

Adhesion of Porphyromonas gingivalis to Hybrid Restorative Materials.

近年, 優れた物性と色調再現性をその主たる特徴とするハイブリッド修復材料が開発され臨床応用されてきているが, 歯周病原性細菌の易付着性という見地からの評価は十分なされていない。そこで, 3種類のハイブリッド修復材料 (エステニア®アートグラス®コンクエスト™), および従来から修復材として用いられている常温重合レジン (即時重合レジン), 20K金合金について, それぞれの表面粗さおよびPorphyromonas gdngivalis (P. gingivalis) 付着量, さらにヒト・モノサイト細胞株THP-1 (THP-1) を用いた細胞傷害性の有無について検討を行った。その結果, 3種類のハイブリッド修復材の表面粗さは20K金合金に次いで滑沢であり, 常温重合レジンが最も粗かった。細菌付着量は, 20K金合金, ハイブリッド歯冠修復材料の順表に増大し, 表面粗さとP. gingivalisの付着量とは相関することが示唆された。また, 細胞傷害性は, どの材料にも認められなかった。これらのことからハイブリッド修復材料は歯周病学的にも臨床での応用が期待される材料であることが示唆された。

Evaluation of Biological Affinities of Several GTR Membranes Using an In Vitro Assay. Technique Assessment of GTR Membranes Using a Human Osteoblastic Cell Line.

歯周組織再生誘導 (Guided Tissue Regeneration, GTR) 法において, そのバリアーに用いられている各種新素材 (Gore-Tex ® 膜とMillipore ®膜) の骨への直接的作用の一端を明らかにするために, ヒト骨芽細胞様細胞 (MG63) を用い, この細胞がGTR膜と接する領域において産生する接着性タンパクの動態を経時的に検討した。すなわち当該部におけるフィプロネクチン, I型およびIII型コラーゲンについて免疫抗体法により蛍光染色を行い, さらにこの蛍光組織化学染色標本を介し, これを蛍光画像解析装置 (ACAS) を用い検討した。結果は, コントロールの実験条件 (膜と共存しない細胞層のみ) 下では, 骨芽細胞MG63は抗フィプロネクチン, I型およびIII型コラーゲンモノクローナル抗体に反応した。また, Gore-Tex ® 膜 (PTFE膜) およびMillipore ®膜 周囲のフィプロネクチンの平均蛍光量やその局在領域は, 培養3日より6日目で減少する傾向が認められたが, Gore-Tex ® 膜およびMillipore ® 膜 周囲のI型およびIII型コラーゲンの平均蛍光量やその局在は, 培養日数の経過とともに上昇する傾向を示した。他方, フィプロネクチン, I型およびIII型コラーゲンの産生において, Gore-Tex ® 膜とMillipore ® 膜 のあいだで差は認められなかった。以上の結果から, 骨芽細胞がGTR膜に接する領域では, フィプロネクチン, I型およびIII型コラーゲン等の接着タンパクが, GTR膜周囲の細胞機能の調節に関与していることが示唆された。

ラット歯槽骨培養上清 (Bone-sup) のラット腹腔マクロファージ走化活性におよぼす影響

tion. Esterase activity and viability of the induced cells was more than 85% and 98% respectively. Rat alveolar bone supernatant (Bone-sup) was prepared by taking out the alveolar bone and incubating it in RPMI 1640 medium at 37•Ž, in 5% CO2 in air. During incubation, the stimulants parathyroid hormone ( PTH) or sonicated Bacteroides gingivalis 381 (BG) , or no stimulant were added. The effect of Bone-sup on chemotactic activity of macrophages was determined by the membrane filter method of bioassay in a multi-well chamber. In all experiments, N-formyl-Lmethionyl-L-leucyl-L-phenylalanine was used as a positive control. Chemotactic activity of macrophages was enhanced by Bone-sup with or without a stimulant. The peak of chemotactic activity was reached in 96 hr of incubation. Factors from rat alveolar bone or fragments of bone accelerated the chemotactic activity of macrophages, suggesting that the factor affects the function of macrophages.