Kyuichi Kamoi

Salivary biomarkers for predicting the progression of chronic periodontitis

OBJECTIVE Predicting the progression of periodontitis would allow for targeted supportive periodontal therapy. The purpose of this study was to determine the usefulness of salivary biomarkers for predicting the progression of periodontitis. DESIGN Eighty-five chronic periodontitis patients were enrolled in an 18-month longitudinal study. Amongst them, 57 experienced progression of periodontitis, indicated at the end of the 18 months by at least one site with >3mm loss of attachment compared with baseline. We determined the levels of aspartate aminotransferase, alanine aminotransferase (ALT), lactate dehydrogenase, alkaline phosphatase and free haemoglobin as biomarkers, as well as the counts of Porphyromonas gingivalis, Prevotella intermedia and Tannerella forsythia, which represented the periodontal bacteria, in the stimulated saliva. The Mann-Whitney U test was used to compare patients with and without progression. After categorising the diagnostic values, the chi-square test was applied. RESULTS Counts and ratios (ratio to total bacteria) of P. gingivalis and P. intermedia were found to be significant predictors of the progression of periodontitis. To increase prediction accuracy, combination analyses were performed. The combination of ALT level and the P. gingivalis ratio showed the highest likelihood (p<0.001, sensitivity 0.40, specificity 0.96, likelihood 11.30). CONCLUSION Our findings suggest that salivary ALT level and the P. gingivalis ratio may be potential indicators for the progression of periodontitis. Such a salivary test could be a useful diagnostic tool for predicting periodontal disease progression.

Large-scale investigation of genomic markers for severe periodontitis

The purpose of the present study was to investigate the genomic markers for periodontitis, using large-scale single-nucleotide polymorphism (SNP) association studies comparing healthy volunteers and patients with periodontitis. Genomic DNA was obtained from 19 healthy volunteers and 22 patients with severe periodontitis, all of whom were Japanese. The subjects were genotyped at 637 SNPs in 244 genes on a large scale, using the TaqMan polymerase chain reaction (PCR) system. Statistically significant differences in allele and genotype frequencies were analyzed with Fisher’s exact test. We found statistically significant differences (P < 0.01) between the healthy volunteers and patients with severe periodontitis in the following genes; gonadotropin-releasing hormone 1 (GNRH1), phosphatidylinositol 3-kinase regulatory 1 (PIK3R1), dipeptidylpeptidase 4 (DPP4), fibrinogen-like 2 (FGL2), and calcitonin receptor (CALCR). These results suggest that SNPs in the GNRH1, PIK3R1, DPP4, FGL2, and CALCR genes are genomic markers for severe periodontitis. Our findings indicate the necessity of analyzing SNPs in genes on a large scale (i.e., genome-wide approach), to identify genomic markers for periodontitis.

The Efficacy of Povidone-Iodine Products against Periodontopathic Bacteria

A total of 8 strains of 6 bacterial species, Porphyromonas gingivalis ATCC33277 and TDC286, Actinobacillus actinomycetemcomitans ATCC29523 and JP2, Fusobacterium nucleatum No. 2, Tannerella forsythensis ATCC43937, Prevotella intermedia ATCC25611 and Streptococcus anginosus ATCC33397, were treated with povidone-iodine (PVP-I) gargle (PVP-I: 0.47 and 0.23% w/v) or chlorhexidine gluconate (CHG) gargle (CHG: 0.002% w/v) for 15, 30 or 60 s, after which they were inoculated into various media, cultured and counted for residual bacteria. At both concentrations, PVP-I gargle reduced the viable cell count of all 8 bacterial strains to below the measurable limit within 15 s. By contrast, there were more than 1,000 viable colonies 60 s following treatment with the CHG gargle. The results demonstrate that povidone-iodine gargle has rapid bactericidal activity against the causative bacteria of periodontal disease.

Studies on the application of bone graft to periodontal therapy. 1. Material and biological studies on hydroxyapatite.

Hydroxyapatite (HA) を歯周治療に応用する基礎的資料を得るために焼成温度の異なるHAの比表面積測定, X線回折および生理的食塩水に対する溶解性試験を行った。さらにラットにおける急性毒性試験およびHAの表面微細構造の検索を行った。得られた結果は次のごとくである。1. 比表面積測定によると加熱焼成温度の上昇とともにその値は低下した。2. X線回折では900℃で典型的なHAのパターンを示した。3. 900℃および1,250℃のHAともに経時的変化がほとんど認められず比較的安定した溶解性を示した。4. 経口, 皮下, 腹腔の3経路で, 900℃ HA, 1,250℃ HAを投与したところ体重において有意の差がみられなかったが, 尿および血清において偶発的と思われる有意の差がみられた。5. 焼成温度の上昇とともに表面構造は滑沢となった。

Proliferation and Tube Formation of Periodontal Endothelial Cells

Angiogenesis is indispensable to guide a regeneration of good periodontal tissue in the wound healing after periodontal surgery. Hepatocyte growth factor is well known for a strong angiogenic factor and it may play important roles in the periodontal tissue during periodontal wound healing. In exploring the promotion of angiogenesis in the periodontal ligament, proliferative and tubulogenic responses of endothelial cells to hepatocyte growth factor and to soluble factors secreted by fibroblasts were investigated. Pavement-shaped cells isolated from a human periodontal ligament were identified as the endothelial cell by their granular immunoreactivity for factor VIII. The proliferation of the endothelial cells was accelerated by the addition of hepatocyte growth factor or fibroblast-conditioned medium, and far more by adding both than either. The endothelial cells seeded on the agar containing both hepatocyte growth factor and fibroblast products formed a dense network in a shorter time than on the agar containing either. The endothelial cells in the dense network took a tube-like structure with lumen and were covered with laminin. These results suggest that hepatocyte growth factor administered into the regenerating periodontal tissue may promote, synergistically with local factors produced by the activated fibroblast, the proliferation and tubulogenesis of the remaining endothelial cells.

Regeneration therapy for oral disease.

The aim of this paper is to provide a review of the current understanding of the mechanisms, cell and factors required for regeneration and restoration of periodontal tissue around natural teeth. Periodontal regeneration is a complex multifactorial process involving cell populations: periodontal ligament cells, bone cells, gingival fibroblasts and epithelial cells. This paper describes bone graft, guided tissue regeneration and enamel matrix derivative with the application of growth factors.

Bone Ceramic・コラーゲンゲル合材移植後の歯周組織再生に関する研究

The aim of this study is to determine the process of periodontal tissue regeneration and the metabolic activity of osteoblasts after implantation of bone ceramic and collagen gel compound materials (BC). Bone defects were artificially prepared in the alveolar septa of the bilateral upper first and second molars of Wistar rats. Subsequently, BC were implanted into the defective sites on the left side, and the gingival flaps were closed. At the defective sites on the right side, as a control, gingival flaps were closed without implantation. Rats were sacrificed 1, 3, 5, 7 or 14 weeks after implantation, and prepared tissue sections were observed both pathologically and autoradiographically using 3H-Proline. The results obtained were as follows: Pathological Findings One week after BC implantation, inflammatory cellular infiltration of the surrounding gingival connective tissue was relatively mild. Three weeks after implantation, BC were present in fibrous connective tissues, and some directly bound to the marices of regenerated bone. Observation 5 weeks after implantation revealed that BC had become embedded in the regenerated bone matrices and that there was giant cell reaction to foreign bodies at the margin of BC located in connective tissue. BC were directly bound to the regenerated bone matrices without intermediary fibrous tissues 7 and 14 weeks after implantation. Connective tissues showed high grade regeneration of collagen fiber bundles, in an arrangement that tended to be fixed in mesial and distal directions. Autoradiographic Findings There was no uptake of 3H-Proline into the regenerated bone matrices or the gingival connective tissue surrounding BC, while uptake of 3H-Proline into the entire area around the root apex and in the vicinity of the alveolar septum was observed with time (weeks) after BC implantation. These results suggest that BC provide nuclei for bone regeneration through inclusion in newly-generated periodontal bone tissue, although it is difficult to produce definite induction of bone tissue by BC alone. It is also apparent that these are useful bone implantation materials for restoration of the physiological morphology of alveolar bone in periodontal surgical treatment.

Abstract of Public Open Special Lecture at Symposium

OF PUBLIC OPEN SPECIAL LECTURE AT SYMPOSIUM

Application of a carbon dioxide laser for early closure of gingival flaps

Abstract The aim of this study was to evaluate the applicability of a carbon dioxide (CO 2 ) laser as a means of achieving early closure of gingival flaps after periodontal surgery. In this study, an incision was made from the maxillary premolar to the canine tooth in dogs. Full thickness flaps were raised, and mucoperiosteal flaps were created. After repositioning the mucoperiosteal flaps in the experiment group, CO 2 lasers were applied to the incision sites, and the flaps were closed with the laser. In the control group, they were closed with simple silk. Animals were sacrificed at postoperative days 7 and 14, and histological examinations were performed. The results showed that although epithelial invasion was observed along the incision areas 7 days after treatment in the control groups, complete closure of the incision area and regular arrangement of fibers in the connective tissue beneath the epithelium were observed in the group treated with lasers. These results suggest that closure of gingival flaps with a CO 2 laser may reduce the possibility of infection at initial incision sites.

Effects of Lipopolysaccharide on Phagocytic Function in Polymorphonuclear Leukocytes Using Flow Cytometry and Confocal Laser Scanning Microscopy.

歯周病の進行は, グラム陰性細菌と感染部位に遊走した多形核白血球 (PMNと略す) との生体応答の結果, 菌体の破壊などによって遊出する菌体内毒素 (LPSと略す) が, 歯周組織の炎症および破壊を惹起し増悪すると考えられている。本研究では, LPSによるPMNへの影響を検討する目的で, PMNをEscherichia coli由来のLPSで前処理し, PMNの貪食能, Fcγ レセプター, C3biレセプターについてフローサイトメーター (FCMと略す) と共焦点レーザー走査顕微鏡 (CLSMと略す) を用いて検索し, 次の結果を得た。1) 貪食率はLPS濃度0.01μg/ml群で対照群と比較して有意に増加し, LPS濃度10μg/ml群で有意に減少した。2) 貪食度はLPS濃度0.01μg/ml, 0.1μg/ml群で対照群と比較して有意に増加した。3) C3biレセプターの発現量はLPS濃度依存的に増加傾向を示した。とくにLPS濃度10μg/ml群で, 対照群と比較して有意に増加した。4) Fcγ レセプターの発現量はLPS濃度依存的に減少傾向を示した。とくにLPS濃度10μg/ml群で, 対照群と比較して有意に減少した。5) CLSMによるビーズ貪食細胞の連続する断層像により, 細胞内と表面とに存在するビーズが観察できた。6) CLSMにより, Fcγ レセプターは細胞膜全体に一様に存在している状態が観察された。また, C3biレセプターは細胞膜の数カ所に局在して観察された。本研究はPMNにLPSを作用させ, FCMとCLSMで貪食能と貪食に関わるレセプターについて詳細に検討した結果, PMNの貪食過程で異物がPMN表面に付着した場合と, PMN内に取り込まれた場合とでは, 重要な差異の起こることを認め, さらにPMNのレセプターの観察によってその存在の様相を明らかにした。