Masao Maeno

A Cohort Study on the Association Between Periodontal Disease and the Development of Metabolic Syndrome

BACKGROUND An association between periodontal disease and metabolic syndrome based on cross-sectional and case-control studies was recently reported, but their causal relationship has not been fully clarified. The objective of this cohort study is to investigate the association between periodontal disease and changes in metabolic-syndrome components to accumulate evidence of the causal relationship between the two conditions. METHODS The study subjects consisted of 1,023 adult employees (727 males and 296 females; mean age: 37.3 years) who underwent medical and dental checkups between 2002 and 2006 and in whom all metabolic-syndrome components were within the standard values in 2002. The association between the presence of periodontal pockets and the positive conversion of metabolic-syndrome components was investigated using multiple logistic-regression analysis, odds ratios (ORs), and 95% confidence intervals (CIs). RESULTS The presence of periodontal pockets was associated with a positive conversion of one or more metabolic components during the 4-year observation period (OR: 1.6; 95% CI: 1.1 to 2.2). The ORs for a positive conversion of one component and two or more components were 1.4 (95% CI: 1.0 to 2.1) and 2.2 (95% CI: 1.1 to 4.1), respectively, and the difference was significant for two or more positive components. Of the metabolic-syndrome components, positive conversions of blood pressure and the blood-lipid index were significantly associated with the presence of periodontal pockets. CONCLUSION The presence of periodontal pockets was associated with a positive conversion of metabolic-syndrome components, suggesting that preventing periodontal disease may prevent metabolic syndrome.

Loop-Mediated Isothermal Amplification Method Targeting the lytA Gene for Detection of Streptococcus pneumoniae

ABSTRACT It is difficult to separate Streptococcus pneumoniae from the genotypically similar species Streptococcus mitis and Streptococcus oralis, which are commensals of the human oral cavity. A novel nucleic acid amplification technique, loop-mediated isothermal amplification (LAMP), which amplifies DNA under isothermal conditions (63°C) with high specificity, efficiency, and rapidity, was examined regarding its applicability for detecting S. pneumoniae. An S. pneumoniae-specific LAMP primer targeting the lytA gene was designed. The primer specificity was validated using 10 Streptococcus and 7 non-Streptococcus species. Within 60 min, the assay could detect 10 or more copies of purified S. pneumoniae DNA with a sensitivity 1,000 times that of conventional PCR. Clinical isolates of 21 other strains (3 S. oralis, 17 S. mitis, and 1 Streptococcus species) that harbor virulence-factor-encoding genes (lytA or ply) were tried to differentiate S. pneumoniae. The detection of S. pneumoniae in clinical isolates was more selective using the LAMP method than using conventional PCR. Therefore, LAMP appears to be a sensitive and reliable means of diagnosing S. pneumoniae infection.

Effect of compressive force on the expression of inflammatory cytokines and their receptors in osteoblastic Saos-2 cells

OBJECTIVE In orthodontic tooth movement, some cytokines released from periodontal ligament fibroblasts and alveolar bone osteoblasts on the pressure side can alter the normal processes of bone remodelling, resulting in physiological bone resorption. We examined the effect of compressive force and interleukin (IL)-1 type I receptor antagonist (IL-1ra) on the expression of inflammatory cytokines that promote osteoclast formation, as well as on their receptors, in osteoblastic Saos-2 cells. DESIGN The cells were cultured in Dulbeccos modified Eagle medium containing 10% fetal bovine serum with or without continuous compressive force (0.5-3.0 g/cm(2)) and/or IL-1ra for up to 24h. The gene expression levels of the cytokines and their receptors were estimated by determining mRNA levels using real-time PCR; the protein levels were determined using ELISA or immunohistochemical staining. RESULTS The expression of IL-1beta, IL-1 receptor, IL-6, IL-6 receptor, IL-8 receptor, IL-11 and tumor necrosis factor-alpha (TNFalpha) increased depending on the strength and duration of the compressive force, whereas the expression of IL-8, IL-11 receptor and TNFalpha receptor did not change with the application of compressive force. The expression of cytokines and their receptors produced by 3.0 g/cm(2) of compressive force decreased with the simultaneous addition of IL-1ra and the decrease was remarkable in IL-8 receptor, IL-11 and TNFalpha. CONCLUSION These results indicate that mechanical stress induces the production of inflammatory cytokines and their receptors in osteoblasts and the phenomenon is enhanced by the autocrine action of IL-1beta, which is increased in amount by mechanical stress.

Association Between Periodontal Disease and Metabolic Syndrome

OBJECTIVES Metabolic syndrome is a complex medical disorder characterized by visceral fat-type obesity involving hypertension, and abnormal glucose and lipid metabolism. The objective of this study was to investigate the relationship between periodontal disease and components of metabolic syndrome (obesity, lipid abnormality, hypertension, and hyperglycemia) in industrial workers of a single company in Tokyo, Japan. METHODS The study subjects consisted of 2478 adult employees (2028 men and 450 women; mean age: 43.3 years). The association between the presence of periodontal pockets and components of metabolic syndrome was investigated cross-sectionally using multiple logistic regression analysis, odds ratios (ORs), and 95 percent confidence intervals (CIs). RESULTS Body mass index, blood pressure, triglycerides, fasting blood glucose, and hemoglobin A1c (HbA1c) were significantly elevated (P < 0.05) in patients with periodontal pockets of 4 mm or more. We found that the OR of the presence of periodontal pockets adjusted for age, gender, and smoking habit was 1.8 (96 percent CI = 1.4-2.3) when the subjects with two positive components and without positive component were compared. And it was 2.4 (96 percent CI = 1.7-2.7) when the subjects with three or four positive components and without positive component were compared. CONCLUSIONS Our findings suggest an association between periodontal disease and metabolic syndrome in Japanese workers between the ages of 20 and 60 years.

Discrimination of Streptococcus pneumoniae from Viridans Group Streptococci by Genomic Subtractive Hybridization

ABSTRACT Two oligonucleotide primer sets for the discrimination of Streptococcus pneumoniae from “pneumococcus-like” oral streptococcal isolates by PCR were developed. Genomic subtractive hybridization was performed to search for differences between Streptococcus pneumoniae strain WU2 and the most closely related oral streptococcus, Streptococcus mitis strain 903. We identified 19 clones that contained S. pneumoniae-specific nucleotide fragments that were absent from the chromosomal DNA of typical laboratory strains of S. mitis and other oral bacteria. Subsequently, oligonucleotide PCR primers for the detection of S. pneumoniae were designed from the sequences of the subtracted DNA fragments, and the specificities of the 19 primer sets were evaluated by PCR using chromosomal DNAs extracted from four S. pneumoniae clinical isolates and from 20 atypical organisms classified as S. mitis or S. oralis, which harbored genes encoding the pneumococcal virulence factors autolysin (lytA) or pneumolysin (ply), as templates. Of the 19 primer sets, two (Spn9802 and Spn9828) did not amplify PCR products from any of the pneumococcus-like streptococcal strains that we examined. The genes containing the Spn9802 and Spn9828 sequences encoded proteins of unknown function that did not correspond to any previously described proteins in other bacteria. These new oligonucleotide primers may be very useful for early and correct diagnosis of S. pneumoniae infections.

Biochemical and Immunocytochemical Characterization of Mineral Binding Proteoglycans in Rat Bone

We examined biochemically and immunocytochemically the type and distribution of mineral binding proteoglycans (PGs) in rat mid-shaft subperiosteal bone using three monoclonal antibodies (MAb 1-B-5, 9-A-2, and 3-B-3) which specifically recognize unsulfated chondroitin, chondroitin 4-sulfate (C4-S) and dermatan sulfate (DS), and chondroitin 6-sulfate. Bone proteins were extracted from fresh specimens with a three-step technique: 4 M guanidine HCl (GdnCl), aqueous EDTA without GdnCl (E-extract), followed by GdnCl. Western blot analysis of SDS-polyacrylamide gel electrophoresis revealed that E-extract after chondroitinase ABC digestion reacted strongly with MAb 9-A-2 but not with MAb 1-B-5 or 3-B-3. After adehyde fixation, ethanolic trimethylammonium EDTA was used as a demineralizing agent for light and electron immunocytochemistry. This provided good retention of water-soluble PGs in the specimens. After chondroitinase ABC pre-treatment of tissue sections, MAb 9-A-2 specifically stained C4-S and/or DS in the walls of osteocyte lacunae and bone canaliculi in the mineralized matrix as well as in the unmineralized matrix such as pre-bone, vascular canals, and pericellular matrix surrounding osteocytes; the remainder of the mineralized matrix lacked staining. These results indicate that mineral binding PGs contain C4-S and/or DS and are exclusively localized in the walls of the bone lacuna and canaliculus.

Compressive force stimulates the gene expression of IL-17s and their receptors in MC3T3-E1 cells

During orthodontic tooth movement, cytokines released from periodontal ligament fibroblasts and alveolar bone osteoblasts can alter the process of bone remodeling. Recently, interleukin-17 (IL-17) was found to stimulate osteoclastic resorption through osteoblasts by inducing receptor activator of nuclear factor κB ligand (RANKL) expression. However, the relationship between mechanical stress and IL-17 production by osteoblasts is not clear. Therefore, we examined the effect of compressive force on the expressions of IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, and their receptors (IL-17RA, IL-17RB, IL-17RC, IL-17RD, and IL‐17RE) using MC3T3-E1 cells as osteoblast-like cells. We also examined the effect of IL‐17A on the expression of IL-17Rs, RANKL, macrophage colony-stimulating factor (M-CSF), and osteoprotegerin (OPG). The cells were cultured with or without continuous compressive force (1.0 and 2.0 g/cm2) for up to 24 hr. The cells were also cultured with or without IL-17A (0.1, 1.0, or 10 ng/ml) for up to 72 hr. The mRNA expressions of IL‐17s and their receptors were estimated by real-time polymerase chain reaction. The expression levels of IL‐17s and their receptors increased depending on the compressive force. The addition of IL-17A increased the expression of IL-17RA, IL-17RB, IL-17RC, IL-17RE, RANKL, and M-CSF, whereas it decreased OPG expression. These results indicate that compressive force induces the expression of IL-17s and their receptors in osteoblast-like cells and that IL-17s and their receptors produced in response to compressive force may affect osteoclastogenesis through the expression of RANKL, M-CSF, and OPG.

Calcium Ions Released from Mineral Trioxide Aggregate Convert the Differentiation Pathway of C2C12 Cells into Osteoblast Lineage

INTRODUCTION The purpose of this study was to examine the effect of mineral trioxide aggregate (MTA) on pluripotent-mesenchymal cell differentiation. METHODS The pluripotent-mesenchymal cell line C2C12 was cultured in a 5% serum medium to induce cell differentiation with or without MTA. The differentiation to myoblasts was analyzed by the immunocytochemical staining of myosin heavy chains. The cellular phenotype-specific markers characterizing the osteoblasts (Runx2 and osterix), chondroblasts (Sox9), myoblasts (MyoD), and adipocytes (LPL) were estimated with mRNA and protein levels by using real-time polymerase chain reaction and Western blot analysis, respectively. To verify that the effect of MTA was caused by the released calcium ions, the mRNA levels were analyzed in the presence or absence of MTA with ethylene glycol tetraacetic acid, calcium chloride, or verapamil. RESULTS C2C12 cells cultured without MTA altered their phenotype to myoblasts, exhibiting positive reactions to myosin heavy chains. However, the cells cultured with MTA were strongly inhibited from developing into myoblasts. The mRNA and protein expressions of Runx2, osterix, and Sox9 significantly increased with MTA; the expressions of MyoD and LPL decreased significantly. Calcium chloride addition without MTA presented a significant increase of mRNA levels of Runx2, osterix, and Sox9; ethylene glycol tetraacetic acid addition with MTA presented a significant increase of mRNA levels of MyoD and LPL. Verapamil blocked the stimulating or suppressing effect of MTA on these transcription factors. CONCLUSIONS Our study showed that MTA converted the differentiation pathway of C2C12 cells into osteoblast and/or chondroblast lineages as a result of elution components such as calcium ions from MTA.

Effects of nicotine and lipopolysaccharide on the expression of matrix metalloproteinases, plasminogen activators, and their inhibitors in human osteoblasts

OBJECTIVE Lipopolysaccharide (LPS) from periodontopathic bacteria can initiate alveolar bone loss through the induction of host-derived cytokines. Smoking increases the risk and severity of periodontitis. We examined the effects of nicotine and LPS on the expression of matrix metalloproteinases (MMPs), plasminogen activators (PAs), and their inhibitors, including tissue inhibitors of metalloproteinases (TIMPs) and PA inhibitor-1 (PAI-1), in osteoblasts. METHODS The cells were cultured with or without 10(-4) M nicotine and 100 ng/ml LPS for 12 days or with 100 microg/ml polymyxin B, 10(-4) M D-tubocurarine, 10 micromol/ml NS398, or 10(-6) M celecoxib in the presence of either nicotine or LPS for 12 days. The gene and protein expression levels for MMPs, PAs, TIMPs, and PAI-1 were examined using real-time PCR and ELISAs, respectively. PGE(2) production was determined using an ELISA. RESULTS The addition of nicotine and/or LPS to the culture medium increased the expression of MMP-1, -2, and -3 and tissue-type PA (tPA); decreased the expression of TIMP-1, -3, and -4; and did not affect expression of TIMP-2 or PAI-1. In the presence of d-tubocurarine or polymyxin B, neither nicotine nor LPS stimulated the expression of MMP-1. In the presence of NS398 or celecoxib, the stimulatory effects of nicotine and LPS on MMP-1 expression were unchanged, but they were unable to stimulate PGE(2) production. CONCLUSION These results suggest that nicotine and LPS stimulate the resorption process that occurs during turnover of osteoid by increasing the production of MMPs and tPA and by decreasing the production of TIMPs. Furthermore, they suggest that the stimulatory effect of nicotine and LPS on PGE(2) production is independent of their stimulatory effect on MMP-1 expression.

Compressive Force Induces Osteoclast Differentiation via Prostaglandin E2 Production in MC3T3-E1 Cells

In orthodontic tooth movement, prostaglandin E2 (PGE2) released from osteoblasts can alter the normal process of bone remodeling. We previously showed that compressive force (CF) controls bone formation by stimulating the production of PGE2 and Ep2 and/or Ep4 receptors in osteoblasts. The present study was undertaken to examine the effect of CF on the production of PGE2, cyclooxygenase-2 (COX-2), macrophage colony-stimulating factor (M-CSF), receptor activator of NF-κB ligand (RANKL), and osteoprotegerin (OPG) using osteoblastic MC3T3-E1 cells and to examine the indirect effect of CF on osteoclast differentiation using RAW264.7 cells as osteoclast precursors. MC3T3-E1 cells were cultured with or without continuous CF (1.0 or 3.0 g/cm2) for 24 hr, and PGE2 production was determined using ELISA. The expression of COX-2, M-CSF, RANKL, and OPG genes and proteins was determined using real-time PCR and ELISA, respectively. Osteoclast differentiation was estimated using tartrate-resistant acid phosphatase (TRAP) staining of RAW 264.7 cells cultured for 10 days with conditioned medium from CF-treated MC3T3-E1 cells and soluble RANKL. As CF increased, PGE2 production and the expression of COX-2, M-CSF, and RANKL increased, whereas OPG expression decreased. The number of TRAP-positive cells increased as CF increased. Celecoxib, a specific inhibitor of COX-2, blocked the stimulatory effect of CF on TRAP staining and the production of PGE2, M-CSF, RANKL, and OPG. These results suggest that CF induces osteoclast differentiation by increasing M-CSF production and decreasing OPG production via PGE2 in osteoblasts.