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Background More than 10 years have passed since the first nationwide study on the reasons for tooth extraction in Japan. In the present study, we conducted the second nationwide survey to update the previous data. Methods This was a descriptive study. A sample population consisting of 5,250 dentists was selected by systematic random sampling using the 2018 membership directory of the Japan Dental Association. The reason for each permanent tooth extraction was documented by each dentist during a period of 1 week from June 4 to June 10, 2018. A questionnaire was provided for documentation. Reasons for tooth extraction were categorised into 6 groups as follows: caries, periodontal disease, fracture, orthodontics, impacted teeth, and others. Results A total of 2345 identified dentists responded to the questionnaire (recovery rate: 44.8%). Information on 7809 extracted teeth from 6398 patients was obtained. Periodontal disease was the main reason for tooth extraction for both sexes (men: 40.4%, women: 34.9%). Caries accounted for 30.2% of tooth extractions among men and 29.0% among women. Periodontal disease was predominant in the groups older than 55 years of age. Dental fracture accounted for 16.8% of tooth extractions among men and 19.2% among women. Conclusions Caries and periodontal disease are still the main reasons for tooth extraction in Japan. Moreover, dentists should note that fractures accounted for approximately one-fifth of permanent tooth extractions after the age of 45 years.

This report describes a validation study of data in the National Database of Health Insurance Claims and Specific Health Checkups of Japan (NDB) obtained by nationwide surveys on tooth extractions. The following 3 data sources on tooth extractions in Japan were compared: (1) the Nationwide Survey of the Reasons for Permanent Tooth Extractions in Japan (a previous survey conducted by the present authors); (2) the Statistics on Medical Care Activities in Public Health Insurance; and (3) the 4th NDB Open Data Japan. Source 1 was a nationwide survey conducted in 2018; sources 2 and 3 comprised data that are freely available for use by the public. In Source 1, 2,345 of 5,250 dentists approached responded to the questionnaire (recovery rate: 44.8%). The number of extracted teeth among younger age groups (aged 25-50 years) reported in Source 1 was lower than that in the other two sources. In contrast, the number of extracted teeth among older age groups reported in Source 1 was higher than that reported in the other two sources. However, when stratified by age group, all differences across the 3 sources regarding the mean annual number of tooth extractions were

Periodontitis is an inflammatory condition that causes the destruction of the supporting tissues of teeth and is a major public health problem affecting more than half of the adult population worldwide. Recently, members of the herpes virus family, such as the Epstein–Barr virus (EBV), have been suggested to be involved in the etiology of periodontitis because bacterial activity alone does not adequately explain the clinical characteristics of periodontitis. However, the role of EBV in the etiology of periodontitis is unknown. This study aimed to examine the effect of inactivated EBV on the expression of inflammatory cytokines in human gingival fibroblasts (HGFs) and the induction of osteoclast differentiation. We found that extremely high levels of interleukin (IL)-6 and IL-8 were induced by inactivated EBV in a copy-dependent manner in HGFs. The levels of IL-6 and IL-8 in HGFs were higher when the cells were treated with EBV than when treated with lipopolysaccharide and lipoteichoic acid. EBV induced IκBα degradation, NF-κB transcription, and RAW264.7 cell differentiation into osteoclast-like cells. These findings suggest that even without infecting the cells, EBV contributes to inflammatory cytokine production and osteoclast differentiation by contact with oral cells or macrophage lineage, resulting in periodontitis onset and progression.

Azithromycin displays immunomodulatory and anti-inflammatory effects in addition to broad-spectrum antimicrobial activity and is used to treat inflammatory diseases, including respiratory and odontogenic infections. Few studies have reported the effect of azithromycin therapy on bone remodeling processes. The aim of this study was to examine the effects of azithromycin on the osteogenic function of osteoblasts using osteoblast-like MC3T3-E1 cells. Cells were cultured in the presence of 0, 0.1, 1, and 10 µg/mL azithromycin, and cell proliferation and alkaline phosphatase (ALPase) activity were determined. In vitro mineralized nodule formation was detected with alizarin red staining. The expression of collagenous and non-collagenous bone matrix protein was determined using real-time PCR or enzyme-linked immunosorbent assays. In cells cultured with 10 µg/mL azithromycin, the ALPase activity and mineralized nodule formation decreased, while the type I collagen, bone sialoprotein, osteocalcin, and osteopontin mRNA expression as well as osteopontin and phosphorylated osteopontin levels increased. These results suggest that a high azithromycin concentration (10 µg/mL) suppresses mineralized nodule formation by decreasing ALPase activity and increasing osteopontin production, whereas low concentrations (≤l.0 µg/mL) have no effect on osteogenic function in osteoblastic MC3T3-E1 cells.

BACKGROUND The interplay between obesity and periodontitis has been widely examined. While obesity was reported as a risk factor for periodontitis, the inverse relationship is still little explored. Therefore, we aimed to determine whether periodontitis and toothbrushing frequency affect the onset of obesity. MATERIAL AND METHODS This cohort study included 1619 employees of a business enterprise headquartered in Tokyo, who in 2002 and 2006 underwent in prescribed annual health checks, both general and dental-specific, and who were not obese in 2002 (body mass index

BACKGROUND Osteoclast precursor cells are constitutively differentiated into mature osteoclasts on bone tissues. We previously reported that the continuous stimulation of RAW264.7 precursor cells with compressive force induces the formation of multinucleated giant cells via receptor activator of nuclear factor kappaB (RANK)-RANK ligand (RANKL) signaling. Here, we examined the bone resorptive function of multinucleated osteoclasts induced by continuous compressive force. MATERIAL AND METHODS Cells were continuously stimulated with 0.3, 0.6, and 1.1 g/cm² compressive force created by increasing the amount of the culture solution in the presence of RANKL. Actin ring organization was evaluated by fluorescence microscopy. mRNA expression of genes encoding osteoclastic bone resorption-related enzymes was examined by quantitative real-time reverse transcription-polymerase chain reaction. Mineral resorption was evaluated using calcium phosphate-coated plates. RESULTS Multinucleated osteoclast-like cells with actin rings were observed for all three magnitudes of compressive force, and the area of actin rings increased as a function of the applied force. Carbonic anhydrase II expression as well as calcium elution from the calcium phosphate plate was markedly higher after stimulation with 0.6 and 1.1 g/cm² force than 0.3 g/cm². Matrix metalloproteinase-9 expression decreased and cathepsin K expression increased slightly by the continuous application of compressive force. CONCLUSIONS Our study demonstrated that multinucleated osteoclast-like cells induced by the stimulation of RAW264.7 cells with continuous compressive force exhibit high dissolution of the inorganic phase of bone by upregulating carbonic anhydrase II expression and actin ring formation. These findings improve our understanding of the role of mechanical load in bone remodeling.

Purpose Circadian rhythm is associated with the pathogenesis of systemic disease and bone mineral metabolism. This study aimed to radiographically evaluate morphological characteristics of the interalveolar septum in circadian rhythm deficient animals. Methods Heads of 10 brain and muscle arnt-like protein-1 (BMAL1)-knockout (KO) mice and 10 wild-type mice sacrificed at 36 weeks were imaged using micro-computed tomography. The mean depth from the cementoenamel junction to the alveolar ridge (virtual bone sounding: VBS) of the interalveolar septum between the first and second molars, and the bone mineral density (BMD) of the interalveolar septum and the mandibular inferior cortex region were calculated. Tooth diameter was also measured. Results The VBS of the interalveolar septum in the BMAL1-KO mice was significantly deeper than that in wild-type mice. The BMD in the BMAL1-KO mice was significantly lower than in the wild-type mice in both regions. No significant difference was observed in tooth diameter between BMAL1-KO and wild-type mice. Conclusion These results suggest that low BMD in the interalveolar septum accelerates bone resorption in the interalveolar septum in BMAL1-KO mice.

This study aimed to determine whether a dental health education program would reduce cardiometabolic risk (obesity, hypertension, dyslipidemia, and hyperglycemia) in people with periodontitis. We used annual check-up data provided by the Japanese company’s health insurance union. Of 182 male employees with cardiometabolic risk and periodontal pockets at baseline, 21 participants of the dental health education program and 21 non-participants matched for age, the presence of obesity, and periodontal pocket at baseline were allocated to the intervention (mean age, 53.3 ± 7.0) and the non-intervention groups (mean age, 52.9 ± 7.0), respectively. The program focused on self-removal of dental plaque with a toothbrush and interdental brush and comprised five sessions over 12 months. In the intervention group, waist circumference (cm) and diastolic blood pressure (mmHg) decreased from 88.4 ± 6.3 to 86.8 ± 6.3 and from 85.7 ± 8.2 to 82.6 ± 8.3, respectively (P

Low-intensity pulsed ultrasound (LIPUS) is used for bone healing in orthopedics. In previous in vivo and in vitro studies, LIPUS has been shown to have promising effects on cellular elements in articular cartilage, particularly chondrocytes in patients with osteoarthritis. However, the effects of LIPUS on the cellular mechanisms through which LIPUS alters extracellular matrix (ECM) synthesis in chondrocytes are unclear. In this study, we investigated the effects of the optimal intensity and cellular mechanisms of LIPUS on the regeneration of cartilage matrix in chondrocytes. LIPUS induced collagen synthesis and the remodeling of aggrecan via the activation of ERK1/2. In contrast, MMP13 expression was decreased in chondrocytes. Additionally, chondrocytes responded optimally to LIPUS at an intensity higher than the clinical setting for bone fracture healing. These results suggested that LIPUS induced ECM regeneration via increases in hypertrophic chondrocytes and delayed endochondral ossification in chondrocytes.

Diabetes mellitus is closely related to oral health. We aimed to determine the relationship between diabetes mellitus and tooth extraction due to periodontal disease and dental caries. Japan’s second nationwide survey data collected from 4 June to 10 June 2018 was used to identify reasons for tooth extraction among patients aged &gt, 40 years. General dentists collected information on patients who underwent tooth extraction procedures, and the presence of diabetes mellitus was determined through interviews. Multivariable logistic regression was performed to investigate the relationship between diabetes mellitus and the reasons for tooth extraction, including periodontal disease and dental caries. In total, 2345 dentists responded to the survey (response rate 44.8%). We analyzed data on 4625 extracted teeth from 3750 patients (1815 males and 1935 females). Among patients with self-reported diabetes mellitus, 55.4% had extractions due to periodontal disease compared to 46.7% of such extractions among those without self-reported diabetes mellitus. Self-reported diabetes mellitus was significantly associated with tooth extraction due to periodontal disease. No significant differences were observed in dental caries according to self-reported diabetes mellitus status. This study provides further evidence of a significant association between diabetes mellitus and tooth extraction due to periodontal disease.

Mechanical stimuli such as fluid shear and cyclic tension force induced extracellular adenosine triphosphate (ATP) release in osteoblasts. In particular, cyclic tension force-induced ATP enhances bone formation through P2X7 activation. Proline-rich tyrosine kinase 2 (PYK2) mediate osteoblasts differentiation is induced by mechanical stimuli. Furthermore, activation of PYK2 also was a response to integrin by mechanical stimuli. Extracellular matrix protein (ECMP)s, which are important factors for bone formation are expressed by osteoblasts. However, the effect of the interaction of 2'(3)-Ο-(4-Benzoylbenzoyl) adenosine-5'-triphosphate (BzATP), which is the agonist of the mechanosensitive receptor P2X7, with PYK2 on ECMP production is poorly understood. Thus, our purpose was to investigate the effects of PYK2 on BzATP-induced ECMP production in osteoblasts. BzATP increased phospho-PYK2 protein expression on days 3 and 7 of culture. Furthermore, the PYK2 inhibitor PF431394 inhibited the stimulatory effect of BzATP on the expression of type I collagen, bone sialoprotein and osteocalcin expression. PF431396 did not inhibit the stimulatory effect of BzATP on osteopontin (OPN) mRNA expression. These results suggest that mechanical stimuli activate P2X7 might induce ECMPs expression through PYK2 except in the case of OPN expression. Altogether, mechanical stimuli-induced ECMPs production might be implicated by extracellular ATP secretion or integrin via PYK2 activation.

Inflammatory cytokines, interleukin (IL)-1, IL-6, and TNF-α, are involved in inflammatory bone diseases such as rheumatoid osteoarthritis and periodontal disease. Particularly, periodontal disease, which destroys alveolar bone, is stimulated by lipopolysaccharide (LPS). Low-intensity pulsed ultrasound (LIPUS) is used for bone healing in orthopedics and dental treatments. However, the mechanism underlying effects of LIPUS on LPS-induced inflammatory cytokine are not well understood. We therefore aimed to investigate the role of LIPUS on LPS-induced IL-1α production. Mouse calvaria osteoblast-like cells MC3T3-E1 were incubated in the presence or absence of LPS (Porphyromonas gingivalis), and then stimulated with LIPUS for 30 min/day. To investigate the role of LIPUS, we determined the expression of IL-1α stimulated with LIPUS and treated with an angiotensin II receptor type 1 (AT1) antagonist, Losartan. We also investigate to clarify the pathway of LIPUS, we transfected siRNA silencing AT1 (siAT1) in MC3T3-E1. LIPUS inhibited mRNA and protein expression of LPS-induced IL-1α. LIPUS also reduced the nuclear translocation of NF-κB by LPS-induced IL-1α. Losartan and siAT1 blocked all the stimulatory effects of LIPUS on IL-1α production and IL-1α-mediated NF-κB translocation induced by LPS. Furthermore, PLCβ inhibitor U73122 recovered NF-κB translocation. These results suggest that LIPUS inhibits LPS-induced IL-1α via AT1-PLCβ in osteoblasts. We exhibit that these findings are in part of the signaling pathway of LIPUS on the anti-inflammatory effects of IL-1α expression.

Periodontal disease is caused by inflammation induced by Porphyromonas gingivalis (P.g.) lipopolysaccharide (LPS) and involves expression of proinflammatory cytokines such as interleukin (IL)-1, IL-6, tumor necrosis factor-α, and receptor activator of nuclear factor kappa B ligand (RANKL), which are implicated in bone resorption. Low-intensity pulsed ultrasound (LIPUS) is commonly used in the treatment of bone fracture. However, the mechanisms by which LIPUS inhibits LPS-induced inflammatory cytokines are poorly understood. Therefore, we investigated the effects of LIPUS on LPS-induced expression of the proinflammatory cytokines IL-6 and RANKL. MC3T3-E1 cells were incubated in the presence or absence of P.g. LPS and then stimulated with LIPUS for 30 min/day for a maximum of 14 days. LPS increased mRNA and protein expressions of IL-6 and RANKL on day 14. In addition, mRNA expression of COX-2 LPS was higher after 3 and 7 days of LIPUS treatment. PGE2 was induced by LPS after 7 and 14 days of culture. LIPUS suppressed all stimulatory effects of LPS. These results suggest that LIPUS inhibits LPS-induced expression of inflammation cytokines by suppressing PGE2 production and might thus have potential applications in the treatment of periodontitis.

This study aimed to examine the oral health status of seniors residing in Providence Health Care (PHC) long-term care facilities in 2002 and 2012.Staff dentists with the University of British Columbia Geriatric Dentistry Program made a complete oral health assessment of 799 elderly residents of 7 long-term care PHC facilities in 2002 and 381 residents in the 5 remaining PHC facilities in 2012. The 2012 data were divided into those for 275 residents who had received treatment in previous years and 106 new residents. All consenting residents were examined by dentists using the clinical oral disorder in elders (CODE) index detailing their medical and oral health status and medications. On completion of the oral health assessment, the dentist documented the need for specific dental treatment and reassessment.Comparing the cohorts from 2002 and 2012, the mean age of the residents who had CODE assessments increased from 85 years to 86 years, the proportion of men increased from 31% to 35%, the mean number of medical conditions per resident remained unchanged (2.6 to 2.5), but the mean number of prescribed medications has increased from 4.0 to 4.6. The percentage of residents with natural teeth increased from 56% to 76%. The proportion of edentulous residents recommended for denture-related treatment decreased from 21% to 10%. The 106 new residents in 2012 had higher treatment needs than the 275 original residents, but fewer required extractions than in 2002. Although the mean number of teeth per resident examined increased from 14.6 to 17.4 over the study period, the need for restorations remained at 20%, and the need for extraction of teeth decreased from 22% to 6%. The proportion of residents with healthy periodontium increased from 14% to 21%, but the need for dental hygiene services increased from 43% to 80%.The profile of long-term care residents who consented to an oral health assessment changed over the first decade of the new millennium, with an increase in mean age and number of prescribed medications, number of retained natural teeth and the need for dental hygiene services, but a decrease in the need for extractions.

TGF-β signaling is one of important function during palatal fusion. Three types of TGF-β receptor (TβR1, TβR2, and TβR3) have been identified, and play essential roles in mechanisms leading to palatal fusion. However, the balance between Smad-dependent/-independent signaling during palatal fusion with inhibited TβR1/2 functions is not fully understood. The objective of this study was to investigate palatal fusion via TGF-β signaling when TβR1 and TβR2, but not TβR3, were inhibited. In addition, the present study examined the functional balance between Smad-dependent/-independent signaling and related gene expression. Palatal organ cultures were treated with TβR1/2 inhibitor in vitro. Control palates were cultured without inhibitor. We observed histological phenotype of palatal fusion, and evaluation of expression pattern by Western blot or real time RT-PCR. Palatal organ cultures treated with the inhibitor did not fuse and the medial edge epithelium remained at embryonic 13 day +72 h in culture. The inhibitor decreased TβR1 and TβR2 expression by approximately 90%, but did not affect TβR3 expression. The expression of p-Smad2 and p-Smad3 was significantly decreased in treated palates compared with controls. The expression of p-Smad4 was slightly decreased in treated palates compared with controls. Smad-independent signaling was also affected by the inhibitor; p-ERK, p-JNK, and p-p38 expressions was significantly reduced in treated palates compared with controls. The expression of transcription factors (Runx1 and Msx1) and extracellular matrix proteins (MMP2/13) was also significantly decreased by inhibitor exposure. Treatment with TβR1/2 inhibitor altered the patterns of the Smad-dependent and -independent signaling pathways during palatal fusion.

Introduction Angiotensin II (Ang II) not only regulates systemic blood pressure through a vasoconstrictive effect, but also promotes bone resorption. We recently reported that Ang II (10–6 M) stimulated the production of matrix metalloproteinases via the AT1 receptor in osteoblastic ROS17/2.8 cells, but suppressed alkaline phosphatase activity. However, the roles of Ang II in osteoblastic differentiation and the function of osteogenesis in osteoblasts are unclear. Therefore, we examined the effect of Ang II on the expression of osteogenesis-related transcription factors and extracellular matrix (ECM) proteins, as well as mineralized nodule formation in ROS17/2.8 cells. Material and methods ROS17/2.8 cells were cultured with 0 (control) or 10–6 M Ang II in the presence or absence of the AT1 receptor blocker losartan. Mineralized nodule formation was detected by Alizarin Red staining. Gene and protein expression levels of transcription factors and ECM proteins were determined using real-time PCR and Western blotting, respectively. Results Runx2, Msx2, and osteocalcin expression significantly decreased with Ang II compared to the control, whereas AJ18 expression significantly increased. Osterix, Dlx5, type I collagen, bone sialoprotein, and osteopontin expression was unaffected. Mineralized nodule formation and calcium content in mineralized nodules decreased with Ang II. Losartan blocked suppressive or stimulatory effects of Ang II on Runx2, Msx2, osteocalcin, and AJ18 expression. Conclusions These results suggest that Ang II suppresses osteoblastic differentiation by altering the expression of osteogenesis-related transcription factors via the AT1 receptor and the function of osteogenesis in ROS17/2.8 cells.

Objective: Matrix metalloproteinases (MMPs), produced by osteoblasts, catalyze the turnover of extracellular matrix (ECM) molecules in osteoid, and the regulation of MMP activity depends on interactions between MMPs and tissue inhibitors of metalloproteinases (TIMPs). We focused on the degradation process of ECM in osteoid that was exposed to mechanical strain, and conducted an in vitro study using MC3T3-E1 osteoblastic cells to examine the effects of tension force (TF) on the expression of MMPs and TIMPs, and activation of mitogen-activated protein kinase (MAPK) pathways. Design: Cells were incubated on flexible-bottomed culture plates and stimulated with or without cyclic TF for 24 hours. The expression of MMPs and TIMPs was examined at mRNA and protein levels by real-time RT-PCR and Western blotting, respectively. The phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, p38 MAPK, and stress-activated protein kinases/c-jun N-terminal kinases (SAPK/JNK) were examined by Western blotting. Results: TF decreased the expression of MMP-1, -3, -13 and phosphorylated ERK1/2. In contrast, TF increased the expression of TIMP-2, -3 and phosphorylated SAPK/JNK. The expression of MMP-2, -14, TIMP-1, -4 and phosphorylated p38 MAPK was unaffected by TF. MMP-1, -3 and -13 expression decreased in cells treated with the ERK inhibitor PD98059 compared with untreated control cells. The JNK inhibitor SP600125 inhibited the TF-induced upregulation of TIMP-2 and -3. Conclusions: The results suggest that TF suppresses the degradation process that occurs during ECM turnover in osteoid via decreased production of MMP-1, -3 and -13, and increased production of TIMP-2 and -3 through the MAPK signaling pathways in osteoblasts.

Aims During orthodontic treatment, facilitating osteoclastic bone resorption in the alveolar bone exposed to the compressive force (CF) is an important factor for tooth movement. The present study investigated the effect of CF stimulation on the differentiation of RAW264.7 cells from precursors to mature osteoclasts. Main methods The cells were continuously stimulated with 0.3, 0.6, or 1.1 g/cm2 CF—which was generated by increasing the volume of culture medium in the wells of a 96-well plate—in the presence or absence of receptor activator of nuclear factor κB (RANK) ligand (RANKL) for 4 days. Key findings In the presence of RANKL, the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and the mRNA levels of dendritic cell-specific transmembrane protein (DC-STAMP) and osteoclast-stimulatory transmembrane protein (OC-STAMP) were increased by application of 0.6 and 1.1 g/cm2 CF as compared to 0.3 g/cm2 CF. The mRNA level of RANK was upregulated whereas that of leucine-rich repeat-containing G-protein-coupled receptor (LGR)4—another RANKL receptor was downregulated by 0.6 and 1.1 g/cm2 CF as compared to 0.3 g/cm2 CF in the absence of RANKL. The proportion of cells with nuclear translocation of the nuclear translocation of nuclear factor of activated T cells (NFAT)c1 was increased by 0.6 and 1.1 g/cm2 CF in the presence of RANKL. Significance Continuous application of CF induced the differentiation of RAW264.7 cells into TRAP-positive multinuclear cells by enhancing the expression of DC- and OC-STAMP and the nuclear translocation of NFATc1. This may result from the CF-induced increase in RANK and decrease in LGR4 expression.

Mineral trioxide aggregate (MTA) has excellent biocompatibility as well as bioactivity, including an ability to induce osteoblast differentiation. We examined the effects of the calcium-sensing receptor (CaSR) on osteogenic gene expression induced by MTA. MC3T3-E1 cells were cultured with or without (control) MTA. The expression levels of Runx2, type I collagen, and CaSR genes were analyzed by real-time polymerase chain reaction and their products were measured using enzyme-linked immunosorbent assays. The levels were increased significantly in cells exposed to MTA compared with control. Next, MC3T3-E1 cells were cultured with MTA and EGTA (a calcium chelator), because calcium ions were released continuously from MTA into the culture. Expression levels were decreased to control levels by MTA plus EGTA. NPS2143 (a CaSR antagonist) also reduced MTA-induced gene expression. These results suggest that MTA induced osteogenic gene expressions of Runx2 and type I collagen via CaSR in MC3T3-E1 cells.

Orthodontic tooth movement induces alveolar bone resorption and formation by mechanical stimuli. Force exerted on the traction side promotes bone formation. Adenosine triphosphate (ATP) is one of the key mediators that respond to bone cells by mechanical stimuli. However, the effect of tension force (TF)-induced ATP on osteogenesis is inadequately understood. Accordingly, we investigated the effect of TF on ATP production and osteogenesis in MC3T3-E1 cells. Cells were incubated in the presence or absence of P2X7 receptor antagonist A438079, and then stimulated with or without cyclic TF (6% or 18%) for a maximum of 24 h using Flexercell Strain Unit 3000. TF significantly increased extracellular ATP release compared to control. Six percent TF had maximum effect on ATP release compared to 18% TF and control. Six percent TF induced the expression of Runx2 and Osterix. Six percent TF also increased the expression of extracellular matrix proteins (ECMPs), ALP activity, and the calcium content in ECM. A438079 blocked the stimulatory effect of 6% TF on the expression of Runx2, Osterix and ECMPs, ALP activity, and calcium content in ECM. This study indicated that TF-induced extracellular ATP is released in osteoblasts, suggesting that TF-induced ATP promotes osteogenesis by autocrine action through P2X7 receptor in osteoblasts. J. Cell. Biochem. 116: 12–21, 2015. © 2014 The Authors. Journal of Cellular Biochemistry published by Wiley Periodicals, Inc.

Angiotensin II (Ang II) plays an important role in the maintenance of bone mass and integrity by activation of the mitogen-activated protein kinases (MAPKs) and by modulation of balance between resorption by osteoclasts and formation by osteoblasts. However, the role of Ang II in the turnover of extracellular matrix (ECM) in osteoid by osteoblasts remains unclear. Therefore, we examined the effect of Ang II on the expression of matrix metalloproteinases (MMPs), plasminogen activators (PAs), and their inhibitors [i.e., tissue inhibitors of metalloproteinases (TIMPs) and PA inhibitor-1 (PAI-1)] using osteoblastic ROS17/2.8 cells. Treatment with Ang II strikingly increased the expressions of MMP-3 and -13 and promoted cell proliferation associated with reduced alkaline phosphatase activity as well as enhanced phosphorylated expression of extracellular signal-regulated kinase (ERK)1/2, p38 MAPK, and stress-activated protein kinases/c-jun N-terminal kinases (SAPK/JNK) in ROS17/2.8 cells. However, Ang II had no effect on the expression of MMP-2, -9, -14, urokinase-type PA, tissue-type PA, TIMP-1, -2, -3, and PAI-1 in cells. Losartan (AT1 receptor blocker) blocked Ang II-induced expression of MMP-3 and -13, whereas PD123319 (AT2 receptor blocker) did not completely block these responses. Losartan also blocked the Ang II-induced phosphorylation of ERK1/2, p38 MAPK, and SAPK/JNK. MAPK kinase 1/2 inhibitor PD98059 and JNK inhibitor SP600125 suppressed Ang II-induced expression of MMP-3 and -13. These results suggested that Ang II stimulated the degradation process that occurs during ECM turnover in osteoid by increasing the production of MMP-3 and -13 through MAPK signaling pathways via the AT1 receptor in osteoblasts. Furthermore, our findings suggest that Ang II does not influence the plasminogen/plasmin pathway in osteoblasts.

The association between obesity and inflammation is well documented in epidemiological studies. Proteolysis of extracellular matrix (ECM) proteins is involved in adipose tissue enlargement, and matrix metalloproteinases (MMPs) collectively cleave all ECM proteins. Here, we examined the effects of C-reactive protein (CRP), an inflammatory biomarker, on the expression of MMPs and tissue inhibitors of metalloproteinases (TIMPs), which are natural inhibitors of MMPs, in adipocyte-differentiated 3T3-L1 cells. We analyzed the expression of Fcγ receptor (FcγR) IIb and FcγRIII, which are candidates for CRP receptors, and the effects of anti-CD16/CD32 antibodies, which can act as FcγRII and FcγRIII blockers on CRP-induced alteration of MMP and TIMP expression. Moreover, we examined the effects of CRP on the activation of mitogen-activated protein kinase (MAPK) signaling, which is involved in MMP and TIMP expression, in the presence or absence of anti-CD16/CD32 antibodies. Stimulation with CRP increased MMP-1, MMP-3, MMP-9, MMP-11, MMP-14, and TIMP-1 expression but did not affect MMP-2, TIMP-2, and TIMP-4 expression; TIMP-3 expression was not detected. Adipocyte-differentiated 3T3-L1cells expressed FcγRIIb and FcγRIII; this expression was upregulated on stimulation with CRP. Anti-CD16/CD32 antibodies inhibited CRP-induced expression of MMPs, except MMP-11, and TIMP-1. CRP induced the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and p38 MAPK but did not affect SAPK/JNK phosphorylation, and Anti-CD16/CD32 attenuated the CRP-induced phosphorylation of p38 MAPK, but not that of ERK1/2. These results suggest that CRP facilitates ECM turnover in adipose tissue by increasing the production of multiple MMPs and TIMP-1 in adipocytes. Moreover, FcγRIIb and FcγRIII are involved in the CRP-induced expression of MMPs and TIMP-1 and the CRP-induced phosphorylation of p38, whereas the FcγR-independent pathway may regulate the CRP-induced MMP-11 expression and the CRP-induced ERK1/2 phosphorylation.

Epidemiological studies have reported that periodontitis and cardiometabolic disease such as cardiovascular disease and type 2 diabetes are associated; however, there have been very few prospective cohort studies on this topic. Therefore, we conducted a 9-year follow-up study to examine the relationship between the duration of periodontitis and cardiometabolic risk factors, including hypertension, hyperglycemia, dyslipidemia, and obesity.The study participants comprised 572 adult industrial workers (417 men and 155 women; mean age, 37.4 years) who had undergone annual medical and dental health examinations from 2003 to 2012; the evaluation of the four cardiometabolic risk factors in 2003 revealed normal values in all the participants. We investigated the relationship between the cumulative duration of the presence of periodontal pockets, which is a major symptom of periodontitis, and the presence of cardiometabolic risk factors after 9 years using multiple logistic regression analysis.The odds ratio (OR) for the presence of ≥1 cardiometabolic risk factor in participants with a cumulative duration of periodontal pockets for ≥6 years was significantly higher than that in participants without pockets. The ORs for the onset of obesity, hypertension, dyslipidemia, and hyperglycemia were higher in participants with a cumulative duration of periodontal pockets for ≥6 years than those in participants without pockets or in participants with a cumulative duration of periodontal pockets for ≤5 years, and all the differences, except dyslipidemia, were significant.Chronic periodontitis was significantly associated with having cardiometabolic risk factors during the 9-year observation period, suggesting that the risk of cardiometabolic disease might increase in people who have untreated periodontitis.

In bone remodeling during orthodontic tooth movement, osteoblasts are closely related to bone matrix formation and osteoclast differentiation. Interleukin (IL)-11 is known to be a regulating factor of bone remodeling. Our previous study showed that compressive force (CF) induced IL-11 production in osteoblastic Saos-2 cells. Therefore, we examined the effect of CF on the expression of IL-11 receptor (IL-11r), IL-11, Runx2, bone morphogenetic protein (BMP)-2, receptor activator of NF-κB ligand (RANKL), macrophage colony-stimulating factor (M-CSF), and osteoprotegerin (OPG) using MC3T3-E1 cells as osteoblasts. To confirm the participation of autocrine mechanisms by CF-induced IL-11, we also examined the effect of CF on expression in the presence and absence of IL-11 antagonist. MC3T3-E1 cells were cultured with or without continuous CF (1.0 or 2.0 g/ cm 2) in the presence or absence of IL-11 antagonist for up to 24 h. The expression of the IL-11r, IL-11, Runx2, BMP-2, RANKL, M-CSF, and OPG genes was estimated by determining mRNA levels by real-time PCR; protein expression was examined by ELISA or Western blotting. CF increased the expression of IL-11r and IL- 11; the maximum level of these expressions was achieved with CF at 2.0 g/cm 2. CF also increased the expression of Runx2 and BMP-2; the maximum level of these expressions was achieved in CF at 1.0 g/cm 2. IL-11 antagonist blocked the stimulatory effects of CF at 1.0 or 2.0 g/cm 2 on the expression of Runx2, BMP-2, RANKL, and M- CSF but not on the expression of IL-11r, IL-11, and OPG. These results suggest that CF at 1.0 g/cm 2 increases the expression of proteins related to bone formation, such as Runx2 and BMP-2, via the autocrine mechanism of CF-induced IL-11 in osteoblasts, whereas CF at 2.0 g/cm 2 increases the expression of RANKL and M-CSF that promotes osteoclast formation via CF-induced IL-11. These results also suggest that the balance of bone remodeling changes according to the strength of CF. © 2012The Hard Tissue Biology Network Association Printed in Japan, All rights reserved.

Objectives: To assess the influence of xylitol chewing gum consumption on mutans streptococci level of 3–4 years old Japanese preschoolers. Methods: 248 participants were examined regarding caries-related factors at baseline and were followed up at 6, 9, and 12 months after the baseline: assessors were blinded, subjects were open labelled and blocked parallel randomised; 142 were selected to use xylitol gum for 3 months (from months 6 to 9) and 106 were controls. Results: 161 participants were analysed (xylitol n = 76, control n = 85). Nineteen caries-related variables, including xylitol gum consumption, were analysed for any association with the main outcome, plaque mutans streptococci scores development within the intervention period, by logistic regression. Six showed statistically significant associations by univariate analysis (P

Objective Sodium butyrate (butyric acid; BA) is a major metabolic by-product of the anaerobic periodontopathic bacteria present in subgingival plaque. We examined the effects of BA and/or indomethacin on cell proliferation, the expression of cyclooxygenases (COXs), prostaglandin (PG) receptors (EP1-4), extracellular matrix proteins, such as type I collagen and osteopontin, and PGE2 production, using ROS17/2.8 cells as osteoblasts. Methods The rat clonal cell line ROS 17/2.8 was cultured with 0, 10−5, 10−4, and 10−3 M BA in the presence or absence of 0.5 μM indomethacin, for up to 7 days. The expression of COX-1, COX-2, EP1, EP2, EP3, EP4, type I collagen, and osteopontin was examined at the mRNA and protein levels using real-time PCR and Western blotting, respectively. The amount of PGE2 in the culture medium was measured by ELISA. Results Proliferation of ROS 17/2.8 cells was not affected by the addition of BA. However, PGE2 production and the expression of COX-1 and COX-2 increased with the addition of BA. In contrast, indomethacin, an inhibitor of COX, blocked the stimulatory effect of BA. Furthermore, EP2 expression increased with BA treatment, whereas EP1 expression was not affected and the expression of EP3 and EP4 was not detected. The addition of BA also increased the expression of type I collagen and osteopontin. Indomethacin blocked about 50% of the stimulatory effect of BA on type I collagen, whereas it did not block the effect on osteopontin. Conclusions These results suggest that BA induces PGE2 production by increasing the expression of COX-1 and COX-2 in osteoblasts, and that an autocrine action of the produced PGE2, via EP1 or BA-induced EP2, is related to an increase in type I collagen expression by BA.

Interleukin-17 (IL-17) is a cytokine secreted primarily by T H -17 cells that can stimulate the development of osteoclasts (osteoclastogenesis) in the presence of osteoblasts. IL-17, through osteoblasts, has indirect effects on the expression of bone resorption-related enzymes in osteoclasts, which have not been well clarified. Here, using MC3T3-E1 cells and RAW264.7 cells as osteoblasts and osteoclast precursors, we aimed to clarify these effects of IL-17A. MC3T3-E1 cells were cultured in the presence or absence of IL-17A for 72 h and the conditioned media collected (in the presence of soluble receptor activator of NF-кB ligand) and used to culture RAW264.7 cells. To assess osteoclast differentiation, adherent cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP). Our analyses demonstrated that the number of TRAP-positive multinucleated cells increases after 3 days of culture in conditioned medium from IL-17A-treated cells compared to untreated controls. In addition, we observed that the levels of cathepsin K and MMP-9 increase in the conditioned medium from IL-17A-treated cells, whereas CA II expression levels remain unaffected. PGE 2 production from MC3T3-E1 cells increased in the presence of IL-17A. Celecoxib, a specific inhibitor of cyclooxygenase-2 (COX-2), blocked both the IL-17A-stimulated increase in TRAP-positive multinucleated cells and the expression of cathepsin K and MMP-9. Furthermore, when MC3T3-E1 cells were transformed with small interfering RNA to silence COX-2 expression before IL-17A treatment, the resulting conditioned medium was less effective at inducing cathepsin K and MMP-9 expression in RAW264.7 cells. These results suggest that IL-17A induces the differentiation and function of osteoclasts via celecoxib-blocked prostaglandin, mainly PGE 2 , in osteoblasts.

Interleukin-17 (IL-17) is a proinflammatory cytokine involved in autoimmune diseases such as rheumatoid arthritis (RA). IL-17 consists of six ligands that signal through five receptors (IL-17Rs). Serine proteinases, such as plasmin and plasminogen activators (PAs), as well as matrix metalloproteinases (MMPs) degrade the extracellular matrix during cartilage remodeling.In contrast, plasminogen activator inhibitor-1 (PAI-1), an endogenous inhibitor of PAs, forms a complex with PA to inhibit plasmin production. In the present study, we examined the effect of IL-17F on the plasminogen/plasmin pathway in cartilage using human articular chondrocytes. We evaluated the effects of IL-17F on the expression of tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), PAI-1, and cyclooxygenases (COXs), as well as on the production of prostaglandin E2 (PGE2). We also examined the indirect effects of PGE2 on the above IL-17F-induced/reduced components using NS-398, a specific inhibitor of COX-2 activity. Cells were cultured with or without IL-17F in the presence or absence of NS-398 for up to 28 days. mRNA and protein expression levels of PAs, PAI-1, and COXs were determined using real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. PGE2 production was determined by ELISA. The addition of IL-17F increased the expression of PAI-1 and COX-2, as well as the production of PGE2, whereas uPA and COX-1 expression decreased and tPA expression was unaffected. NS-398 did not block the reducing/stimulating effects of IL-17F on the expression of COX-1 and COX-2. In contrast, NS-398 may be, in part, responsible for IL-17F-induced/reduced uPA and PAI-1 expression. Our results suggest that IL-17F suppresses the plasminogen/plasmin pathway by increasing PAI-1 expression and decreasing uPA expression in chondrocytes.

Interleukin (IL)-17 plays an important role in many autoimmune and inflammatory diseases. We recently demonstrated the various direct and indirect effects of IL-17A on osteoclastogenesis. However, the effect of IL-17A on bone matrix formation by osteoblasts is unclear. Therefore, we examined the effect of IL-17A on IL-17 receptor (IL-17R), extracellular matrix protein (ECMP), and cyclooxygenase (COX) expression; prostaglandin (PG) E2 production; alkaline phosphatase (ALPase) activity; and mineralized nodule formation in osteoblastic ROS17/2.8 cells. We also examined the indirect effect of PGE2 on IL-17-induced ECMP expression using NS398, a specific inhibitor of COX-2 activity. Cells were cultured with 0 (control), 1, 10, or 100 ng/mL IL-17A in the presence or absence of 50 ng/mL neutralizing anti-IL-17A antibodies or 1 μM NS398. IL-17RA, IL-17RC, type I collagen, bone sialoprotein (BSP), osteocalcin, osteopontin, COX-1, and COX-2 expression was examined by real-time PCR and Western blotting. PGE2 production was examined by ELISA. The expression of IL-17RA, type I collagen, BSP, osteocalcin, osteopontin, and COX-2, and PGE2 production were increased significantly in the presence of IL-17A, whereas the expression of IL-17RC and COX-1, cell proliferation, ALPase activity, and mineralized nodule formation were unaffected. Anti-IL-17 antibodies blocked IL-17Ainduced ECMP expression. NS398 blocked IL-17A-induced PGE2 production, but it did not affect IL-17A-induced ECMP expression. These results suggest that IL-17A stimulates ECMP expression via IL-17RC and/or IL-17A-induced IL-17RA in osteoblasts; however, mineralized nodule formation by the cells was unaffected by the addition of IL-17A. Furthermore, our results indicate that the stimulatory effect of IL-17A on ECMP expression is independent of the indirect effect of IL-17A-induced PGE2.

Interleukin-17 (IL-17) is produced exclusively by activated T cells and neutrophils, and stimulates osteoclastic bone resorption via osteoblasts by inducing the expression of "receptor activator of NF-kappaB (RANK) ligand" (RANKL). However, the direct effects of IL-17 on the differentiation of osteoclast precursors into osteoclasts and on the function of osteoclasts have not been clarified. Therefore, we examined the effects of IL-17A on the differentiation of osteoclast precursors using RAW264.7 cells and also on the expression of carbonic anhydrase II (CA II), cathepsin K, matrix metalloproteinases-9 (MMP-9), RANK, c-fms, and IL-17 receptors in these cells. The cells were cultured with or without 0.1, 1.0, 10 or 50 ng/mL IL-17 in the presence of soluble RANKL for up to 10 days. The CA II, cathepsin K, and MMP-9 mRNA and protein expression levels were examined using real-time PCR and Western blotting, respectively. The mRNA expression levels of RANK, c-fms, and IL-17 receptors were monitored by real-time PCR. Osteoclast differentiation was estimated using tartrate-resistant acid phosphatase (TRAP) staining of the cells. TRAP-positive cells were observed after day 5 of culture, and the number of cells decreased in the presence of 10 and 50 ng/mL IL-17A at days 5 and 7. In the presence of IL-17A, the expressions of cathepsin K, MMP-9 and c-fms decreased markedly on days 5 and/or 7 of culture, whereas the expression of CA II and IL-17 receptor (type A) increased remarkably at days 3 and 7, respectively. The expression of RANK and IL-17 receptor (type C) was not affected by the addition of IL-17A. These results suggest that the differentiation of osteoclast precursors into osteoclasts is suppressed at high concentrations of IL-17A. Furthermore, IL-17A suppresses the hydrolysis of matrix proteins during bone resorption by decreasing the production of cathepsin K and MMP-9 in osteoclasts.

This study concerns the use of low-energy pulsed ultrasound as nondestructive photodynamic antimicrobial therapy for controlling dental plaque. We examined the antibacterial and bactericidal effects of low-energy pulsed ultrasound on mutans streptococci and its inhibitory effects on bacterial cell adhesion of Streptococcus mutans . The results indicated weak antibacterial and bactericidal effects. However, ultrasonic stimulation for less than 20 min markedly decreased bacterial cell adhesion. To analyze the mechanism underlying the inhibitory effect, we examined cell surface protein antigen C (PAc) and glucosyltransferase I (GTF-I) expression in S. mutans . The levels of PAc gene and protein expression were markedly decreased by ultrasonic stimulation for 20 min. However, no change in GTF-I expression was observed. The expression of stress response heat shock proteins GroEL and DnaK was also examined. GroEL and DnaK levels were significantly decreased by ultrasonic stimulation, and the expression of the PAc protein was also diminished upon the addition of GroEL or DnaK inhibitors without ultrasonic stimulation. These observations suggest that the expression of the PAc protein in S. mutans may be dependent on heat shock proteins. Thus, low-energy pulsed ultrasound decreases bacterial adhesion by the inhibitory effect on the PAc protein and heat shock protein expression and may be useful as photodynamic antimicrobial chemotherapy in controlling dental plaque.

Background/aims: The cell surface protein antigen (PAg) and glucosyltransferases (GTFs) produced by Streptococcus sobrinus are considered to be major colonization factors of the organism. Methods: We constructed a fusion gene encoding a protein composed of the alanine-rich region of PAg (PAgA) and the glucan-binding domain (GB) of GTF-I, which catalyzes the synthesis of water-insoluble glucan in S. sobrinus. The fusion protein PAgA-GB was purified from cell extracts of Escherichia coli harboring the fusion gene, and antibodies against the fusion protein were prepared in rabbits. Results: In the presence of sucrose, the antibody against PAgA-GB significantly inhibited the adhesion of both S. sobrinus MT8145 and Streptococcus mutans Xc to saliva-coated hydroxyapatite beads, and the inhibitory effect on S. sobrinus was stronger than that on S. mutans. In the absence of sucrose, the antibody against PAgA-GB significantly inhibited the adhesion of both S. sobrinus and S. mutans, however the inhibitory effect on S. sobrinus was unexpectedly weaker than that on S. mutans. A similar result was observed with the antibody against the intact recombinant PAg protein (rPAg), while the same antibody reacted more strongly against S. sobrinus than against S. mutans cells. Conclusion: Taken together, these results show that the antibody against S. sobrinus GTF-I may be useful for effective inhibition of the sucrose-dependent adhesion of S. sobrinus. However, PAg of S. sobrinus may not function primarily as a receptor for acquired pellicles, and other cell surface proteins may be involved in the sucrose-independent adhesion of S. sobrinus.

Elevated concentrations of interleukin (IL)-6 and soluble IL-6 receptor (sIL-6Ralpha) in synovial fluid have been implicated in joint cartilage destruction. We examined the effect of IL-6 and sIL-6Ralpha on cell growth, alkaline phosphatase (ALPase) activity, and the expression of Sox-9, type II collagen, aggrecan core, link protein, BMP-7, and BMP receptors in human chondrocytes. Cell proliferation increased slightly in the presence of both IL-6 and sIL-6Ralpha, whereas ALPase activity decreased markedly. The expression of Sox-9 and aggrecan core did not change in the presence or absence of IL-6 and sIL-6Ralpha, whereas the expression of type II collagen, link protein, BMP-7, and BMP receptors increased in the presence of both IL-6 and sIL-6Ralpha. These results suggest that IL-6 and sIL-6Ralpha suppress the differentiation of chondrocytes and induce the repair of arthrodial cartilage through an increase in the expression of cartilage matrix proteins, BMP-7, and BMP receptors in the cells.

This study used in vivo microdialysis to examine the effects of intragingival application of lipopolysaccharide (LPS) derived from Porphyromonas gingivalis (Pg-LPS) on gingival tumour necrosis factor (TNF)-α and interleukin (IL)-6 levels in rats. A microdialysis probe with an injection needle attached to the surface of the dialysis membrane was implanted into the gingiva of the upper incisor. For comparison, the effects of LPS derived from Escherichia coli (Ec-LPS) on IL-6 and TNF-α levels were also analysed. Pg-LPS (1 μg/1 μL) or Ec-LPS (1 or 6 μg/1 μL) was applied by microsyringe, with gingival dialysates collected every hour. Enzyme-linked immunosorbent assay (ELISA) revealed that gingival dialysates contained approximately 389 pg·mL−1 of IL-6 basally; basal TNF-α levels were lower than the detection limit of the ELISA. Pg-LPS failed to alter IL-6 levels but markedly increased TNF-α levels, which remained elevated for 2 h after treatment. Neither IL-6 nor TNF-α were affected by Ec-LPS. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that the gingiva expresses Toll-like receptor (TLR) 2 and TLR4 mRNA. Immunohistochemical examination showed that TLR2 and TLR4 are expressed by gingival epithelial cells. The present study provides in vivo evidence that locally applied Pg-LPS, but not Ec-LPS, into the gingiva transiently increases gingival TNF-α without affecting IL-6. The present results suggest that TLR2 but not TLR4 expressed on gingival epithelial cells may mediate the Pg-LPS-induced increase in gingival TNF-α in rats.