Impact of dental health intervention on cardiovascular metabolic risk: a pilot study of Japanese adults.

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 < 0.05). Intergroup comparison showed significant improvement of systolic blood pressure (mmHg) in the intervention group (-3.7 ± 12.5) compared with the non-intervention group (4.0 ± 15.9) (P < 0.05) with no significant differences in the other parameters. The intervention group had a decrease in plaque accumulation and periodontitis symptoms, such as the depth of periodontal pocket and the presence of periodontal pocket and bleeding on probing, but an increase in the frequency of interdental brushing and duration of tooth brushing. Our findings show that dental self-care may improve blood pressure in people with cardiometabolic risk factors and periodontitis.

Effects of interleukin-6 and tumor necrosis factor-α on the expression of angiogenic and collagenolytic factors in premature and mature adipocytes.

OBJECTIVE: The remodeling of the vascular network and collagen in the extracellular matrix is closely associated with the expansion and dysfunction of adipose tissue. In the present study, we investigated the effects of interleukin (IL)-6 and tumor necrosis factor (TNF)-α on the expression of angiogenic factors, collagen, and collagenase and its endogenous inhibitor in premature and mature adipocytes. METHODS: Premature and mature adipocytes were differentiated from 3T3-L1 cells and stimulated with IL-6 or TNF-α to mimic the early and late phases of obesity development. The levels of expression of angiogenic factors, including vascular endothelial cell growth factor a (Vegfa), hepatocyte growth factor (Hgf), angiopoietin (Angpt)1, and Angpt2, as well as type I collagen, matrix metallopeptidase (Mmp) 13, and tissue inhibitor of Mmp (Timp) 1, were determined using real-time reverse transcription polymerase chain reaction or enzyme-linked immunosorbent assay. Human umbilical vein endothelial cells were grown with the culture supernatant of adipocytes stimulated with/without IL-6 or TNF-α, and the formation of tube structures was evaluated. RESULTS: IL-6 and TNF-α induced the expression of Vegfa, Hgf, and Angpt2 and decreased the expression of Angpt1 in premature adipocytes, whereas, they decreased the expression of Vegfa and Hgf in mature adipocytes. The culture supernatant of IL-6- or TNF-α-stimulated premature adipocytes induced the formation of tube structures. IL-6 and TNF-α had no effects on type I collagen expression in both premature and mature adipocytes but suppressed the expression of Mmp13 and Timp1 in mature and premature adipocytes, respectively. CONCLUSION: The effects of IL-6 and TNF-α on the expression of angiogenic and collagenolytic factors differed between premature and mature adipocytes. This finding suggests that these inflammatory cytokines induce expansion and dysfunction of adipose tissue via angiogenesis and collagen turnover in premature and mature adipocytes.

Effect of Periodontitis and Toothbrushing Frequency on Obesity Onset: A Cohort Study.

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 <25). The response variable was obesity (or absence) at 4 years, while the explanatory variables were presence/absence of periodontal pockets and toothbrushing frequency in 2002; their relationships were examined by multiple logistic regression analysis. RESULTS Subjects with periodontal pockets ≥4 mm showed a significantly higher odds ratio (OR) for onset of obesity at 4 years than those without periodontal pockets [OR: 1.59, 95% CI (confidence interval): 1.08-2.35, p<0.05]. Similarly, subjects who brushed their teeth ≥3 times/day had a significantly lower obesity OR than those who brushed ≤1 time/day (OR: 0.49, 95% CI: 0.28-0.85, p<0.01). CONCLUSIONS The presence of periodontal pockets and toothbrushing frequency are significantly associated with the onset of obesity. Periodontal pockets ≥4 mm are associated with increased risk of obesity, while frequent toothbrushing (≥3 times/day) appears to reduce the risk of obesity.

Continuous Compressive Force Induces Differentiation of Osteoclasts with High Levels of Inorganic Dissolution.

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.

Low-intensity pulsed ultrasound induces cartilage matrix synthesis and reduced MMP13 expression in chondrocytes.

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.

Continuous application of compressive force induces fusion of osteoclast-like RAW264.7 cells via upregulation of RANK and downregulation of LGR4.

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.

PYK2 mediates BzATP-induced extracellular matrix proteins synthesis.

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.

A collagen membrane containing osteogenic protein-1 facilitates bone regeneration in a rat mandibular bone defect.

OBJECTIVES: Osteogenic protein-1 (OP-1) has shown osteoinductive activities and is useful for clinical treatments, including bone regeneration. Regenerative procedures using a bioabsorbable collagen membrane (BCM) are well established in periodontal and implant dentistry. We evaluated the subsequent effects of the BCM in combination with OP-1 on bone regeneration in a rat mandibular circular critical-sized bone defect in vivo. DESIGN: We used 8 rats that received surgery in both sides of the mandible, and created the total 16 defects which were divided into 4 groups: Group 1; no treatment, as a control, Group 2; BCM alone, Group 3; BCM containing low dose 0.5µg of OP-1 (L-OP-1), and Group 4; BCM containing high dose 2.0µg of OP-1 (H-OP-1). Newly formed bone was evaluated by micro computed tomography (micro-CT) and histological analyses at 8 weeks postoperatively. In quantitative and qualitative micro-CT analyses of the volume of new bone formation, bone density, and percentage of new bone area was evaluated. RESULTS: BCM with rhOP-1 significantly increased and accelerated bone volume, bone mineral density, and percentage of new bone area compared to control and BCM alone at 8 weeks after surgery; these enhancements in bone regeneration in the OP-1-treated groups were dose-dependent. CONCLUSIONS: OP-1 delivered with a BCM may have effective osteoinductive potency and be a good combination for bone regeneration. The use of such a combination device for osteogenesis may result in safer and more predictable bone regenerative outcomes in the future.

Low-intensity pulsed ultrasound inhibits lipopolysaccharide-induced IL-6 and RANKL expression in osteoblasts.

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.

Effects of C-reactive protein on the expression of matrix metalloproteinases and their inhibitors via Fcγ receptors on 3T3-L1 adipocytes.

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.

Involvement of the calcium-sensing receptor in mineral trioxide aggregate-induced osteogenic gene expression in murine MC3T3-E1 cells.

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.

LIPUS suppressed LPS-induced IL-1α through the inhibition of NF-κB nuclear translocation via AT1-PLCß pathway in MC3T3-E1 cells.

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, PLCß inhibitor U73122 recovered NF-κB translocation. These results suggest that LIPUS inhibits LPS-induced IL-1α via AT1-PLCß 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.

Association Between the Duration of Periodontitis and Increased Cardiometabolic Risk Factors: A 9-Year Cohort Study.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Tension Force Downregulates Matrix Metalloproteinase Expression and Upregulates the Expression of Their Inhibitors through MAPK Signaling Pathways in MC3T3-E1 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.

Angiotensin II suppresses osteoblastic differentiation and mineralized nodule formation via AT1 receptor in ROS17/2.8 cells.

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.

Low-intensity pulsed ultrasound-induced ATP increases bone formation via the P2X7 receptor in osteoblast-like MC3T3-E1 cells.

Low-intensity pulsed ultrasound (LIPUS) is used for bone healing in orthopedics and dentistry. It has been shown that LIPUS induces the secretion of extracellular adenosine triphosphate (ATP), a key mediator of osteoblast response to mechanical stimuli. However, the detailed mechanism of LIPUS-induced osteogenesis has been elusive. In this study, we investigated the role of the P2X7 receptor in LIPUS-induced osteogenesis. LIPUS induced the release of extracellular ATP, differentiation of osteoblasts and osteogenesis via the P2X7 receptor, without affecting the activity of alkaline phosphatase (ALPase). These results suggest that LIPUS-induced extracellular ATP promotes bone formation via the osteoblast P2X7 receptor independently of ALPase.

Tension force-induced ATP promotes osteogenesis through P2X7 receptor in osteoblasts.

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.

Serum γ-glutamyltransferase level is associated with periodontal disease independent of drinking habits in Japanese adults.

Background Non-alcoholic fatty liver disease is considered a hepatic manifestation of metabolic syndrome. Periodontal disease is a mild chronic inflammatory disease with systemic effects, and many studies have indicated an association between metabolic syndrome and periodontitis. In the present study, we investigated the relationship between periodontitis and liver biochemical parameters according to alcohol drinking habits through a cross-sectional study based on data from Japanese people in occupational settings. Material and Methods The subjects were 1510 employees (1218 males, 292 females, mean age 50.4 years) who underwent dental and medical checkups in 2012. Associations between the presence of periodontal pockets and serum levels of liver biochemical parameters were assessed. Results Alanine aminotransferase (ALT) and γ-glutamyltransferase (GGT) levels were higher in subjects with than without periodontal pockets. Multiple logistic regression analysis (adjusting for age, gender, cigarette smoking, and alcohol drinking habits, and components of metabolic syndrome) with GGT or ALT as the dependent variable revealed that there was a significant association between periodontal pockets and GGT (odds ratio, OR=1.48), but not ALT. Similar associations were observed when an analysis was performed according to the presence or absence of alcohol drinking habits; the OR was higher in subjects without (OR=1.84) than with drinking habits (OR=1.41). Conclusions The presence of periodontal pockets was associated with serum levels of GGT, a liver biochemical parameter, in Japanese adults with no drinking habit, suggesting that periodontal disease is associated with liver function, independent of alcohol ingestion.

Functional role of TGF-ß receptors during palatal fusion in vitro.

OBJECTIVE: Reported expression patterns for TGF-ß receptors (TßR-I, -II, and -III) during palatogenesis suggest that they play essential roles in the mechanisms leading to palatal fusion. The purpose of this study was to compare the functions of the three TßRs during palatal fusion. METHODS: Using organ culture of mouse palatal shelves, expression levels of TßR-I, -II, and -III were suppressed by transfecting the siRNAs siTßR-I, -II, and -III, respectively. Phosphorylation of SMAD2 was examined as an indicator of downstream signalling via each TßR. Linkage between TGF-ß signalling and critical events in palatal fusion led to the use of, MMP-13 expression as an outcome measure for the function of the TGF-ß receptors. RESULTS: The siRNA treatment decreased the expression level of each receptor by more than 85%. When treated with either siTßR-I or -II, palatal shelves at E13+72 h were not fused, with complete clefting in the anterior and posterior regions. The middle palatal region following treatment with either siTßR-I or -II had fusion from one-half or one-third of the palatal region. Treatment with siTßR-III resulted in a persistent midline seam of medial edge epithelium (MEE) in the anterior region with islands of persistent MEE in the middle and posterior regions of the midline. Treatment with all three siTßRs altered the pattern of SMAD2 phosphorylation. Palatal shelf cultures treated with siTßR-I or -II, but not -III, showed altered MMP-13 expression levels. CONCLUSION: The ability to identify and recover MEE and palatal mesenchymal cells during palatal fusion will aid in the evaluation of the different mechanistic events regulated by each TßR during palatogenesis.

Nicotine affects bone resorption and suppresses the expression of cathepsin K, MMP-9 and vacuolar-type H(+)-ATPase d2 and actin organization in osteoclasts.

Tobacco smoking is an important risk factor for the development of several cancers, osteoporosis, and inflammatory diseases such as periodontitis. Nicotine is one of the major components of tobacco. In previous study, we showed that nicotine inhibits mineralized nodule formation by osteoblasts, and the culture medium from osteoblasts containing nicotine and lipopolysaccharide increases osteoclast differentiation. However, the direct effect of nicotine on the differentiation and function of osteoclasts is poorly understood. Thus, we examined the direct effects of nicotine on the expression of nicotine receptors and bone resorption-related enzymes, mineral resorption, actin organization, and bone resorption using RAW264.7 cells and bone marrow cells as osteoclast precursors. Cells were cultured with 10(-5), 10(-4), or 10(-3) M nicotine and/or 50 µM α-bungarotoxin (btx), an 7 nicotine receptor antagonist, in differentiation medium containing the soluble RANKL for up 7 days. 1-5, 7, 9, and 10 nicotine receptors were expressed on RAW264.7 cells. The expression of 7 nicotine receptor was increased by the addition of nicotine. Nicotine suppressed the number of tartrate-resistant acid phosphatase positive multinuclear osteoclasts with large nuclei(≥10 nuclei), and decreased the planar area of each cell. Nicotine decreased expression of cathepsin K, MMP-9, and V-ATPase d2. Btx inhibited nicotine effects. Nicotine increased CA II expression although decreased the expression of V-ATPase d2 and the distribution of F-actin. Nicotine suppressed the planar area of resorption pit by osteoclasts, but did not affect mineral resorption. These results suggest that nicotine increased the number of osteoclasts with small nuclei, but suppressed the number of osteoclasts with large nuclei. Moreover, nicotine reduced the planar area of resorption pit by suppressing the number of osteoclasts with large nuclei, V-ATPase d2, cathepsin K and MMP-9 expression and actin organization.