Co-Stimulation of AGEs and LPS Induces Inflammatory Mediators through PLCγ1/JNK/NF-κB Pathway in MC3T3-E1 Cells.

Advanced glycation end-products (AGEs) are increased under hyperglycemia in vivo and are associated with the onset of diabetes. According to previous studies, AGEs exacerbate inflammatory diseases. However, the mechanism by which AGEs aggravate osteoblast inflammation remains unknown. Therefore, the aim of this study was to determine the effects of AGEs on the production of inflammatory mediators in MC3T3-E1 cells and the underlying molecular mechanisms. Co-stimulation with AGEs and lipopolysaccharide (LPS) was found to increase the mRNA and protein levels of cyclooxygenase 2 (COX2), interleukin-1α (IL-1α), S100 calcium-binding protein A9 (S100A9), and the production of prostaglandin E2 (PGE2) compared to no stimulation (untreated control) or individual stimulation with LPS or AGEs. In contrast, the phospholipase C (PLC) inhibitor, U73122, inhibited these stimulatory effects. Co-stimulation with AGEs and LPS also increased the nuclear translocation of nuclear factor-kappa B (NF-κB) compared to no stimulation (untreated control) or individual stimulation with LPS or AGE. However, this increase was inhibited by U73122. Co-stimulation with AGEs and LPS-induced phosphorylated phospholipase Cγ1 (p-PLCγ1) and phosphorylated c-Jun N-terminal kinase (p-JNK) expression compared to no stimulation or individual stimulation with LPS or AGEs. U73122 inhibited the effects induced by co-stimulation. siPLCγ1 did not increase the expression of p-JNK and the translocation of NF-κB. Overall, co-stimulation with AGEs and LPS may promote inflammation mediators in MC3T3-E1 cells by activating the nuclear translocation of NF-κB via PLCγ1-JNK activation.

Human Gingival Fibroblast Attachment to Smooth Titanium Disks with Different Surface Roughnesses.

Peri-implantitis is a significant problem associated with dental implants. It has been hypothesized that creating a soft-tissue seal around the implant neck prevents peri-implantitis. This study aims to clarify the effects of the surface smoothness of titanium disks on soft tissues. Thus, titanium disks were prepared through electrolytic composite polishing (ECP), sisal buffing (SB), hairline polishing (HP), and laser cutting (LC). The surface roughness values of seven items was measured. For ECP, SB, HP, and LC samples, the Ra values were 0.075, 0.217, 0.671, and 1.024 µm and the Sa values were 0.005, 0.115, 0.500, and 0.676, respectively, indicating that the surface roughness was remarkably lower with ECP. Moreover, the Wsk values for ECP, SB, HP, and LC were 0.521, 1.018, -0.678, and -0.558, respectively. The smooth surfaces produced by ECP and SB were biased toward the concave surface, whereas those produced by HP and LC were biased toward the convex surface. The Rku values for ECP, SB, HP, and LC were 2.984, 11.774, 14.182, and 26.232, respectively. Only the ECP exhibited a moderate bias peak and produced an extremely smooth surface. The contact angles in the cases of ECP, SB, HP, and LC were 60.1°, 66.3°, 68.4°, and 79.3°, respectively, indicating the hydrophobicity of the titanium disks. Human oral fibroblasts were then incubated on each disk for 24 and 48 h to measure cell attachment, and no significant differences were observed. The differences in Ra and Sa did not affect cell attachment. Therefore, by applying ECP to the abutment or implant neck, the cell attachment required for soft-tissue formation while preventing bacterial adhesion can be achieved.

Effects of Electric-Toothbrush Vibrations on the Expression of Collagen and Non-Collagen Proteins through the Focal Adhesion Kinase Signaling Pathway in Gingival Fibroblasts.

Electric-toothbrush vibrations, which remove plaque, are transmitted to the gingival connective tissue via epithelial cells. Physical energy affects cell function; however, the effects of electric-toothbrush vibrations on gingival extracellular matrix (ECM) protein expression remain unknown. We aimed to examine the effects of these vibrations on the expression of ECM proteins-type I collagen (col I), type III collagen (col III), elastin, and fibronectin (FN)-using human gingival fibroblasts (HGnFs). HGnFs were seeded for 5 days in a six-well plate with a hydrophilic surface, exposed to electric-toothbrush vibrations, and cultured for 7 days. Subsequently, the mRNA and protein levels of col I, col III, elastin, and FN were examined. To investigate the role of focal adhesion kinase (FAK) signaling on ECM protein expression in vibration-stimulated cells, the cells were treated with siRNA against protein tyrosine kinase (PTK). Electric-toothbrush vibrations increased col I, col III, elastin, and FN expression; promoted collagen and non-collagen protein production; and enhanced FAK phosphorylation in HGnFs. Moreover, PTK2 siRNA completely blocked the effects of these vibrations on the expression of col I, col III and elastin mRNA. The results suggest that electric-toothbrush vibrations increase collagen, elastin, and FN production through the FAK-signaling pathway in fibroblasts.

Epstein-Barr Virus Promotes the Production of Inflammatory Cytokines in Gingival Fibroblasts and RANKL-Induced Osteoclast Differentiation in RAW264.7 Cells.

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.

Reasons for Tooth Extractions in Japan: The Second Nationwide Survey.

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.

A Validation Study of the National Database of Health Insurance Claims and Specific Health Checkups of Japan Regarding the Annual Number of Tooth Extractions Performed Across the Entire Japanese Population.

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 <0.05 teeth/year. The present results suggest that the NDB is a reliable resource for data on tooth extractions performed across the entire Japanese population.

Effect of Azithromycin on Mineralized Nodule Formation in MC3T3-E1 Cells.

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.

Self-Reported Diabetes Mellitus and Tooth Extraction Due to Periodontal Disease and Dental Caries in the Japanese Population.

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 > 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.

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.

Morphological characteristics of interalveolar septum and mandible in BMAL1 gene knockout mice.

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.

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.

Identification of Smad-dependent and -independent signaling with transforming growth factor-ß type 1/2 receptor inhibition in palatogenesis.

TGF-ß signaling is one of important function during palatal fusion. Three types of TGF-ß receptor (TßR1, TßR2, and TßR3) 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 TßR1/2 functions is not fully understood. The objective of this study was to investigate palatal fusion via TGF-ß signaling when TßR1 and TßR2, but not TßR3, 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 TßR1/2 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 TßR1 and TßR2 expression by approximately 90%, but did not affect TßR3 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 TßR1/2 inhibitor altered the patterns of the Smad-dependent and -independent signaling pathways during palatal fusion.

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.

Changes in Oral Health and Treatment Needs for Elderly Residents of Long-Term Care Facilities Over 10 Years.

PURPOSE: 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. METHODS: 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. RESULTS: 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%. CONCLUSIONS: 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.

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.

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.