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Sample records for affecting osteoblastic activity

  1. A proteome study of secreted prostatic factors affecting osteoblastic activity: identification and characterisation of cyclophilin A

    DEFF Research Database (Denmark)

    Andersen, H; Jensen, Ole Nørregaard; Eriksen, E F

    2003-01-01

    )] of control. IGF-I did not significantly affect these decreases. Cyclophilin A alone or in combination with IGF-I did not have any effect on differentiation (assessed by measuring the activity of alkaline phosphatase (ALP)). In conclusion, these results suggest cyclophilin A is not involved in the......Prostate cancer cells metastasise to bone causing a predominantly osteosclerotic response. It has previously been shown that PC3 cells secrete factors which stimulate the mitogenic activity of human bone marrow stromal (hBMS) cells. Some of these mitogens have been found to be proteins with a...... molecular weight between 20 and 30 kDa. Even though a number of investigations have been performed to identify the osteoblastic mitogenic factor or factors produced by prostate cancer cells, it is still unknown what causes the mitogenic activation of osteoblasts. Therefore, the aim of this study was to...

  2. Urokinase plasminogen activator receptor affects bone homeostasis by regulating osteoblast and osteoclast function

    DEFF Research Database (Denmark)

    Furlan, Federico; Galbiati, Clara; Jørgensen, Niklas R;

    2007-01-01

    reorganization in mature osteoclasts. INTRODUCTION: Urokinase receptor (uPAR) is actively involved in the regulation of important cell functions, such as proliferation, adhesion, and migration. It was previously shown that the major players in bone remodeling, osteoblasts and osteoclasts, express uPAR and...... to mechanical tests. UPAR KO calvaria osteoblasts were characterized by proliferation assays, RT-PCR for important proteins secreted during differentiation, and immunoblot for activator protein 1 (AP-1) family members. In vitro osteoclast formation was tested with uPAR KO bone marrow monocytes in the...... osteoblasts showed a proliferative advantage with no difference in apoptosis, higher matrix mineralization, and earlier appearance of alkaline phosphatase (ALP). Surface RANKL expression at different stages of differentiation was not altered. AP-1 components, such as JunB and Fra-1, were upregulated in u...

  3. Urokinase plasminogen activator receptor affects bone homeostasis by regulating osteoblast and osteoclast function

    DEFF Research Database (Denmark)

    Furlan, Federico; Galbiati, Clara; Jørgensen, Niklas R;

    2007-01-01

    The uPAR and its ligand uPA are expressed by both osteoblasts and osteoclasts. Their function in bone remodeling is unknown. We report that uPAR-lacking mice display increased BMD, increased osteogenic potential of osteoblasts, decreased osteoclasts formation, and altered cytoskeletal reorganizat...

  4. Activation of Nfatc2 in osteoblasts causes osteopenia.

    Science.gov (United States)

    Zanotti, Stefano; Canalis, Ernesto

    2015-07-01

    Nuclear factor of activated T-cells (Nfat) c1 to c4 are transcription factors that play an undisputable role in osteoclastogenesis. However, Nfat function in osteoblastic cells is controversial. Constitutive activation of Nfatc1 and c2 in osteoblasts suppresses cell function, although the study of Nfat in vivo has yielded conflicting results. To establish the consequences of Nfatc2 activation in osteoblasts, we generated transgenic mice where a 3.6 kb fragment of the collagen type I α1 promoter directs expression of a constitutively active Nfatc2 mutant (Col3.6-Nfatc2). The skeletal phenotype of Col3.6-Nfatc2 mice of both sexes and of sex-matched littermate controls was investigated by microcomputed tomography and histomorphometry. Col3.6- Nfatc2 mice were born at the expected Mendelian ratio and appeared normal. Nfatc2 expression was confirmed in parietal bones from 1 and 3 month old transgenic mice. One month old Col3.6-Nfatc2 female mice exhibited cancellous bone compartment osteopenia secondary to a 30% reduction in bone formation. In contrast, cancellous femoral bone volume and bone formation were not altered in male transgenics, whereas osteoblast number was higher, suggesting incomplete osteoblast maturation. Indices of bone resorption were not affected in either sex. At 3 months of age, the skeletal phenotype evolved; and Col3.6-Nfatc2 male mice exhibited vertebral osteopenia, whereas femoral cancellous bone was not affected in either sex. Nfatc2 activation in osteoblasts had no impact on cortical bone structure. Nfatc2 activation inhibited alkaline phosphatase activity and mineralized nodule formation in bone marrow stromal cell cultures. In conclusion, Nfatc2 activation in osteoblasts inhibits bone formation and causes cancellous bone osteopenia. PMID:25573264

  5. Osteoblast differentiation and migration are regulated by Dynamin GTPase activity

    OpenAIRE

    Pierre P. Eleniste; Huang, Su; Wayakanon, Kornchanok; Largura, Heather W.; Bruzzaniti, Angela

    2013-01-01

    Bone formation is controlled by osteoblasts but the signaling proteins that control osteoblast differentiation and function are still unclear. We examined if the dynamin GTPase, which is associated with actin remodeling and migration in other cells, plays a role in osteoblast differentiation and migration. Dynamin mRNA was expressed in primary osteoblasts throughout differentiation (0–21 days). However, alkaline phosphatase (ALP) activity, a marker of osteoblast differentiation, was decreased...

  6. A proteome study of secreted prostatic factors affecting osteoblastic activity: galectin-1 is involved in differentiation of human bone marrow stromal cells

    DEFF Research Database (Denmark)

    Andersen, H; Jensen, Ole N; Moiseeva, Elena P;

    2003-01-01

    , which induced hBMS cell proliferation by 3-fold. This effect was abolished by IGF-I. PC3 CM and galectin-1 in concentrations of 10 and 1000 ng/ml increased the alkaline phosphatase (ALP) activity of hBMS cells up to 175 +/- 27%, 137 +/- 8%, and 131 +/- 11%, respectively, compared with ALP activity of......Prostate cancer cells metastasize to bone causing a predominantly osteosclerotic response. It has been shown that cells from the human prostate cancer cell line PC3 secrete factors that influence the behavior of osteoblast-like cells. Some of these factors with mitogenic activity have been found to...

  7. Inhibition of osteoblast activity by zoledronic acid

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    Fernanda Gonçalves Basso

    2013-10-01

    Full Text Available INTRODUCTION: Patients treated with nitrogen-containing bisphosphonates, such as zoledronic acid (ZA, have frequently shown oral bone exposure areas, termed osteonecrosis. In addition, these patients may also present low repair and regeneration potential, mainly after tooth extractions. These side-effects caused by bisphosphonates may be due to their inhibitory effects on oral mucosa and local bone cells. OBJECTIVE: To evaluate the effects of ZA on the mineralization capacity of cultured osteoblasts. MATERIALS AND METHODS: Human immortalized osteoblasts (SaOs-2 were grown in plain culture medium (Dulbecco's Modified Eagle Medium [DMEM] + 10% fetal bovine serum [FBS] in wells of 24-well plates. After 48-hour incubation, the plain DMEM was replaced by a solution with ZA at 5 µM which was maintained in contact with cells for seven, 14 or 21 days. After these periods, cells were evaluated regarding alkaline phosphatase (ALP activity and mineral nodule formation (alizarin red. Data were statistically analyzed by Mann-Whitney test, at 5% of significance level. RESULTS: ZA caused significant reduction on ALP activity and mineral nodules formation by cultured osteoblasts in all evaluated periods (p < 0.05. CONCLUSION: These data indicate that ZA causes inhibition on the osteogenic phenotype of cultured human osteoblasts, which, in turn, may reduce bone repair in patients subjected to ZA therapy.

  8. Osteoblast differentiation and migration are regulated by dynamin GTPase activity.

    Science.gov (United States)

    Eleniste, Pierre P; Huang, Su; Wayakanon, Kornchanok; Largura, Heather W; Bruzzaniti, Angela

    2014-01-01

    Bone formation is controlled by osteoblasts, but the signaling proteins that control osteoblast differentiation and function are still unclear. We examined if the dynamin GTPase, which is associated with actin remodeling and migration in other cells, plays a role in osteoblast differentiation and migration. Dynamin mRNA was expressed in primary osteoblasts throughout differentiation (0-21 days). However, alkaline phosphatase (ALP) activity, a marker of osteoblast differentiation, was decreased in osteoblasts over-expressing dynamin. Conversely, ALP activity was increased following shRNA-mediated knockdown of dynamin and in osteoblasts treated with the dynamin inhibitor, dynasore. Dynasore also reduced c-fos and osterix expression, markers of early osteoblasts, suggesting a role for dynamin in pre-osteoblast to osteoblast differentiation. Since dynamin GTPase activity is regulated by tyrosine phosphorylation, we examined the mechanism of dynamin dephosphorylation in osteoblasts. Dynamin formed a protein complex with the tyrosine phosphatase PTP-PEST and inhibition of phosphatase activity increased the level of phosphorylated dynamin. Further, PTP-PEST blocked the Src-mediated increase in the phosphorylation and GTPase activity of wild-type dynamin but not the phosphorylation mutant dynY231F/Y597F. Although ALP activity was increased in osteoblasts expressing GTPase-defective dynK44A, and to a lesser extent dynY231F/Y597F, osteoblast migration was significantly inhibited by dynK44A and dynY231F/Y597F. These studies demonstrate a novel role for dynamin GTPase activity and phosphorylation in osteoblast differentiation and migration, which may be important for bone formation. PMID:24387844

  9. Intermittent exposure to ethanol vapor affects osteoblast behaviour more severely than estrogen deficiency does

    International Nuclear Information System (INIS)

    With rising rates of alcohol consumption acute and chronic damage from alcohol is expected to increase all over the world. Habitual excessive alcohol consumption is associated with pathological effects on bone. The aim of the present in vitro study was to investigate comparatively the proliferation and synthetic activity of osteoblasts (OB) isolated from the trabecular bone of rats previously exposed to 7-week intermittent exposure to ethanol vapor, sham-aged rats and long-term estrogen deficient rats. Cell proliferation (WST1) and synthesis of alkaline phosphatase (ALP), osteocalcin (OC), collagen I (CICP), transforming growth factor beta1 (TGF-β1), interleukin-6 (IL-6), tumor necrosis factor alfa (TNFα) were measured at 3, 7 and 14 days of culture. Osteoblast proliferation rate and TGF-β1, IL-6 and TNFα syntheses were significantly affected by alcohol exposure. Estrogen deficiency and alcohol consumption share many common pathophysiological mechanisms of damage to bone, but alcohol affects OB proliferation and TNFα synthesis significantly more than menopause does. Therefore, these in vitro data suggest that alcohol has even more deleterious effects on bone than estrogen deficiency does

  10. Carbon nanohorns allow acceleration of osteoblast differentiation via macrophage activation

    Science.gov (United States)

    Hirata, Eri; Miyako, Eijiro; Hanagata, Nobutaka; Ushijima, Natsumi; Sakaguchi, Norihito; Russier, Julie; Yudasaka, Masako; Iijima, Sumio; Bianco, Alberto; Yokoyama, Atsuro

    2016-07-01

    Carbon nanohorns (CNHs), formed by a rolled graphene structure and terminating in a cone, are promising nanomaterials for the development of a variety of biological applications. Here we demonstrate that alkaline phosphatase activity is dramatically increased by coculture of human monocyte derived macrophages (hMDMs) and human mesenchymal stem cells (hMSCs) in the presence of CNHs. CNHs were mainly localized in the lysosome of macrophages more than in hMSCs during coculturing. At the same time, the amount of Oncostatin M (OSM) in the supernatant was also increased during incubation with CNHs. Oncostatin M (OSM) from activated macrophage has been reported to induce osteoblast differentiation and matrix mineralization through STAT3. These results suggest that the macrophages engulfed CNHs and accelerated the differentiation of mesenchymal stem cells into the osteoblast via OSM release. We expect that the proof-of-concept on the osteoblast differentiation capacity by CNHs will allow future studies focused on CNHs as ideal therapeutic materials for bone regeneration.Carbon nanohorns (CNHs), formed by a rolled graphene structure and terminating in a cone, are promising nanomaterials for the development of a variety of biological applications. Here we demonstrate that alkaline phosphatase activity is dramatically increased by coculture of human monocyte derived macrophages (hMDMs) and human mesenchymal stem cells (hMSCs) in the presence of CNHs. CNHs were mainly localized in the lysosome of macrophages more than in hMSCs during coculturing. At the same time, the amount of Oncostatin M (OSM) in the supernatant was also increased during incubation with CNHs. Oncostatin M (OSM) from activated macrophage has been reported to induce osteoblast differentiation and matrix mineralization through STAT3. These results suggest that the macrophages engulfed CNHs and accelerated the differentiation of mesenchymal stem cells into the osteoblast via OSM release. We expect that the

  11. Osteoclasts but not osteoblasts are affected by a calcified surface treated with zoledronic acid in vitro

    International Nuclear Information System (INIS)

    Bisphosphonates are potent inhibitors of osteoclast-mediated bone resorption. Recent interest has centered on the effects of bisphosphonates on osteoblasts. Chronic dosing of osteoblasts with solubilized bisphosphonates has been reported to enhance osteogenesis and mineralization in vitro. However, this methodology poorly reflects the in vivo situation, where free bisphosphonate becomes rapidly bound to mineralized bone surfaces. To establish a more clinically relevant cell culture model, we cultured bone cells on calcium phosphate coated quartz discs pre-treated with the potent nitrogen-containing bisphosphonate, zoledronic acid (ZA). Binding studies utilizing [14C]-labeled ZA confirmed that the bisphosphonate bound in a concentration-dependent manner over the 1-50 μM dose range. When grown on ZA-treated discs, the viability of bone-marrow derived osteoclasts was greatly reduced, while the viability and mineralization of the osteoblastic MC3T3-E1 cell line were largely unaffected. This suggests that only bone resorbing cells are affected by bound bisphosphonate. However, this system does not account for transient exposure to unbound bisphosphonate in the hours following a clinical dosing. To model this event, we transiently treated osteoblasts with ZA in the absence of a calcified surface. Osteoblasts proved highly resistant to all transitory treatment regimes, even when utilizing ZA concentrations that prevented mineralization and/or induced cell death when dosed chronically. This study represents a pharmacologically more relevant approach to modeling bisphosphonate treatment on cultured bone cells and implies that bisphosphonate therapies may not directly affect osteoblasts at bone surfaces

  12. Mechanical stress activates Smad pathway through PKCδ to enhance interleukin-11 gene transcription in osteoblasts.

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    Shinsuke Kido

    Full Text Available BACKGROUND: Mechanical stress rapidly induces ΔFosB expression in osteoblasts, which binds to interleukin (IL-11 gene promoter to enhance IL-11 expression, and IL-11 enhances osteoblast differentiation. Because bone morphogenetic proteins (BMPs also stimulate IL-11 expression in osteoblasts, there is a possibility that BMP-Smad signaling is involved in the enhancement of osteoblast differentiation by mechanical stress. The present study was undertaken to clarify whether mechanical stress affects BMP-Smad signaling, and if so, to elucidate the role of Smad signaling in mechanical stress-induced enhancement of IL-11 gene transcription. METHODOLOGY/PRINCIPAL FINDINGS: Mechanical loading by fluid shear stress (FSS induced phosphorylation of BMP-specific receptor-regulated Smads (BR-Smads, Smad1/5, in murine primary osteoblasts (mPOBs. FSS rapidly phosphorylated Y311 of protein kinase C (PKCδ, and phosphorylated PKCδ interacted with BR-Smads to phosphorylate BR-Smads. Transfection of PKCδ siRNA or Y311F mutant PKCδ abrogated BR-Smads phosphorylation and suppressed IL-11 gene transcription enhanced by FSS. Activated BR-Smads bound to the Smad-binding element (SBE of IL-11 gene promoter and formed complex with ΔFosB/JunD heterodimer via binding to the C-terminal region of JunD. Site-directed mutagenesis in the SBE and the AP-1 site revealed that both SBE and AP-1 sites were required for full activation of IL-11 gene promoter by FSS. CONCLUSIONS/SIGNIFICANCE: These results demonstrate that PKCδ-BR-Smads pathway plays an important role in the intracellular signaling in response to mechanical stress, and that a cross-talk between PKCδ-BR-Smads and ΔFosB/JunD pathways synergistically stimulates IL-11 gene transcription in response to mechanical stress.

  13. Stem cell factor (SCF) protects osteoblasts from oxidative stress through activating c-Kit-Akt signaling

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    Yang, Lei [Department of Orthopedics, Changzhou Wujin People’s Hospital-South Division, Affiliated Hospital of Jiangsu University, Changzhou (China); Wu, Zhong [Department of Orthopedics, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai (China); Yin, Gang; Liu, Haifeng; Guan, Xiaojun; Zhao, Xiaoqiang [Department of Orthopedics, Changzhou Wujin People’s Hospital-South Division, Affiliated Hospital of Jiangsu University, Changzhou (China); Wang, Jianguang, E-mail: jianguangwang@163.com [Department of Orthopedics, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai (China); Zhu, Jianguo, E-mail: gehujianguo68@163.com [Department of Orthopedics, Changzhou Wujin People’s Hospital-South Division, Affiliated Hospital of Jiangsu University, Changzhou (China)

    2014-12-12

    Highlights: • SCF receptor c-Kit is functionally expressed in primary and transformed osteoblasts. • SCF protects primary and transformed osteoblasts from H{sub 2}O{sub 2}. • SCF activation of c-Kit in osteoblasts, required for its cyto-protective effects. • c-Kit mediates SCF-induced Akt activation in cultured osteoblasts. • Akt activation is required for SCF-regulated cyto-protective effects in osteoblasts. - Abstract: Osteoblasts regulate bone formation and remodeling, and are main target cells of oxidative stress in the progression of osteonecrosis. The stem cell factor (SCF)-c-Kit pathway plays important roles in the proliferation, differentiation and survival in a range of cell types, but little is known about its functions in osteoblasts. In this study, we found that c-Kit is functionally expressed in both osteoblastic-like MC3T3-E1 cells and primary murine osteoblasts. Its ligand SCF exerted significant cyto-protective effects against hydrogen peroxide (H{sub 2}O{sub 2}). SCF activated its receptor c-Kit in osteoblasts, which was required for its cyto-protective effects against H{sub 2}O{sub 2}. Pharmacological inhibition (by Imatinib and Dasatinib) or shRNA-mediated knockdown of c-Kit thus inhibited SCF-mediated osteoblast protection. Further investigations showed that protection by SCF against H{sub 2}O{sub 2} was mediated via activation of c-Kit-dependent Akt pathway. Inhibition of Akt activation, through pharmacological or genetic means, suppressed SCF-mediated anti-H{sub 2}O{sub 2} activity in osteoblasts. In summary, we have identified a new SCF-c-Kit-Akt physiologic pathway that protects osteoblasts from H{sub 2}O{sub 2}-induced damages, and might minimize the risk of osteonecrosis caused by oxidative stress.

  14. P53 and Beta-Catenin Activity during Estrogen treatment of Osteoblasts

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    Kolman Kevin

    2005-07-01

    Full Text Available Abstract Background This study was undertaken to examine the relationship between the tumor suppressor gene p53 and the nuclear signaling protein beta-catenin during bone differentiation. Cross talk between p53 and beta-catenin pathways has been demonstrated and is important during tumorigenesis and DNA damage, where deregulation of beta catenin activates p53. In this study, we used estrogen treatment of osteoblasts as a paradigm to study the relationship between the two proteins during osteoblast differentiation. Results We exposed osteoblast-like ROS17/2.8 cells to 17-beta estradiol (E2, in a short term assay, and studied the cellular distribution and expression of beta-catenin. We found beta-catenin to be up regulated several fold following E2 treatment. Levels of p53 and its functional activity mirrored the quantitative changes seen in beta-catenin. Alkaline phosphatase, an early marker of osteoblast differentiation, was increased in a manner similar to beta-catenin and p53. In order to determine if there was a direct relationship between alkaline phosphatase expression and beta-catenin, we used two different approaches. In the first approach, treatment with LiCl, which is known to activate beta-catenin, caused a several fold increase in alkaline phosphatase activity. In the second approach, transient transfection of wild type beta-catenin into osteoblasts increased alkaline phosphatase activity two fold over basal levels, showing that beta catenin expression can directly affect alkaline phosphatase expression. However increase in beta catenin activity was not associated with an increase in its signaling activity through TCF/LEF mediated transcription. Immunofluorescence analyses of p53 and beta-catenin localization showed that E2 first caused an increase in cytosolic beta-catenin followed by the accumulation of beta-catenin in the nucleus. Nuclear p53 localization was detected in several cells. Expression of p53 was accompanied by

  15. Factors affecting growth, differentiation and apoptosis of osteoblastic and osteosarcoma cells

    OpenAIRE

    Li, Yan

    2008-01-01

    Osteoblasts play a fundamental role in determining bone structure and function. These cells originate from mesenchymal stem cells (MSCs) and through proliferation and differentiation develop into preosteoblasts and then into mature cells. Most of these cells undergo apoptosis before reaching their terminal differentiated stages of either osteocytes or bone lining cells. These processes, i.e. proliferation, differentiation, and apoptosis, are affected by systemic hormones and...

  16. Comparison of black, green and rooibos tea on osteoblast activity.

    Science.gov (United States)

    Nash, L A; Ward, W E

    2016-02-17

    Globally, tea is the second most consumed beverage after water. Habitual tea intake has been associated with higher bone mineral density, particularly in postmenopausal women. This association may be due to its polyphenols and resulting protective antioxidant effects. While in vivo studies have shown improved bone outcomes with a consumption of individual purified tea polyphenols, it is unclear if a particular tea - due to its different profiles of polyphenols - is more beneficial than others. Therefore, we compared three different types of commercially available teas on osteoblasts: green, black and rooibos tea. Tea was normalized to 1 or 10 μg per mL gallic acid equivalents to assess differences in outcomes based on tea profiles rather than the quantity of polyphenol naturally present. The lower level of polyphenols (1 μg per mL gallic acid equivalents) - regardless of tea type and thus polyphenol profile - resulted in greater mineral content as well as cellular and alkaline phosphatase activity in Saos2 cells. Moreover, this was associated with higher markers of differentiation (osteopontin, sclerostin) and reduced cellular toxicity and pro-inflammatory markers (IL6, TNFα). Green, black and rooibos tea improved osteoblast activity at the low level and support epidemiological evidence suggesting tea consumption may benefit bone heath. PMID:26885714

  17. The role of osteoblasts in regulating hematopoietic stem cell activity and tumor metastasis

    Directory of Open Access Journals (Sweden)

    Neiva K.

    2005-01-01

    Full Text Available Bone marrow stromal cells are critical regulators of hematopoiesis. Osteoblasts are part of the stromal cell support system in bone marrow and may be derived from a common precursor. Several studies suggested that osteoblasts regulate hematopoiesis, yet the entire mechanism is not understood. It is clear, however, that both hematopoietic precursors and osteoblasts interact for the production of osteoclasts and the activation of resorption. We observed that hematopoietic stem cells (HSCs regulate osteoblastic secretion of various growth factors, and that osteoblasts express some soluble factors exclusively in the presence of HSCs. Osteoblasts and hematopoietic cells are closely associated with each other in the bone marrow, suggesting a reciprocal relationship between them to develop the HSC niche. One critical component regulating the niche is stromal-derived factor-1 (SDF-1 and its receptor CXCR4 which regulates stem cell homing and, as we have recently demonstrated, plays a crucial role in facilitating those tumors which metastasize to bone. Osteoblasts produce abundant amounts of SDF-1 and therefore osteoblasts play an important role in metastasis. These findings are discussed in the context of the role of osteoblasts in marrow function in health and disease.

  18. Parathyroid Hormone Increases Activating Transcription Factor 4 Expression and Activity in Osteoblasts: Requirement for Osteocalcin Gene Expression

    OpenAIRE

    Yu, Shibing; Franceschi, Renny T; Luo, Min; Zhang, Xiaoyan; Jiang, Di; Lai, Yumei; Jiang, Yu; Zhang, Jian; Xiao, Guozhi

    2008-01-01

    PTH is an important peptide hormone regulator of calcium homeostasis and osteoblast function. However, its mechanism of action in osteoblasts is poorly understood. Our previous study demonstrated that PTH activates mouse osteocalcin (Ocn) gene 2 promoter through the osteoblast-specific element 1 site, a recently identified activating transcription factor-4 (ATF4) -binding element. In the present study, we examined effects of PTH on ATF4 expression and activity as well as the requirement for A...

  19. Strontium ranelate affects signaling from mechanically-stimulated osteocytes towards osteoclasts and osteoblasts.

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    Bakker, Astrid D; Zandieh-Doulabi, Behrouz; Klein-Nulend, Jenneke

    2013-03-01

    Strontium Ranelate (SrRan) is used to decrease the risk of bone fractures. Any factor that alters the release of paracrine signals by osteocytes in response to mechanical stimuli potentially affects bone mass and structure, and thus fracture resistance. We hypothesized that SrRan affects paracrine signaling from mechanically-stimulated osteocytes towards osteoclast-precursors and osteoblasts. MLO-Y4 osteocytes were cultured for 24h with SrRan (0.1-3mM) and either or not mechanically stimulated by pulsating fluid flow (PFF; 0.7 ± 0.3 Pa, 5 Hz) for 60 min. Nitric oxide (NO) and prostaglandin E(2) (PGE(2)) release, and expression of mechanoresponsive genes were quantified. Conditioned medium (CM) from osteocytes was added to mouse bone marrow cells for 7 days to assess osteoclastogenesis, or MC3T3-E1 osteoblasts for 4-16 days to measure osteogenic gene expression. SrRan (3mM) enhanced NO and PGE(2) release to the same extent in static osteocytes (NO: 1.6-fold; PGE(2): 2.8-fold) and PFF-stimulated osteocytes (NO: 1.3-fold; PGE(2): 2.6-fold). CM from PFF-treated osteocytes without SrRan enhanced Ki67 expression but reduced Runx2 and Ocn expression in osteoblasts. This effect on gene expression was not observed with CM obtained from osteocytes treated with the combination of PFF and 3mM SrRan. CM from PFF-treated osteocytes inhibited osteoclastogenesis by 1.9-fold. The combination of PFF and 3mM SrRan reduced osteocyte-stimulated osteoclastogenesis even more strongly (4.3-fold). In conclusion, SrRan affects paracrine signaling between mechanically-stimulated MLO-Y4 osteocytes and both osteoblasts and osteoclast precursors. The positive effects of SrRan on bone fracture resistance may thus be partly explained by altered paracrine signaling by osteocytes. PMID:23234812

  20. Nanofiber diameter-dependent MAPK activity in osteoblasts.

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    Jaiswal, Devina; Brown, Justin L

    2012-11-01

    The major challenge for bone tissue engineering lies in the fabrication of scaffolds that can mimic the extracellular matrix and promote osteogenesis. Electrospun fibers are being widely researched for this application due to high porosity, interconnectivity, and mechanical strength of the fibrous scaffolds. Electrospun poly methyl methacrylate (PMMA, 2.416 ± 0.100 μm) fibers were fabricated and etched using a 60% propylene glycol methyl ether acetate (PGMEA)/limonene (vol/vol) solution to obtain fiber diameters ranging from 2.5 to 0.5 μm in a time-dependent manner. The morphology of the fibrous scaffolds was evaluated using scanning electron microscopy and cellular compatibility with etchant-treated scaffold was assessed using immunoflurescence. Mitogen-activated protein kinases (MAPK) activation in response to different fiber diameter was evaluated with western blot as well as quantitative in-cell western. We report that electrospun micro-fibers can be etched to 0.552 ± 0.047 μm diameter without producing beads. Osteoblasts adhered to the fibers and a change in fiber diameter played a major role in modulating the activation of extracellular signal-regulated kinase (ERK) and p38 kinases with 0.882 ± 0.091 μm diameter fibers producing an inverse effect on ERK and p38 phosphorylation. These results indicate that nanofibers produced by wet etching can be effectively utilized to produce diameters that can differentially modulate MAPK activation patterns. PMID:22700490

  1. Effect of retinoic acid and vitamin D3 on osteoblast differentiation and activity in aging.

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    Bosetti, Michela; Sabbatini, Maurizio; Calarco, Anna; Borrone, Alessia; Peluso, Gianfranco; Cannas, Mario

    2016-01-01

    Several studies have evidenced that in aging, osteoblast functional activity is impaired: osteoblast proliferation is slower and matrix deposition is less efficient. Because peroxisome-proliferator-activated receptor γ2 (PPARγ2) and fatty acids are important inhibitory signals in osteoblast development, we have investigated in human primary osteoblasts obtained from patients of different ages, the influence of retinoic acid and calcitriol on enzymes involved in differentiative (PPARγ2, β-catenin, and insulin-like growth factor 1) and metabolic (carnitine palmitoyltransferase 1) intracellular pathways, and on transglutaminase 2, as enzyme fundamental for stabilizing the newly deposited extracellular matrix in bone. Retinoic acid and calcitriol influenced, respectively, proliferation and differentiation of osteoblasts, and an increase in PPARγ2 expression was observed following retinoic acid administration, whereas a decrease was observed following calcitriol administration. Aging widely influenced all parameters analyzed (the proliferation, differentiation, and new matrix deposition are significantly reduced in aged osteoblasts), with the exception of PPARγ2, which we found to be constitutively overexpressed and not modulated by retinoic acid or calcitriol administration. Our findings show the impaired ability of aged osteoblasts to perform adequate functional response and draw attention to the therapeutic approaches for bone healing in elderly patients. PMID:25691285

  2. Factors circulating in the blood of type 2 diabetes mellitus patients affect osteoblast maturation – Description of a novel in vitro model

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    Ehnert, Sabrina, E-mail: sabrina.ehnert@gmail.com [BG Trauma Center, Eberhard Karls Universität Tübingen, Schnarrenbergstr. 95, D-72076 Tübingen (Germany); Freude, Thomas, E-mail: tfreude@bgu-tuebingen.de [BG Trauma Center, Eberhard Karls Universität Tübingen, Schnarrenbergstr. 95, D-72076 Tübingen (Germany); Ihle, Christoph, E-mail: cihle@bgu-tuebingen.de [BG Trauma Center, Eberhard Karls Universität Tübingen, Schnarrenbergstr. 95, D-72076 Tübingen (Germany); Mayer, Larissa, E-mail: lara.nk@gmail.com [BG Trauma Center, Eberhard Karls Universität Tübingen, Schnarrenbergstr. 95, D-72076 Tübingen (Germany); Braun, Bianca, E-mail: bianca.braun@med.uni-tuebingen.de [BG Trauma Center, Eberhard Karls Universität Tübingen, Schnarrenbergstr. 95, D-72076 Tübingen (Germany); Graeser, Jessica, E-mail: jessica.graeser@student.reutlingen-university.de [BG Trauma Center, Eberhard Karls Universität Tübingen, Schnarrenbergstr. 95, D-72076 Tübingen (Germany); Flesch, Ingo, E-mail: iflesch@bgu-tuebingen.de [BG Trauma Center, Eberhard Karls Universität Tübingen, Schnarrenbergstr. 95, D-72076 Tübingen (Germany); and others

    2015-03-15

    Type 2 diabetes mellitus (T2DM) is one of the most frequent metabolic disorders in industrialized countries. Among other complications, T2DM patients have an increased fracture risk and delayed fracture healing. We have demonstrated that supraphysiological glucose and insulin levels inhibit primary human osteoblasts' maturation. We aimed at developing a more physiologically relevant in vitro model to analyze T2DM-mediated osteoblast changes. Therefore, SCP-1-immortalized pre-osteoblasts were differentiated with T2DM or control (non-obese and obese) sera. Between both control groups, no significant changes were observed. Proliferation was significantly increased (1.69-fold), while AP activity and matrix mineralization was significantly reduced in the T2DM group. Expression levels of osteogenic marker genes and transcription factors were altered, e.g. down-regulation of RUNX2 and SP-7 or up-regulation of STAT1, in the T2DM group. Active TGF-β levels were significantly increased (1.46-fold) in T2DM patients' sera. SCP-1 cells treated with these sera showed significantly increased TGF-β signaling (2.47-fold). Signaling inhibition effectively restored osteoblast maturation in the T2DM group. Summarizing our data, SCP-1 cells differentiated in the presence of T2DM patients' serum exhibit reduced osteoblast function. Thus, this model has a high physiological impact, as it can identify circulating factors in T2DM patients' blood that may affect bone function, e.g. TGF-β. - Highlights: • We present here a physiologically relevant in vitro model for diabetic osteopathy. • Blood of T2DM patients contains factors that affect osteoblasts' function. • The model developed here can be used to identify these factors, e.g. TGF-β. • Blocking TGF-β signaling partly rescues the osteoblasts' function in the T2DM group. • The model is useful to demonstrate the role of single factors in diabetic osteopathy.

  3. Factors circulating in the blood of type 2 diabetes mellitus patients affect osteoblast maturation – Description of a novel in vitro model

    International Nuclear Information System (INIS)

    Type 2 diabetes mellitus (T2DM) is one of the most frequent metabolic disorders in industrialized countries. Among other complications, T2DM patients have an increased fracture risk and delayed fracture healing. We have demonstrated that supraphysiological glucose and insulin levels inhibit primary human osteoblasts' maturation. We aimed at developing a more physiologically relevant in vitro model to analyze T2DM-mediated osteoblast changes. Therefore, SCP-1-immortalized pre-osteoblasts were differentiated with T2DM or control (non-obese and obese) sera. Between both control groups, no significant changes were observed. Proliferation was significantly increased (1.69-fold), while AP activity and matrix mineralization was significantly reduced in the T2DM group. Expression levels of osteogenic marker genes and transcription factors were altered, e.g. down-regulation of RUNX2 and SP-7 or up-regulation of STAT1, in the T2DM group. Active TGF-β levels were significantly increased (1.46-fold) in T2DM patients' sera. SCP-1 cells treated with these sera showed significantly increased TGF-β signaling (2.47-fold). Signaling inhibition effectively restored osteoblast maturation in the T2DM group. Summarizing our data, SCP-1 cells differentiated in the presence of T2DM patients' serum exhibit reduced osteoblast function. Thus, this model has a high physiological impact, as it can identify circulating factors in T2DM patients' blood that may affect bone function, e.g. TGF-β. - Highlights: • We present here a physiologically relevant in vitro model for diabetic osteopathy. • Blood of T2DM patients contains factors that affect osteoblasts' function. • The model developed here can be used to identify these factors, e.g. TGF-β. • Blocking TGF-β signaling partly rescues the osteoblasts' function in the T2DM group. • The model is useful to demonstrate the role of single factors in diabetic osteopathy

  4. The shunt from the cyclooxygenase to lipoxygenase pathway in human osteoarthritic subchondral osteoblasts is linked with a variable expression of the 5-lipoxygenase-activating protein.

    Science.gov (United States)

    Maxis, Kelitha; Delalandre, Aline; Martel-Pelletier, Johanne; Pelletier, Jean-Pierre; Duval, Nicolas; Lajeunesse, Daniel

    2006-01-01

    Osteoarthritis (OA) is characterized by articular cartilage degradation and hypertrophic bone changes with osteophyte formation and abnormal bone remodeling. Two groups of OA patients were identified via the production of variable and opposite levels of prostaglandin E2 (PGE2) or leukotriene B4 (LTB4) by subchondral osteoblasts, PGE2 levels discriminating between low and high subgroups. We studied whether the expression of 5-lipoxygenase (5-LO) or 5-LO-activating protein (FLAP) is responsible for the shunt from prostaglandins to leukotrienes. FLAP mRNA levels varied in low and high OA groups compared with normal, whereas mRNA levels of 5-LO were similar in all osteoblasts. Selective inhibition of cyclooxygenase-2 (COX-2) with NS-398-stimulated FLAP expression in the high OA osteoblasts subgroup, whereas it was without effect in the low OA osteoblasts subgroup. The addition of PGE2 to the low OA osteoblasts subgroup decreased FLAP expression but failed to affect it in the high OA osteoblasts subgroup. LTB4 levels in OA osteoblasts were stimulated about twofold by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) plus transforming growth factor-beta (TGF-beta), a situation corresponding to their effect on FLAP mRNA levels. Treatments with 1,25(OH)2D3 and TGF-beta also modulated PGE2 production. TGF-beta stimulated PGE2 production in both OA osteoblast groups, whereas 1,25(OH)2D3 alone had a limited effect but decreased the effect of TGF-beta in the low OA osteoblasts subgroup. This modulation of PGE2 production was mirrored by the synthesis of COX-2. IL-18 levels were only slightly increased in a subgroup of OA osteoblasts compared with normal; however, no relationship was observed overall between IL-18 and PGE2 levels in normal and OA osteoblasts. These results suggest that the shunt from the production of PGE2 to LTB4 is through regulation of the expression of FLAP, not 5-LO, in OA osteoblasts. The expression of FLAP in OA osteoblasts is also modulated differently by 1,25(OH

  5. Irisin promotes osteoblast proliferation and differentiation via activating the MAP kinase signaling pathways.

    Science.gov (United States)

    Qiao, Xiaoyong; Yong Qiao, Xiao; Nie, Ying; Ma, Yaxian; Xian Ma, Ya; Chen, Yan; Cheng, Ran; Yin, Weiyao; Yao Yinrg, Wei; Hu, Ying; Xu, Wenming; Ming Xu, Wen; Xu, Liangzhi; Zhi Xu, Liang

    2016-01-01

    Physical exercise is able to improve skeletal health. However, the mechanisms are poorly known. Irisin, a novel exercise-induced myokine, secreted by skeletal muscle in response to exercise, have been shown to mediate beneficial effects of exercise in many disorders. In the current study, we demonstrated that irisin promotes osteoblast proliferation, and increases the expression of osteoblastic transcription regulators, such as Runt-related transcription factor-2, osterix/sp7; and osteoblast differentiation markers, including alkaline phosphatase, collagen type 1 alpha-1, osteocalcin, and osteopontin in vitro. Irisin also increase ALP activity and calcium deposition in cultured osteoblast. These osteogenic effects were mediated by activating the p38 mitogen-activated protein kinase (p-p38 MAPK) and extracellular signal-regulated kinase (ERK). Inhibition of p38 MAPK by SB023580 or pERK by U0126 abolished the proliferation and up-regulatory effects of irisin on Runx2 expression and ALP activity. Together our observation suggest that irisin directly targets osteoblast, promoting osteoblast proliferation and differentiation via activating P38/ERK MAP kinase signaling cascades in vitro. Whether irisin can be utilized as the therapeutic agents for osteopenia and osteoporosis is worth to be further pursued. PMID:26738434

  6. Telomerase activity promotes osteoblast differentiation by modulating IGF-signaling pathway

    DEFF Research Database (Denmark)

    Saeed, Hamid; Qiu, Weimin; Chen, Li;

    2015-01-01

    The contribution of deficient telomerase activity to age-related decline in osteoblast functions and bone formation is poorly studied. We have previously demonstrated that telomerase over-expression led to enhanced osteoblast differentiation of human bone marrow skeletal (stromal) stem cells (h......MSC) in vitro and in vivo. Here, we investigated the signaling pathways underlying the regulatory functions of telomerase in osteoblastic cells. Comparative microarray analysis and Western blot analysis of telomerase-over expressing hMSC (hMSC-TERT) versus primary hMSC revealed significant up....... In addition, telomerase deficiency caused significant reduction in IGF signaling proteins in osteoblastic cells cultured from telomerase deficient mice (Terc (-/-)). The low bone mass exhibited by Terc (-/-) mice was associated with significant reduction in serum levels of IGF1 and IGFBP3 as well as...

  7. Photodynamic activation as a molecular switch to promote osteoblast cell differentiation via AP-1 activation.

    Science.gov (United States)

    Kushibiki, Toshihiro; Tu, Yupeng; Abu-Yousif, Adnan O; Hasan, Tayyaba

    2015-01-01

    In photodynamic therapy (PDT), cells are impregnated with a photosensitizing agent that is activated by light irradiation, thereby photochemically generating reactive oxygen species (ROS). The amounts of ROS produced depends on the PDT dose and the nature of the photosensitizer. Although high levels of ROS are cytotoxic, at physiological levels they play a key role as second messengers in cellular signaling pathways, pluripotency, and differentiation of stem cells. To investigate further the use of photochemically triggered manipulation of such pathways, we exposed mouse osteoblast precursor cells and rat primary mesenchymal stromal cells to low-dose PDT. Our results demonstrate that low-dose PDT can promote osteoblast differentiation via the activation of activator protein-1 (AP-1). Although PDT has been used primarily as an anti-cancer therapy, the use of light as a photochemical "molecular switch" to promote differentiation should expand the utility of this method in basic research and clinical applications. PMID:26279470

  8. An Oligodeoxynucleotide with Promising Modulation Activity for the Proliferation and Activation of Osteoblast

    Directory of Open Access Journals (Sweden)

    Xinhua Sun

    2011-04-01

    Full Text Available The paper explored the regulatory role of oligodeoxynucleotides (ODNs with specific sequences in the proliferation and activation of osteoblast, using human osteoblast-like cell line MG 63 as the model. Through the administration of ODNs to MG 63 cells at a concentration of 1.0 µg/mL, ODN MT01 with positive effects on proliferation and activation of osteoblast was selected from 11 different ODNs by methyl thiazolyl tetrazolium (MTT assay and alkaline phosphatase (ALP activity measurement. To get a deeper insight into the molecular mechanism, effects of ODN MT01 treatment on the expression level of Sp7, runx-2, collagen-I, osteoprotegerin (OPG and RANK ligand (RANKL were determined using quantitative real time PCR and Western blotting. Remarkably, the mRNA and protein expression levels of Sp7, runx-2, collagen-I and OPG were improved after ODN MT01 treatment. Meanwhile, the protein expression level of RANKL was dramatically decreased. These results suggested that ODN MT01 had a significant impact in facilitating osteogenic proliferation and activation, and provided a direct evidence for the notion that single strand ODN could regulate the balance of bone formation and resorption, and thus was of great potential in the rebuilding of alveolar bone.

  9. Tissue transglutaminase (TG2 activity regulates osteoblast differentiation and mineralization in the SAOS-2 cell line

    Directory of Open Access Journals (Sweden)

    Xiaoxue Yin

    2012-08-01

    Full Text Available Tissue transglutaminase (type II, TG2 has long been postulated to directly promote skeletal matrix calcification and play an important role in ossification. However, limited information is available on the expression, function and modulating mechanism of TG2 during osteoblast differentiation and mineralization. To address these issues, we cultured the well-established human osteosarcoma cell line SAOS-2 with osteo-inductive conditioned medium and set up three time points (culture days 4, 7, and 14 to represent different stages of SAOS-2 differentiation. Osteoblast markers, mineralization, as well as TG2 expression and activity, were then assayed in each stage. Furthermore, we inhibited TG activity with cystamine and then checked SAOS-2 differentiation and mineralization in each stage. The results showed that during the progression of osteoblast differentiation SAOS-2 cells presented significantly high levels of osteocalcin (OC mRNA, bone morphogenetic protein-2 (BMP-2 and collagen I, significantly high alkaline phosphatase (ALP activity, and the increased formation of calcified matrix. With the same tendency, TG2 expression and activity were up-regulated. Furthermore, inhibition of TG activity resulted in a significant decrease of OC, collagen I, and BMP-2 mRNA and of ALP activity and mineralization. This study demonstrated that TG2 is involved in osteoblast differentiation and may play a role in the initiation and regulation of the mineralization processes. Moreover, the modulating effects of TG2 on osteoblasts may be related to BMP-2.

  10. Sphingosine 1-phosphate receptor activation enhances BMP-2-induced osteoblast differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Sato, Chieri [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan); Iwasaki, Tsuyoshi, E-mail: tsuyo-i@huhs.ac.jp [Division of Pharmacotherapy, Department of Pharmacy, School of Pharmacy, Hyogo University of Health Sciences, 1-3-6 Minatojima, Chuo-ku, Kobe 650-8530 (Japan); Kitano, Sachie; Tsunemi, Sachi; Sano, Hajime [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan)

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer We investigated the role of S1P signaling for osteoblast differentiation. Black-Right-Pointing-Pointer Both S1P and FTY enhanced BMP-2-stimulated osteoblast differentiation by C2C12 cells. Black-Right-Pointing-Pointer S1P signaling enhanced BMP-2-stimulated Smad and ERK phosphorylation by C2C12 cells. Black-Right-Pointing-Pointer MEK/ERK signaling is a pathway underlying S1P signaling for osteoblast differentiation. -- Abstract: We previously demonstrated that sphingosine 1-phosphate (S1P) receptor-mediated signaling induced proliferation and prostaglandin productions by synovial cells from rheumatoid arthritis (RA) patients. In the present study we investigated the role of S1P receptor-mediated signaling for osteoblast differentiation. We investigated osteoblast differentiation using C2C12 myoblasts, a cell line derived from murine satellite cells. Osteoblast differentiation was induced by the treatment of bone morphogenic protein (BMP)-2 in the presence or absence of either S1P or FTY720 (FTY), a high-affinity agonist of S1P receptors. Osteoblast differentiation was determined by osteoblast-specific transcription factor, Runx2 mRNA expression, alkaline phosphatase (ALP) activity and osteocalcin production by the cells. Smad1/5/8 and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation was examined by Western blotting. Osteocalcin production by C2C12 cells were determined by ELISA. Runx2 expression and ALP activity by BMP-2-stimulated C2C12 cells were enhanced by addition of either S1P or FTY. Both S1P and FTY enhanced BMP-2-induced ERK1/2 and Smad1/5/8 phosphorylation. The effect of FTY was stronger than that of S1P. S1P receptor-mediated signaling on osteoblast differentiation was inhibited by addition of mitogen-activated protein kinase/ERK kinase (MEK) 1/2 inhibitor, indicating that the S1P receptor-mediated MEK1/2-ERK1/2 signaling pathway enhanced BMP-2-Smad signaling. These results indicate that S1P

  11. Sphingosine 1-phosphate receptor activation enhances BMP-2-induced osteoblast differentiation

    International Nuclear Information System (INIS)

    Highlights: ► We investigated the role of S1P signaling for osteoblast differentiation. ► Both S1P and FTY enhanced BMP-2-stimulated osteoblast differentiation by C2C12 cells. ► S1P signaling enhanced BMP-2-stimulated Smad and ERK phosphorylation by C2C12 cells. ► MEK/ERK signaling is a pathway underlying S1P signaling for osteoblast differentiation. -- Abstract: We previously demonstrated that sphingosine 1-phosphate (S1P) receptor-mediated signaling induced proliferation and prostaglandin productions by synovial cells from rheumatoid arthritis (RA) patients. In the present study we investigated the role of S1P receptor-mediated signaling for osteoblast differentiation. We investigated osteoblast differentiation using C2C12 myoblasts, a cell line derived from murine satellite cells. Osteoblast differentiation was induced by the treatment of bone morphogenic protein (BMP)-2 in the presence or absence of either S1P or FTY720 (FTY), a high-affinity agonist of S1P receptors. Osteoblast differentiation was determined by osteoblast-specific transcription factor, Runx2 mRNA expression, alkaline phosphatase (ALP) activity and osteocalcin production by the cells. Smad1/5/8 and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation was examined by Western blotting. Osteocalcin production by C2C12 cells were determined by ELISA. Runx2 expression and ALP activity by BMP-2-stimulated C2C12 cells were enhanced by addition of either S1P or FTY. Both S1P and FTY enhanced BMP-2-induced ERK1/2 and Smad1/5/8 phosphorylation. The effect of FTY was stronger than that of S1P. S1P receptor-mediated signaling on osteoblast differentiation was inhibited by addition of mitogen-activated protein kinase/ERK kinase (MEK) 1/2 inhibitor, indicating that the S1P receptor-mediated MEK1/2-ERK1/2 signaling pathway enhanced BMP-2-Smad signaling. These results indicate that S1P receptor-mediated signaling plays a crucial role for osteoblast differentiation.

  12. Voriconazole Enhances the Osteogenic Activity of Human Osteoblasts In Vitro through a Fluoride-Independent Mechanism.

    Science.gov (United States)

    Allen, Kahtonna C; Sanchez, Carlos J; Niece, Krista L; Wenke, Joseph C; Akers, Kevin S

    2015-12-01

    Periostitis, which is characterized by bony pain and diffuse periosteal ossification, has been increasingly reported with prolonged clinical use of voriconazole. While resolution of clinical symptoms following discontinuation of therapy suggests a causative role for voriconazole, the biological mechanisms contributing to voriconazole-induced periostitis are unknown. To elucidate potential mechanisms, we exposed human osteoblasts in vitro to voriconazole or fluconazole at 15 or 200 μg/ml (reflecting systemic or local administration, respectively), under nonosteogenic or osteogenic conditions, for 1, 3, or 7 days and evaluated the effects on cell proliferation (reflected by total cellular DNA) and osteogenic differentiation (reflected by alkaline phosphatase activity, calcium accumulation, and expression of genes involved in osteogenic differentiation). Release of free fluoride, vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) was also measured in cell supernatants of osteoblasts exposed to triazoles, with an ion-selective electrode (for free fluoride) and enzyme-linked immunosorbent assays (ELISAs) (for VEGF and PDGF). Voriconazole but not fluconazole significantly enhanced the proliferation and differentiation of osteoblasts. In contrast to clinical observations, no increases in free fluoride levels were detected following exposure to either voriconazole or fluconazole; however, significant increases in the expression of VEGF and PDGF by osteoblasts were observed following exposure to voriconazole. Our results demonstrate that voriconazole can induce osteoblast proliferation and enhance osteogenic activity in vitro. Importantly, and in contrast to the previously proposed mechanism of fluoride-stimulated osteogenesis, our findings suggest that voriconazole-induced periostitis may also occur through fluoride-independent mechanisms that enhance the expression of cytokines that can augment osteoblastic activity. PMID:26324277

  13. Effects of fluoridation of porcine hydroxyapatite on osteoblastic activity of human MG63 cells

    Science.gov (United States)

    Li, Zhipeng; Huang, Baoxin; Mai, Sui; Wu, Xiayi; Zhang, Hanqing; Qiao, Wei; Luo, Xin; Chen, Zhuofan

    2015-06-01

    Biological hydroxyapatite, derived from animal bones, is the most widely used bone substitute in orthopedic and dental treatments. Fluorine is the trace element involved in bone remodeling and has been confirmed to promote osteogenesis when administered at the appropriate dose. To take advantage of this knowledge, fluorinated porcine hydroxyapatite (FPHA) incorporating increasing levels of fluoride was derived from cancellous porcine bone through straightforward chemical and thermal treatments. Physiochemical characteristics, including crystalline phases, functional groups and dissolution behavior, were investigated on this novel FPHA. Human osteoblast-like MG63 cells were cultured on the FPHA to examine cell attachment, cytoskeleton, proliferation and osteoblastic differentiation for in vitro cellular evaluation. Results suggest that fluoride ions released from the FPHA play a significant role in stimulating osteoblastic activity in vitro, and appropriate level of fluoridation (1.5 to 3.1 atomic percents of fluorine) for the FPHA could be selected with high potential for use as a bone substitute.

  14. Polycythemia is associated with bone loss and reduced osteoblast activity in mice

    NARCIS (Netherlands)

    Oikonomidou, P R; Casu, C; Yang, Z; Crielaard, B; Shim, J H; Rivella, S; Vogiatzi, M G

    2015-01-01

    Increased fragility has been described in humans with polycythemia vera (PV). Herein, we describe an osteoporotic phenotype associated with decreased osteoblast activity in a mouse model of PV and another mouse of polycythemia and elevated circulating erythropoietin (EPO). Our results are important

  15. Dexamethasone-induced apoptosis of osteocytic and osteoblastic cells is mediated by TAK1 activation.

    Science.gov (United States)

    Ding, Heyuan; Wang, Tao; Xu, Dongli; Cha, Bingbing; Liu, Jun; Li, Yiming

    2015-05-01

    Increased apoptosis of osteoblasts and osteocytes is the main mechanism of glucocorticoid (GC)-induced osteonecrosis. In the current study, we investigated whether dexamethasone (Dex)-induced osteoblastic and osteocytic cell apoptosis is mediated through activation of transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1), and whether TAK1 inhibition could promote survival opposing the deleterious effects of Dex. We found that TAK1 was activated by Dex in both osteocytic MLO-Y4 and osteoblastic OB-6 cells, which was prevented by two known anti-oxidants N-acetylcysteine (NAC) and ebselen. TAK1 inhibitors, including LYTAK1 and 5Z-7-oxozeaenol (57-OZ), inhibited Dex-induced apoptosis of MLO-Y4 and OB-6 cells. Meanwhile shRNA-mediated knockdown of TAK1 also suppressed Dex-induced damages to MLO-Y4 and OB-6 cells. On the other hand, exogenously over-expressing TAK1 enhanced Dex-induced MLO-Y4 and OB-6 cell apoptosis. At the molecular level, we found that TAK1 mediated Dex-induced pro-apoptotic Pyk2-JNK activation. Inhibition or silencing of TAK1 almost abolished Pyk2-JNK phosphorylations by Dex in MLO-Y4 and OB-6 cells. TAK1 over-expression, on the other hand, increased Dex's activity on Pyk2-JNK phosphorylations in above cells. We conclude that part of the pro-apoptotic actions of Dex on osteoblastic and osteocytic cells are mediated through TAK1 activation, and that inhibition of TAK1 might protect from GC-induced damages to osteoblasts and osteocytes. PMID:25753204

  16. Induced Microcracking Affects Osteoblast Attachment on Hydroxyapatite Scaffolds for Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Ian O. Smith

    2006-01-01

    Full Text Available Bone microdamage caused by routine activity plays an important role in triggering targeted and nontargeted bone remodeling. Targeted remodeling occurs near localized areas of microdamage[1-4]. We hypothesize that bone remodeling may be directly and positively influenced by inducing microcracks in hydroxyapatite (HA scaffolds for bone tissue engineering. A study by Case et al. showed microcracking in HA discs having >98% theoretical density[5]. These microcracks occurred without the application of external stress, likely as a result of thermal expansion anisotropy (TEA. TEA generates microcracks only when the specimen grain size exceeds a critical value (GCR. Due to conflicting data in the literature on the thermal expansion along the crystallographic axes of HA, it is difficult to estimate GCR precisely for HA, but GCR likely ranges from a few tenths of microns to several microns[6]. As the grain size of a polycrystalline specimen increases above the critical grain size, the number of microcracks also increases but the number density of microcracks is difficult to control. Therefore, in this study we used Vickers microindentation to induce controlled microcracks in >99% dense HA discs. The effect of microcracking on the osteoblast (OB attachment was then quantified. Control HA discs and microcracked HA were seeded with MC3T3-E1 OBs and cultured for 4 hours. The OBs were then stained with Rhodamine-Phalloidin and Hoechst fluorescent dyes to identify the actin fibers and nuclei, respectively. OB attachment was quantified using fluorescent light microscopy. OB attachment at 4 hours was 29% ± 7.6% of the initial seeded OBs for the microcracked HA specimens and 18% ± 6.1% of initial seeded OBs for the non-microcracked control HA specimens. The difference in these OB attachment values was statistically significant as determined via the Student t-test (p = 0.004, with p < 0.05 taken to indicate significance.

  17. The aryl hydrocarbon receptor suppresses osteoblast proliferation and differentiation through the activation of the ERK signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Haitao; Du, Yuxuan; Zhang, Xulong; Sun, Ying; Li, Shentao; Dou, Yunpeng [Department of Immunology, School of Basic Medical Sciences, Capital Medical University, No. 10 Xitoutiao, You An Men, Beijing 100069 (China); Li, Zhanguo [Department of Rheumatology and Immunology, Clinical Immunology Center, Peking University People' s Hospital, No. 11 Xizhimen South Street, Beijing 100044 (China); Yuan, Huihui, E-mail: huihui_yuan@163.com [Department of Immunology, School of Basic Medical Sciences, Capital Medical University, No. 10 Xitoutiao, You An Men, Beijing 100069 (China); Zhao, Wenming, E-mail: zhao-wenming@163.com [Department of Immunology, School of Basic Medical Sciences, Capital Medical University, No. 10 Xitoutiao, You An Men, Beijing 100069 (China)

    2014-11-01

    Ahr activation is known to be associated with synovitis and exacerbated rheumatoid arthritis (RA), but its contributions to bone loss have not been completely elucidated. Osteoblast proliferation and differentiation are abnormal at the erosion site in RA. Here, we reported that the expression of Ahr was increased in the hind paws' bone upon collagen-induced arthritis (CIA) in mice, and the levels of Ahr were negatively correlated with bone mineral density (BMD). In addition, immunofluorescent staining showed that the high expression of Ahr was mainly localized in osteoblasts from the CIA mice compared to normal controls. Moreover, the luciferase intensity of Ahr in the nucleus increased by 12.5% in CIA osteoblasts compared to that in normal controls. In addition, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) activation of the Ahr inhibited pre-osteoblast MC3T3-E1 cellular proliferation and differentiation in a dose-dependent manner. Interestingly, the levels of alkaline phosphatase (ALP) mRNA expression in the osteoblasts of CIA mice were reduced compared to normal controls. In contrast, decreased ALP expression by activated Ahr was completely reversed after pretreatment with an Ahr inhibitor (CH-223191) in MC3T3-E1 cell lines and primary osteoblasts on day 5. Our data further showed that activation of Ahr promoted the phosphorylation of ERK after 5 days. Moreover, Ahr-dependent activation of the ERK signaling pathway decreased the levels of proliferation cells and inhibited ALP activity in MC3T3-E1 cells. These results demonstrated that the high expression of Ahr may suppress osteoblast proliferation and differentiation through activation of the ERK signaling pathway, further enabling bone erosion in CIA mice. - Highlights: • The upregulation of Ahr was localized in osteoblasts of CIA mice. • The overexpression of Ahr suppressed osteoblast development. • The Ahr activated ERK signaling pathway to exacerbate bone erosion.

  18. The aryl hydrocarbon receptor suppresses osteoblast proliferation and differentiation through the activation of the ERK signaling pathway

    International Nuclear Information System (INIS)

    Ahr activation is known to be associated with synovitis and exacerbated rheumatoid arthritis (RA), but its contributions to bone loss have not been completely elucidated. Osteoblast proliferation and differentiation are abnormal at the erosion site in RA. Here, we reported that the expression of Ahr was increased in the hind paws' bone upon collagen-induced arthritis (CIA) in mice, and the levels of Ahr were negatively correlated with bone mineral density (BMD). In addition, immunofluorescent staining showed that the high expression of Ahr was mainly localized in osteoblasts from the CIA mice compared to normal controls. Moreover, the luciferase intensity of Ahr in the nucleus increased by 12.5% in CIA osteoblasts compared to that in normal controls. In addition, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) activation of the Ahr inhibited pre-osteoblast MC3T3-E1 cellular proliferation and differentiation in a dose-dependent manner. Interestingly, the levels of alkaline phosphatase (ALP) mRNA expression in the osteoblasts of CIA mice were reduced compared to normal controls. In contrast, decreased ALP expression by activated Ahr was completely reversed after pretreatment with an Ahr inhibitor (CH-223191) in MC3T3-E1 cell lines and primary osteoblasts on day 5. Our data further showed that activation of Ahr promoted the phosphorylation of ERK after 5 days. Moreover, Ahr-dependent activation of the ERK signaling pathway decreased the levels of proliferation cells and inhibited ALP activity in MC3T3-E1 cells. These results demonstrated that the high expression of Ahr may suppress osteoblast proliferation and differentiation through activation of the ERK signaling pathway, further enabling bone erosion in CIA mice. - Highlights: • The upregulation of Ahr was localized in osteoblasts of CIA mice. • The overexpression of Ahr suppressed osteoblast development. • The Ahr activated ERK signaling pathway to exacerbate bone erosion

  19. Histone H4-related osteogenic growth peptide (OGP): a novel circulating stimulator of osteoblastic activity.

    OpenAIRE

    Bab, I; Gazit, D.; Chorev, M; Muhlrad, A; Shteyer, A; Greenberg, Z; Namdar, M; Kahn, A.

    1992-01-01

    It has been established that regenerating marrow induces an osteogenic response in distant skeletal sites and that this activity is mediated by factors released into the circulation by the healing tissue. In the present study we have characterized one of these factors, a 14 amino acid peptide named osteogenic growth peptide (OGP). Synthetic OGP, identical in structure to the native molecule, stimulates the proliferation and alkaline phosphatase activity of osteoblastic cells in vitro and incr...

  20. Antiosteoporotic Activity of Anthraquinones from Morinda officinalis on Osteoblasts and Osteoclasts

    Directory of Open Access Journals (Sweden)

    Cheng-Jian Zheng

    2009-01-01

    Full Text Available Bioactivity-guided fractionation led to the successful isolation of antiosteoporotic components, i.e. physicion (1, rubiadin-1-methyl ether (2, 2-hydroxy-1-methoxy- anthraquinone (3, 1,2-dihydroxy-3-methylanthraquinone (4, 1,3,8-trihydroxy-2-methoxy- anthraquinone (5, 2-hydroxymethyl-3-hydroxyanthraquinone (6, 2-methoxyanthraquinone (7 and scopoletin (8 from an ethanolic extract of the roots of Morinda officinalis. Compounds 4-8 are isolated for the first time from M. officinalis. Among them, compounds 2 and 3 promoted osteoblast proliferation, while compounds 4, 5 increased osteoblast ALP activity. All of the isolated compounds inhibited osteoclast TRAP activity and bone resorption, and the inhibitory effects on osteoclastic bone resorption of compounds 1 and 5 were stronger than that of other compounds. Taken together, antiosteoporotic activity of M. officinalis and its anthraquinones suggest therapeutic potential against osteoporosis.

  1. Antiosteoporotic activity of anthraquinones from Morinda officinalis on osteoblasts and osteoclasts.

    Science.gov (United States)

    Wu, Yan-Bin; Zheng, Cheng-Jian; Qin, Lu-Ping; Sun, Lian-Na; Han, Ting; Jiao, Lei; Zhang, Qiao-Yan; Wu, Jin-Zhong

    2009-01-01

    Bioactivity-guided fractionation led to the successful isolation of antiosteoporotic components, i.e. physicion (1), rubiadin-1-methyl ether (2), 2-hydroxy-1-methoxy- anthraquinone (3), 1,2-dihydroxy-3-methylanthraquinone (4), 1,3,8-trihydroxy-2-methoxy- anthraquinone (5), 2-hydroxymethyl-3-hydroxyanthraquinone (6), 2-methoxyanthraquinone (7) and scopoletin (8) from an ethanolic extract of the roots of Morinda officinalis. Compounds 4-8 are isolated for the first time from M. officinalis. Among them, compounds 2 and 3 promoted osteoblast proliferation, while compounds 4, 5 increased osteoblast ALP activity. All of the isolated compounds inhibited osteoclast TRAP activity and bone resorption, and the inhibitory effects on osteoclastic bone resorption of compounds 1 and 5 were stronger than that of other compounds. Taken together, antiosteoporotic activity of M. officinalis and its anthraquinones suggest therapeutic potential against osteoporosis. PMID:19169204

  2. Zinc may increase bone formation through stimulating cell proliferation, alkaline phosphatase activity and collagen synthesis in osteoblastic MC3T3-E1 cells

    OpenAIRE

    Seo, Hyun-Ju; Cho, Young-Eun; Kim, Taewan; Shin, Hong-In; Kwun, In-Sook

    2010-01-01

    Zinc is an essential trace element required for bone formation, however not much has been clarified yet for its role in osteoblast. We hypothesized that zinc would increase osteogenetic function in osteoblasts. To test this, we investigated whether zinc treatment enhances bone formation by stimulating osteoblast proliferation, bone marker protein alkaline phosphatase activity and collagen synthesis in osteoblastic MC3T3-E1 cells. MC3T3-E1 cells were cultured and treated with various concentra...

  3. Calcium influx through stretch-activated channels mediates microfilament reorganization in osteoblasts under simulated weightlessness

    Science.gov (United States)

    Luo, Mingzhi; Yang, Zhouqi; Li, Jingbao; Xu, Huiyun; Li, Shengsheng; Zhang, Wei; Qian, Airong; Shang, Peng

    2013-06-01

    We have explored the role of Ca2+ signaling in microfilament reorganization of osteoblasts induced by simulated weightlessness using a random positioning machine (RPM). The RPM-induced alterations of cell morphology, microfilament distribution, cell proliferation, cell migration, cytosol free calcium concentration ([Ca2+]i), and protein expression in MG63 osteoblasts were investigated. Simulated weightlessness reduced cell size, disrupted microfilament, inhibited cellular proliferation and migration, and induced an increase in [Ca2+]i in MG63 human osteosarcoma cells. Gadolinium chloride (Gd), an inhibitor for stretch-activated channels, attenuated the increase in [Ca2+]i and microfilament disruption. Further, the expression of calmodulin was significantly increased by simulated weightlessness, and an inhibitor of calmodulin, W-7, aggravated microfilament disruption. Our findings demonstrate that simulated weightlessness induces Ca2+ influx through stretch-activated channels, then results in microfilament disruption.

  4. Yam (Dioscorea batatas) Root and Bark Extracts Stimulate Osteoblast Mineralization by Increasing Ca and P Accumulation and Alkaline Phosphatase Activity

    OpenAIRE

    Kim, Suji; Shin, Mee-Young; Son, Kun-Ho; Sohn, Ho-Yong; Lim, Jae-Hwan; Lee, Jong-Hwa; Kwun, In-Sook

    2014-01-01

    Yam (Dioscorea batatas) is widely consumed as functional food for health promotion mainly in East Asia countries. We assessed whether yam root (tuber) or bark (peel) extracts stimulated the activity of osteoblasts for osteogenesis. MC3T3-E1 cells (mouse osteoblasts) were treated with yam root extracts (water or methanol) (study I) or bark extracts (water or hexane) (study II) within 0~10 μg/mL during the periods of osteoblast proliferation (5~10 day), matrix maturation (11~15 day) and mineral...

  5. Effect of acceleration on osteoblastic and osteoclastic activities: Analysis of bone metabolism using goldfish scale as a model for bone

    Science.gov (United States)

    Suzuki, S.; Kitamura, K.; Nemoto, N.; Shimizu, S.; Wada, W.; Kondo, K.; Tabata, T.; Sodeyama, S.; Ijiri, I.; Hattori, H.

    It is well known that hypo-gravity and hyper-gravity influence bone metabolism However basic data concerning the mechanism are a few because no in vitro model system of human bone is available Human bone consists of osteoblasts osteoclasts and the bone matrix No technique for the co-culture of these components has ever been developed Fish scale is a calcified tissue that contains osteoblasts osteoclasts and bone matrix all of which are similar to those found in human bone Recently we developed a new in vitro model system using goldfish scale This system can simultaneously detect the activities of both scale osteoclasts and osteoblasts with tartrate-resistant acid phosphatase and alkaline phosphatase as the respective markers Using this system we analyzed the bone metabolism under acceleration with a custom-made G-load apparatus Osteoclastic activity in the goldfish scales was suppressed under low-acceleration 0 5-G while osteoblastic activity did not change under this acceleration Under high-acceleration 6-G however the osteoblastic activity of the scales increased In addition the osteoclastic activity of the scales decreased These results suggest that both osteoblastic and osteoclastic activities are regulated by the strength of acceleration Therefore we strongly believe that our in vitro system is useful for analysis of bone metabolism under acceleration

  6. Mechanical Stimulation and IGF-1 Enhance mRNA Translation Rate in Osteoblasts Via Activation of the AKT-mTOR Pathway.

    Science.gov (United States)

    Bakker, Astrid D; Gakes, Tom; Hogervorst, Jolanda M A; de Wit, Gerard M J; Klein-Nulend, Jenneke; Jaspers, Richard T

    2016-06-01

    Insulin-like growth factor-1 (IGF-1) is anabolic for muscle by enhancing the rate of mRNA translation via activation of AKT and subsequent activation of the mammalian target of rapamycin complex 1 (mTOR), thereby increasing cellular protein production. IGF-1 is also anabolic for bone, but whether the mTOR pathway plays a role in the rate of bone matrix protein production by osteoblasts is unknown. We hypothesized that anabolic stimuli such as mechanical loading and IGF-1 stimulate protein synthesis in osteoblasts via activation of the AKT-mTOR pathway. MC3T3-E1 osteoblasts were either or not subjected for 1 h to mechanical loading by pulsating fluid flow (PFF) or treated with or without human recombinant IGF-1 (1-100 ng/ml) for 0.5-6 h, to determine phosphorylation of AKT and p70S6K (downstream of mTOR) by Western blot. After 4 days of culture with or without the mTOR inhibitor rapamycin, total protein, DNA, and gene expression were quantified. IGF-1 (100 ng/ml) reduced IGF-1 gene expression, although PFF enhanced IGF-1 expression. IGF-1 did not affect collagen-I gene expression. IGF-1 dose-dependently enhanced AKT and p70S6K phosphorylation at 2 and 6 h. PFF enhanced phosphorylation of AKT and p70S6K already within 1 h. Both IGF-1 and PFF enhanced total protein per cell by ∼30%, but not in the presence of rapamycin. Our results show that IGF-1 and PFF activate mTOR, thereby stimulating the rate of mRNA translation in osteoblasts. The known anabolic effect of mechanical loading and IGF-1 on bone may thus be partly explained by mTOR-mediated enhanced protein synthesis in osteoblasts. PMID:26505782

  7. Oxygen plasma surface modification augments poly(L-lactide-co-glycolide) cytocompatibility toward osteoblasts and minimizes immune activation of macrophages.

    Science.gov (United States)

    Scislowska-Czarnecka, Anna; Szmigiel, Dariusz; Genet, Michel; Dupont-Gillain, Christine; Pamula, Elzbieta; Kolaczkowska, Elzbieta

    2015-12-01

    Here, we report on modification of one of the model biomedical polymers, poly L-lactide-co-glycolide (PLGA; 85:15), by reactive ion etching (RIE) oxygen plasma treatment. PLGA's major disadvantage is high hydrophobicity which restrains binding of cell-adhesive proteins and host cells. In the current approach, we aimed to answer two questions: (1) will only short (10 s) and moderate (20-200 mTorr, 45-90 W) RIE oxygen plasma treatment, leading to decrease of water contact angle by only up to 10°, sufficiently improve PLGA adherence to cells, and (2) how will this affect osteoblasts and activation of the immune system? All obtained modified PLGAs had improved hydrophilicity but unaltered roughness (as revealed by water contact angle measurements, X-ray photoelectron spectroscopy, and atomic force microscopy) resulting in significantly improved adhesion of osteoblasts (MG-63) and their low activation. Importantly, macrophages (RAW 264.7), one of the key cells initiating inflammation and bone resorption, responded significantly less vigorously to the modified polymers, expressing/releasing lower amounts of nitric oxide, matrix metalloproteinases (MMP-9), and pro-inflammatory cytokines (TNF-α, IL-6, IL-12p70, IFN-γ, IL-10). We conclude that already slight RIE oxygen plasma modification of PLGA is sufficient to improve its surface properties, and enhance cytocompatibility. Most importantly, this type of modification prevents excessive immune response. PMID:26014580

  8. Orbital fluid shear stress promotes osteoblast metabolism, proliferation and alkaline phosphates activity in vitro.

    Science.gov (United States)

    Aisha, M D; Nor-Ashikin, M N K; Sharaniza, A B R; Nawawi, H; Froemming, G R A

    2015-09-10

    Prolonged disuse of the musculoskeletal system is associated with reduced mechanical loading and lack of anabolic stimulus. As a form of mechanical signal, the multidirectional orbital fluid shear stress transmits anabolic signal to bone forming cells in promoting cell differentiation, metabolism and proliferation. Signals are channeled through the cytoskeleton framework, directly modifying gene and protein expression. For that reason, we aimed to study the organization of Normal Human Osteoblast (NHOst) cytoskeleton with regards to orbital fluid shear (OFS) stress. Of special interest were the consequences of cytoskeletal reorganization on NHOst metabolism, proliferation, and osteogenic functional markers. Cells stimulated at 250 RPM in a shaking incubator resulted in the rearrangement of actin and tubulin fibers after 72 h. Orbital shear stress increased NHOst mitochondrial metabolism and proliferation, simultaneously preventing apoptosis. The ratio of RANKL/OPG was reduced, suggesting that orbital shear stress has the potential to inhibit osteoclastogenesis and osteoclast activity. Increase in ALP activity and OCN protein production suggests that stimulation retained osteoblast function. Shear stress possibly generated through actin seemed to hold an anabolic response as osteoblast metabolism and functional markers were enhanced. We hypothesize that by applying orbital shear stress with suitable magnitude and duration as a non-drug anabolic treatment can help improve bone regeneration in prolonged disuse cases. PMID:26163894

  9. Orbital fluid shear stress promotes osteoblast metabolism, proliferation and alkaline phosphates activity in vitro

    International Nuclear Information System (INIS)

    Prolonged disuse of the musculoskeletal system is associated with reduced mechanical loading and lack of anabolic stimulus. As a form of mechanical signal, the multidirectional orbital fluid shear stress transmits anabolic signal to bone forming cells in promoting cell differentiation, metabolism and proliferation. Signals are channeled through the cytoskeleton framework, directly modifying gene and protein expression. For that reason, we aimed to study the organization of Normal Human Osteoblast (NHOst) cytoskeleton with regards to orbital fluid shear (OFS) stress. Of special interest were the consequences of cytoskeletal reorganization on NHOst metabolism, proliferation, and osteogenic functional markers. Cells stimulated at 250 RPM in a shaking incubator resulted in the rearrangement of actin and tubulin fibers after 72 h. Orbital shear stress increased NHOst mitochondrial metabolism and proliferation, simultaneously preventing apoptosis. The ratio of RANKL/OPG was reduced, suggesting that orbital shear stress has the potential to inhibit osteoclastogenesis and osteoclast activity. Increase in ALP activity and OCN protein production suggests that stimulation retained osteoblast function. Shear stress possibly generated through actin seemed to hold an anabolic response as osteoblast metabolism and functional markers were enhanced. We hypothesize that by applying orbital shear stress with suitable magnitude and duration as a non-drug anabolic treatment can help improve bone regeneration in prolonged disuse cases. - Highlights: • OFS stress transmits anabolic signals to osteoblasts. • Actin and tubulin fibers are rearranged under OFS stress. • OFS stress increases mitochondrial metabolism and proliferation. • Reduced RANKL/OPG ratio in response to OFS inhibits osteoclastogenesis. • OFS stress prevents apoptosis and stimulates ALP and OCN

  10. Orbital fluid shear stress promotes osteoblast metabolism, proliferation and alkaline phosphates activity in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Aisha, M.D. [Institute of Medical Molecular Biotechnology and Faculty of Medicine, Universiti Teknologi MARA, Sungai Buloh 47000, Selangor (Malaysia); Nor-Ashikin, M.N.K. [Institute of Medical Molecular Biotechnology and Faculty of Medicine, Universiti Teknologi MARA, Sungai Buloh 47000, Selangor (Malaysia); DDH, Universiti Teknologi MARA, ShahAlam 40450, Selangor (Malaysia); Sharaniza, A.B.R. [DDH, Universiti Teknologi MARA, ShahAlam 40450, Selangor (Malaysia); Nawawi, H. [Center for Pathology Diagnostic and Research Laboratories, Clinical Training Center, Universiti Teknologi MARA, Sungai Buloh 47000, Selangor (Malaysia); I-PPerForM, Universiti Teknologi MARA, Selayang 47000 Selangor (Malaysia); Froemming, G.R.A., E-mail: gabriele@salam.uitm.edu.my [Institute of Medical Molecular Biotechnology and Faculty of Medicine, Universiti Teknologi MARA, Sungai Buloh 47000, Selangor (Malaysia); I-PPerForM, Universiti Teknologi MARA, Selayang 47000 Selangor (Malaysia)

    2015-09-10

    Prolonged disuse of the musculoskeletal system is associated with reduced mechanical loading and lack of anabolic stimulus. As a form of mechanical signal, the multidirectional orbital fluid shear stress transmits anabolic signal to bone forming cells in promoting cell differentiation, metabolism and proliferation. Signals are channeled through the cytoskeleton framework, directly modifying gene and protein expression. For that reason, we aimed to study the organization of Normal Human Osteoblast (NHOst) cytoskeleton with regards to orbital fluid shear (OFS) stress. Of special interest were the consequences of cytoskeletal reorganization on NHOst metabolism, proliferation, and osteogenic functional markers. Cells stimulated at 250 RPM in a shaking incubator resulted in the rearrangement of actin and tubulin fibers after 72 h. Orbital shear stress increased NHOst mitochondrial metabolism and proliferation, simultaneously preventing apoptosis. The ratio of RANKL/OPG was reduced, suggesting that orbital shear stress has the potential to inhibit osteoclastogenesis and osteoclast activity. Increase in ALP activity and OCN protein production suggests that stimulation retained osteoblast function. Shear stress possibly generated through actin seemed to hold an anabolic response as osteoblast metabolism and functional markers were enhanced. We hypothesize that by applying orbital shear stress with suitable magnitude and duration as a non-drug anabolic treatment can help improve bone regeneration in prolonged disuse cases. - Highlights: • OFS stress transmits anabolic signals to osteoblasts. • Actin and tubulin fibers are rearranged under OFS stress. • OFS stress increases mitochondrial metabolism and proliferation. • Reduced RANKL/OPG ratio in response to OFS inhibits osteoclastogenesis. • OFS stress prevents apoptosis and stimulates ALP and OCN.

  11. Activation of L-type calcium channels is required for gap junction-mediated intercellular calcium signaling in osteoblastic cells

    Science.gov (United States)

    Jorgensen, Niklas Rye; Teilmann, Stefan Cuoni; Henriksen, Zanne; Civitelli, Roberto; Sorensen, Ole Helmer; Steinberg, Thomas H.

    2003-01-01

    The propagation of mechanically induced intercellular calcium waves (ICW) among osteoblastic cells occurs both by activation of P2Y (purinergic) receptors by extracellular nucleotides, resulting in "fast" ICW, and by gap junctional communication in cells that express connexin43 (Cx43), resulting in "slow" ICW. Human osteoblastic cells transmit intercellular calcium signals by both of these mechanisms. In the current studies we have examined the mechanism of slow gap junction-dependent ICW in osteoblastic cells. In ROS rat osteoblastic cells, gap junction-dependent ICW were inhibited by removal of extracellular calcium, plasma membrane depolarization by high extracellular potassium, and the L-type voltage-operated calcium channel inhibitor, nifedipine. In contrast, all these treatments enhanced the spread of P2 receptor-mediated ICW in UMR rat osteoblastic cells. Using UMR cells transfected to express Cx43 (UMR/Cx43) we confirmed that nifedipine sensitivity of ICW required Cx43 expression. In human osteoblastic cells, gap junction-dependent ICW also required activation of L-type calcium channels and influx of extracellular calcium.

  12. Effect of vibration on osteoblastic and osteoclastic activities: Analysis of bone metabolism using goldfish scale as a model for bone

    Science.gov (United States)

    Suzuki, N.; Kitamura, K.; Nemoto, T.; Shimizu, N.; Wada, S.; Kondo, T.; Tabata, M. J.; Sodeyama, F.; Ijiri, K.; Hattori, A.

    In osteoclastic activity during space flight as well as hind limb unloading by tail suspension, inconsistent results have been reported in an in vivo study. The bone matrix plays an important role in the response to physical stress. However, there is no suitable in vitro co-culture system of osteoblasts and osteoclasts including bone matrix. On the other hand, fish scale is a calcified tissue that contains osteoblasts, osteoclasts, and bone matrix, all of which are similar to those found in human bones. Recently, we developed a new in vitro model system using goldfish scale. This system can detect the activities of osteoclasts and osteoblasts with tartrate-resistant acid phosphatase and alkaline phosphatase as the respective markers and precisely analyze the co-relationship between osteoblasts and osteoclasts. Using this system, we analyzed the bone metabolism under various degrees of acceleration (0.5-, 1-, 2-, 4-, and 6-G) by vibration with a G-load apparatus. After loading for 5 and 10 min, the scales were incubated for 6 and 24 h. The osteoblastic and osteoclastic activities were then measured. The osteoblastic activities gradually increased corresponding to 1-G to 6-G acceleration. In addition, ER mRNA expression was the highest under 6-G acceleration. On the other hand, the osteoclastic activity decreased at 24 h of incubation under low acceleration (0.5- and 1-G). This change coincided with TRAP mRNA expression. Under 2-G acceleration, the strength of suppression in osteoclastic activity was the highest. The strength of the inhibitory action under 4- and 6-G acceleration was lower than that under 2-G acceleration. In our co-culture system, osteoblasts and osteoclasts in the scale sensitively responded to several degrees of acceleration. Therefore, we strongly believe that our in vitro co-culture system is useful for the analysis of bone metabolism under loading or unloading.

  13. Optimization of extraction and purification of active fractions from Schisandra chinensis (Turcz.) and its osteoblastic proliferation stimulating activity.

    Science.gov (United States)

    Caichompoo, Wanida; Zhang, Qiao-Yan; Hou, Ting-Ting; Gao, Hua-Juan; Qin, Lu-Ping; Zhou, Xiu-Jia

    2009-02-01

    Extraction and purification conditions of lignans from the fruits and seeds of Schisandra chinensis (Turcz.) were investigated through an orthogonal design of L(9)(3(4)) assay and macroporous resin technology. The extraction was optimized using 95% ethanol. For purification, the extract was dissolved in 30% ethanol, then adsorbed on a AB-8 macroporous resin and eluted with 30% ethanol and 70% ethanol successively, the latter resulting in a residue containing 65.2% of lignans. By HPLC analysis schisandrin, deoxyschisandrin and gamma-schisandrin were quantitatively determined. UMR 106 cells were used to examine the stimulatory activity of the lignans on osteoblasts in vitro. The lignans stimulated the proliferation of and the activity of alkaline phosphatase in the osteoblasts indicating their potential activity against osteoporosis. PMID:18698667

  14. Alterations of metabolic activity in human osteoarthritic osteoblasts by lipid peroxidation end product 4-hydroxynonenal

    OpenAIRE

    Shi, Qin; Vaillancourt, France; Côté, Véronique; Fahmi, Hassan; Lavigne, Patrick; Afif, Hassan; Di Battista, John A.; Fernandes, Julio C; Benderdour, Mohamed

    2006-01-01

    4-Hydroxynonenal (HNE), a lipid peroxidation end product, is produced abundantly in osteoarthritic (OA) articular tissues, but its role in bone metabolism is ill-defined. In this study, we tested the hypothesis that alterations in OA osteoblast metabolism are attributed, in part, to increased levels of HNE. Our data showed that HNE/protein adduct levels were higher in OA osteoblasts compared to normal and when OA osteoblasts were treated with H2O2. Investigating osteoblast markers, we found t...

  15. Porphyromonas gingivalis induces receptor activator of NF-kappaB ligand expression in osteoblasts through the activator protein 1 pathway.

    Science.gov (United States)

    Okahashi, Nobuo; Inaba, Hiroaki; Nakagawa, Ichiro; Yamamura, Taihei; Kuboniwa, Masae; Nakayama, Koji; Hamada, Shigeyuki; Amano, Atsuo

    2004-03-01

    Porphyromonas gingivalis, an important periodontal pathogen, is closely associated with inflammatory alveolar bone resorption, and several components of the organism such as lipopolysaccharides have been reported to stimulate production of cytokines that promote inflammatory bone destruction. We investigated the effect of infection with viable P. gingivalis on cytokine production by osteoblasts. Reverse transcription-PCR and real-time PCR analyses revealed that infection with P. gingivalis induced receptor activator of nuclear factor kappaB (NF-kappaB) ligand (RANKL) mRNA expression in mouse primary osteoblasts. Production of interleukin-6 was also stimulated; however, osteoprotegerin was not. SB20350 (an inhibitor of p38 mitogen-activated protein kinase), PD98059 (an inhibitor of classic mitogen-activated protein kinase kinase, MEK1/2), wortmannin (an inhibitor of phosphatidylinositol 3 kinase), and carbobenzoxyl-leucinyl-leucinyl-leucinal (an inhibitor of NF-kappaB) did not prevent the RANKL expression induced by P. gingivalis. Degradation of inhibitor of NF-kappaB-alpha was not detectable; however, curcumin, an inhibitor of activator protein 1 (AP-1), prevented the RANKL production induced by P. gingivalis infection. Western blot analysis revealed that phosphorylation of c-Jun, a component of AP-1, occurred in the infected cells, and an analysis of c-Fos binding to an oligonucleotide containing an AP-1 consensus site also demonstrated AP-1 activation in infected osteoblasts. Infection with P. gingivalis KDP136, an isogenic deficient mutant of arginine- and lysine-specific cysteine proteinases, did not stimulate RANKL production. These results suggest that P. gingivalis infection induces RANKL expression in osteoblasts through AP-1 signaling pathways and cysteine proteases of the organism are involved in RANKL production. PMID:14977979

  16. Use of green fluorescent fusion protein to track activation of the transcription factor osterix during early osteoblast differentiation

    International Nuclear Information System (INIS)

    Osterix (Osx) is a transcription factor required for the differentiation of preosteoblasts into fully functioning osteoblasts. However, the pattern of Osx activation during preosteoblast differentiation and maturation has not been clearly defined. Our aim was to study Osx activation during these processes in osteoblasts differentiating from murine and human embryonic stem cells (ESC). To do this, we constructed an Osx-GFP fusion protein reporter system to track Osx translocation within the cells. The distribution of Osx-GFP at representative stages of differentiation was also investigated by screening primary osteoblasts, mesenchymal stem cells, synoviocytes, and pre-adipocytes. Our experiments revealed that Osx-GFP protein was detectable in the cytoplasm of cultured, differentiated ESC 4 days after plating of enzymatically dispersed embryoid bodies. Osterix-GFP protein became translocated into the nucleus on day 7 following transfer of differentiated ESC to osteogenic medium. After 14 days of differentiation, cells showing nuclear translocation of Osx-GFP formed rudimentary bone nodules that continued to increase in number over the following weeks (through day 21). We also found that Osx translocated into the nuclei of mesenchymal stem cells (C3H10T1/2) and pre-osteoblasts (MC3T3-E1) and showed partial activation in pre-adipocytes (MC3T3-L1). These data suggest that Osx activation occurs at a very early point in the differentiation of the mesenchymal-osteoblastic lineage

  17. Class I PI-3-Kinase Signaling Is Critical for Bone Formation Through Regulation of SMAD1 Activity in Osteoblasts.

    Science.gov (United States)

    Gámez, Beatriz; Rodríguez-Carballo, Edgardo; Graupera, Mariona; Rosa, José Luis; Ventura, Francesc

    2016-08-01

    Bone formation and homeostasis is carried out by osteoblasts, whose differentiation and activity are regulated by osteogenic signaling networks. A central mediator of these inputs is the lipid kinase phosphatidylinositol 3-kinase (PI3K). However, at present, there are no data on the specific role of distinct class IA PI3K isoforms in bone biology. Here, we performed osteoblast-specific deletion in mice to show that both p110α and p110β isoforms are required for survival and differentiation and function of osteoblasts and thereby control bone formation and postnatal homeostasis. Impaired osteogenesis arises from increased GSK3 activity and a depletion of SMAD1 protein levels in PI3K-deficient osteoblasts. Accordingly, pharmacological inhibition of GSK3 activity or ectopic expression of SMAD1 or SMAD5 normalizes bone morphogenetic protein (BMP) transduction and osteoblast differentiation. Together, these results identify the PI3K-GSK3-SMAD1 axis as a central node integrating multiple signaling networks that govern bone formation and homeostasis. © 2016 American Society for Bone and Mineral Research. PMID:26896753

  18. Osteoblast response (initial adhesion and alkaline phosphatase activity following exposure to a barrier membrane/enamel matrix derivative combination

    Directory of Open Access Journals (Sweden)

    Thangakumaran S

    2009-01-01

    Full Text Available Background and Objective: The enamel matrix derivative (EMD has been used in combination with barrier membranes to optimize regeneration in vertical osseous defects. However, the osteoblast response when exposed to the EMD/barrier membrane combination has not yet been evaluated. The osteoblast behavior when exposed to a combination of regenerative materials must be evaluated to fully understand their effect on bone regeneration. Therefore, the present study was undertaken to estimate the initial adhesion and alkaline phosphatase (ALP activity of an osteoblast cell line (SaOS-2 when exposed to four commercially available resorbable membranes and determine if the addition of EMD had any modulatory effect on osteoblast behavior. Materials and Methods: 5 x 104 SaOS-2 cells between passages 7-10 were cultured in two 24-well culture plates. Plate A was used for the adhesion assay and Plate B was used for the ALP assay. A MTT (3-[4, 5-dimethylthiazolyl-2]-2, 5-diphenyltetrazolium bromide assay was done after 24 hours to determine the adhesion of the osteoblastic cells to four barrier membranes: 1 a non cross-linked porcine Type I and III collagen membrane (BG, 2 a weakly cross-linked Type I collagen membrane (HG, 3 a glutaraldehyde cross-linked bovine Type I collagen (BM, and 4 a resorbable polymer membrane (CP. Osteoblast differentiation was studied using an ALP assay with p-nitro phenyl phosphate as the substrate at 24 hours, 72 hours, and 1 week. A total of 50 µg/ml of EMD dissolved in 10 mM acetic acid was added into each well and the entire experimental protocol outlined above was repeated. Results: The osteoblast adhesion to collagen barriers showed a statistically insignificant reduction following the addition of EMD. Adhesion to the polymer barrier, although significantly lower when compared with collagen barriers, was unaffected by the addition of EMD. ALP activity after 1 week among the various groups was as follows: EMD alone (75.59±2

  19. Activation of L-type calcium channels is required for gap junction-mediated intercellular calcium signaling in osteoblastic cells

    DEFF Research Database (Denmark)

    Jørgensen, Niklas Rye; Teilmann, Stefan Cuoni; Henriksen, Zanne;

    2003-01-01

    The propagation of mechanically induced intercellular calcium waves (ICW) among osteoblastic cells occurs both by activation of P2Y (purinergic) receptors by extracellular nucleotides, resulting in "fast" ICW, and by gap junctional communication in cells that express connexin43 (Cx43), resulting in...... extracellular calcium, plasma membrane depolarization by high extracellular potassium, and the L-type voltage-operated calcium channel inhibitor, nifedipine. In contrast, all these treatments enhanced the spread of P2 receptor-mediated ICW in UMR rat osteoblastic cells. Using UMR cells transfected to express Cx......43 (UMR/Cx43) we confirmed that nifedipine sensitivity of ICW required Cx43 expression. In human osteoblastic cells, gap junction-dependent ICW also required activation of L-type calcium channels and influx of extracellular calcium....

  20. Intercellular calcium signaling occurs between human osteoblasts and osteoclasts and requires activation of osteoclast P2X7 receptors

    DEFF Research Database (Denmark)

    Jørgensen, Niklas R; Henriksen, Zanne; Sørensen, Ole;

    2002-01-01

    Signaling between osteoblasts and osteoclasts is important in bone homeostasis. We previously showed that human osteoblasts propagate intercellular calcium signals via two mechanisms: autocrine activation of P2Y receptors, and gap junctional communication. In the current work we identified...... mechanically induced intercellular calcium signaling between osteoblasts and osteoclasts and among osteoclasts. Intercellular calcium responses in osteoclasts required P2 receptor activation but not gap junctional communication. Pharmacological studies and reverse transcriptase-PCR amplification demonstrated...... that human osteoclasts expressed functional P2Y1 receptors, but, unexpectedly, desensitization of P2Y1 did not block calcium signaling to osteoclasts. We also found that osteoclasts expressed functional P2X7 receptors and showed that pharmacological inhibition of these receptors blocked calcium...

  1. Strontium Ranelate affects signaling from mechanically-stimulated osteocytes towards osteoclasts and osteoblasts

    NARCIS (Netherlands)

    A.D. Bakker; B. Zandieh-Doulabi; J. Klein-Nulend

    2013-01-01

    Strontium Ranelate (SrRan) is used to decrease the risk of bone fractures. Any factor that alters the release of paracrine signals by osteocytes in response to mechanical stimuli potentially affects bone mass and structure, and thus fracture resistance. We hypothesized that SrRan affects paracrine s

  2. α-Melanocyte stimulating hormone attenuates dexamethasone-induced osteoblast damages through activating melanocortin receptor 4-SphK1 signaling.

    Science.gov (United States)

    Guo, Shiguang; Xie, Yue; Fan, Jian-bo; Ji, Feng; Wang, Shouguo; Fei, Haodong

    2016-01-01

    Long-term glucocorticoid (GC) usage may cause non-traumatic femoral head osteonecrosis. Dexamethasone (Dex) is shown to exert potent cytotoxic effect to osteoblasts. Here, we investigated the potential activity of α-melanocyte stimulating hormone (α-MSH) against the process. Our data revealed that pretreatment of α-MSH significantly inhibited Dex-induced apoptosis and necrosis in both osteoblastic-like MC3T3-E1 cells and primary murine osteoblasts. Melanocortin receptor 4 (MC4R) acts as the receptor of α-MSH in mediating its actions in osteoblasts. The MC4R antagonist SHU9119, or shRNA-mediated knockdown of MC4R, almost abolished α-MSH-induced activation of downstream signalings (Akt and Erk1/2) and its pro-survival effect in osteoblasts. Further studies showed that α-MSH activated MC4R downstream sphingosine kinase 1 (SphK1) and increased cellular sphingosine-1-phosphate (S1P) content in MC3T3-E1 cells and primary murine osteoblasts, which were blocked by SHU9119 or MC4R shRNAs. SphK1 inhibition by the its inhibitor N,N-dimethylsphingosine (DMS), or SphK1 knockdown by targeted-shRNAs, largely attenuated α-MSH-mediated osteoblast protection against Dex. Together, these results suggest that α-MSH alleviates Dex-induced damages to cultured osteoblasts through activating MC4R-SphK1 signaling. PMID:26631960

  3. Dioxin-induced up-regulation of the active form of vitamin D is the main cause for its inhibitory action on osteoblast activities, leading to developmental bone toxicity

    International Nuclear Information System (INIS)

    Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD) is known to cause bone toxicity, particularly during animal development, although its action mechanism to cause this toxicity has yet to be elucidated. Mouse pups were exposed to TCDD via dam's milk that were administered orally with 15 μg TCDD/kg b.w. on postnatal day 1. Here we report that TCDD causes up-regulation of vitamin D 1α-hydroxylase in kidney, resulting in a 2-fold increase in the active form of vitamin D, 1,25-dihydroxyvitamin D3, in serum. This action of TCDD is not caused by changes in parathyroid hormone, a decrease in vitamin D degrading enzyme, vitamin D 24-hydroxylase, or alterations in serum Ca2+ concentration. Vitamin D is known to affect bone mineralization. Our data clearly show that TCDD-exposed mice exhibit a marked decrease in osteocalcin and collagen type 1 as well as alkaline phosphatase gene expression in tibia by postnatal day 21, which is accompanied with a mineralization defect in the tibia, lowered activity of osteoblastic bone formation, and an increase in fibroblastic growth factor-23, a sign of increased vitamin D effect. Despite these significant effects of TCDD on osteoblast activities, none of the markers of osteoclast activities was found to be affected. Histomorphometry confirmed that osteoblastic activity, but not bone resorption activity, was altered by TCDD. A prominent lesion commonly observed in these TCDD-treated mice was impaired bone mineralization that is characterized by an increased volume and thickness of osteoids lining both the endosteum of the cortical bone and trabeculae. Together, these data suggest that the impaired mineralization resulting from reduction of the osteoblastic activity, which is caused by TCDD-induced up-regulation of vitamin D, is responsible for its bone developmental toxicity.

  4. Affecting osteoblastic responses with in vivo engineered potato pectin fragments

    DEFF Research Database (Denmark)

    Kokkonen, Hanna; Verhoef, Renè; Kauppinen, Kyösti;

    2012-01-01

    on Petri dishes coated with RG-Is from native or genetically engineered potato tubers. Uncoated tissue culture polystyrene (TCPS) and aminated (AMI) dishes served as controls. MHRPTR_GAL sample was depleted of galactose (9 mol % galactose; 23 mol % arabinose) and MHRPTR_ARA of arabinose (61 mol % galactose...... subtler. However, tetracycline-stained calcium-containing mineral was detected merely only on uncoated TCPS. These results indicate the possibility to affect bone cell growth with in vivo-modified pectin fragments, consecutively providing information on the significance of certain monosaccharides...

  5. Induced Microcracking Affects Osteoblast Attachment on Hydroxyapatite Scaffolds for Tissue Engineering

    OpenAIRE

    Smith, Ian O; Melissa J. Baumann; Case, Eldon D.

    2006-01-01

    Bone microdamage caused by routine activity plays an important role in triggering targeted and nontargeted bone remodeling. Targeted remodeling occurs near localized areas of microdamage[1-4]. We hypothesize that bone remodeling may be directly and positively influenced by inducing microcracks in hydroxyapatite (HA) scaffolds for bone tissue engineering. A study by Case et al. showed microcracking in HA discs having >98% theoretical density[5]. These microcracks occurred without the app...

  6. Calcite as a bone substitute. Comparison with hydroxyapatite and tricalcium phosphate with regard to the osteoblastic activity

    Energy Technology Data Exchange (ETDEWEB)

    Monchau, F., E-mail: Francine.monchau@univ-artois.fr [Laboratoire Genie Civil et geo-Environnement (EA 4515, Universite Lille Nord de France), Equipe Biomateriaux Artois (Universite d' Artois), IUT/GMP, 1230, rue de l' Universite, BP 819, 62408 Bethune cedex (France); Hivart, Ph.; Genestie, B. [Laboratoire Genie Civil et geo-Environnement (EA 4515, Universite Lille Nord de France), Equipe Biomateriaux Artois (Universite d' Artois), IUT/GMP, 1230, rue de l' Universite, BP 819, 62408 Bethune cedex (France); Chai, F. [Laboratoire Medicaments et Biomateriaux a Liberation Controlee (INSERM U 1008, Universite Lille Nord de France), Groupe de Recherche sur les Biomateriaux (Universite Lille-2), Faculte de Medecine, 1, place de Verdun, 59045 Lille cedex (France); and others

    2013-01-01

    Close to the bone mineral phase, the calcic bioceramics, such as hydroxyapatite (HA) and {beta}-tricalcium phosphate ({beta}-TCP), are commonly used as substitutes or filling materials in bone surgery. Besides, calcium carbonate (CaCO{sub 3}) is also used for their excellent biocompatibility and bioactivity. However, the problem with the animal-origin aragonite demands the new technique to synthesize pure calcite capable of forming 3D bone implant. This study aims to manufacture and evaluate a highly-pure synthetic crystalline calcite with good cytocompatibility regarding to the osteoblasts, comparing to that of HA and {beta}-TCP. After the manufacture of macroporous bioceramic scaffolds with the identical internal architecture, their cytocompatibility is studied through MC3T3-E1 osteoblasts with the tests of cell viability, proliferation, vitality, etc. The results confirmed that the studied process is able to form a macroporous material with a controlled internal architecture, and this synthesized calcite is non-cytotoxic and facilitate the cell proliferation. Indeed requiring further improvement, the studied calcite is definitely an interesting alternative not only to coralline aragonite but also to calcium phosphate ceramics, particularly in bone sites with the large bone remodelling. Highlights: Black-Right-Pointing-Pointer Macroporous calcite manufacturing with controlled architecture as bone substitute Black-Right-Pointing-Pointer Cytotoxicity: adaptation of the colony-forming method with the target cells: MC3T3-E1 osteoblasts Black-Right-Pointing-Pointer Study of osteoblast proliferation and activity on calcite, HA and TCP.

  7. [Research on effect of Sargentodoxae caulis on activity of osteoclasts and proliferation differentiation of osteoblasts].

    Science.gov (United States)

    Chen, Li-zhen; Zhou, Ying; Huang, Jun-fei; Zhang, Xue; Feng, Ting-ting

    2015-11-01

    Through morphological observation, HE staining, TRAP staining and toluidine blue staining of bone resorption pits to identify osteoclasts which obtained by 1α, 25-(OH)2 VitD3 inducing rabbit bone marrow cells. Three indicators-TRAP staining, TRAP enzyme activity detecting and the number and area of bone resorption pits were adapted to detect the effect of Sargentodoxae caulis on the activity of osteoclasts. Culturing MC3T3-E1 Subclong 14 cells and detecting the effect of S. caulis on differentiation and proliferation of them by MTT and detecting the alkaline phosphatase in cells. The results show that all of the low, middle and high doses of water and alcohol extracts of S. caulis have significant inhibition on osteoclast differentiation and bone resorption ability in a dose-dependent manner. The low and middle doses of water and alcohol extracts of S. caulis can stimulate differentiation and proliferation of MC3T3-ElSubclone 14 cells, which indicates S. caulis can prevent osteoporosis and the function could be achieved by inhibiting osteoclast activity and promoting the proliferation and differentiation of osteoblasts. PMID:27097425

  8. Similar healthy osteoclast and osteoblast activity on nanocrystalline hydroxyapatite and nanoparticles of tri-calcium phosphate compared to natural bone

    Directory of Open Access Journals (Sweden)

    MacMillan AK

    2014-12-01

    Full Text Available Adam K MacMillan,1 Francis V Lamberti,1 Julia N Moulton,2 Benjamin M Geilich,2 Thomas J Webster2,3 1RTI Surgical, Alachua, FL, USA; 2Department of Chemical Engineering, Northeastern University, Boston, MA, USA; 3Center of Excellence for Advanced Materials Research, King Abdulaziz University, Jeddah, Saudi Arabia Abstract: While there have been numerous studies to determine osteoblast (bone forming cell functions on nanocrystalline compared to micron crystalline ceramics, there have been few studies which have examined osteoclast activity (including tartrate-resistant acid phosphatase, formation of resorption pits, size of resorption pits, and receptor activator of nuclear factor κB [RANK]. This is despite the fact that osteoclasts are an important part of maintaining healthy bone since they resorb bone during the bone remodeling process. Moreover, while it is now well documented that bone formation is enhanced on nanoceramics compared to micron ceramics, some have pondered whether osteoblast functions (such as osteoprotegerin and RANK ligand [RANKL] are normal (ie, non-diseased on such materials compared to natural bone. For these reasons, the objective of the present in vitro study was to determine various functions of osteoclasts and osteoblasts on nanocrystalline and micron crystalline hydroxyapatite as well as tri-calcium phosphate materials and compare such results to cortical and cancellous bone. Results showed for the first time similar osteoclast activity (including tartrate-resistant acid phosphatase, formation of resorption pits, size of resorption pits, and RANK and osteoblast activity (osteoprotegerin and RANKL on nanocrystalline hydroxyapatite compared to natural bone, whereas osteoclast and osteoblast functions on micron crystalline versions of these ceramics were much different than natural bone. In this manner, this study provides additional evidence that nanocrystalline calcium phosphates can serve as suitable synthetic

  9. Caveolin-1 is critical in the proliferative effect of leptin on osteoblasts through the activation of Akt.

    Science.gov (United States)

    Zou, Lin; Zhang, Guichun; Liu, Lifeng; Chen, Chen; Cao, Xuecheng; Cai, Jinfang

    2016-09-01

    Osteoblasts are critical in bone remodeling and the repair of bone fractures. Leptin is involved in bone metabolism and osteoblast survival through the downstream signaling pathway, however, the exact mechanism of the effect of leptin on osteoblasts remains to be fully elucidated. In the present study, hFOB 1.19 cells were used to observe the effects of leptin on cell proliferation and apoptosis, and to investigate the underlying mechanism. The results confirmed that treatment of hFOB 1.19 cells with leptin significantly induced cell proliferation. Western blot analysis showed that the expression of caveolin‑1 and the activation of Akt in the cells treated with leptin were significantly increased, compared with the control cells. Additionally, inhibiting Akt activation eliminated the effects on cell proliferation induced by leptin. The rates of cell apoptosis and cell cycle distribution were examined using flow cytometry, which revealed a decrease in the apoptotic rate and an increase in the proportion of cells in the S phase. This indicated that leptin was capable of inducing cell proliferation by inhibiting apoptosis and stimulating cell progression to the S phase. Transfection of the cells with caveolin‑1 small interfering RNA showed that the activation of Akt induced by leptin was significantly inhibited. Furthermore, caveolin‑1 knockdown and inhibiting Akt activation eliminated the increased proliferation, increased proportion of cells in the S phase and increased anti‑apoptotic effects induced by leptin. Taken together, the data obtained in the present study demonstrated that caveolin‑1 was critical in the proliferative effect of leptin on osteoblasts via the activation of Akt. PMID:27430651

  10. Communication between osteoblasts stimulated by electromagnetic fields

    Institute of Scientific and Technical Information of China (English)

    ZHANG JianBao; ZHANG XiaoJun

    2007-01-01

    Pulsed electromagnetic field can affect the proliferation of osteoblasts, but the mechanism is obscure yet. The communication between osteoblasts, isolated from calvaria bone of newborn SD rats and stimulated with the rectangular electromagnetic field of 15 Hz and 4 mT, was studied. Our results showed that the osteoblasts radiated a kind of light after they were stimulated with the electromagnetic field and it is the light that promotes the proliferation of un-stimulated osteoblasts.

  11. The thiocarbamate disulphide drug, disulfiram induces osteopenia in rats by inhibition of osteoblast function due to suppression of acetaldehyde dehydrogenase activity.

    Science.gov (United States)

    Mittal, Monika; Khan, Kainat; Pal, Subhashis; Porwal, Konica; China, Shyamsundar Pal; Barbhuyan, Tarun K; Baghel, Khemraj S; Rawat, Tara; Sanyal, Sabyasachi; Bhadauria, Smrati; Sharma, Vishnu L; Chattopadhyay, Naibedya

    2014-05-01

    Dithiocarbamates (DTC), a sulfhydryl group containing compounds, are extensively used by humans that include metam and thiram due to their pesticide properties, and disulfiram (DSF) as an alcohol deterrent. We screened these DTC in an osteoblast viability assay. DSF exhibited the highest cytotoxicity (IC50 488nM). Loss in osteoblast viability and proliferation was due to induction of apoptosis via G1 arrest. DSF treatment to osteoblasts reduced glutathione (GSH) levels and exogenous addition of GSH prevented DSF-induced reactive oxygen species generation and osteoblast apoptosis. DSF also inhibited osteoblast differentiation in vitro and in vivo, and the effect was associated with inhibition of aldehyde dehydrogenase (ALDH) activity. Out of various ALDH isozymes, osteoblasts expressed only ALDH2 and DSF downregulated its transcript as well as activity. Alda-1, a specific activator of ALDH2, stimulated osteoblast differentiation. Subcutaneous injection of DSF over the calvarium of new born rats reduced the differentiation phenotype of calvarial osteoblasts but increased the mRNA levels of Runx-2 and osteocalcin. DSF treatment at a human-equivalent dose of 30 mg/kg p.o. to adult Sprague Dawley rats caused trabecular osteopenia and suppressed the formation of mineralized nodule by bone marrow stromal cells. Moreover, DSF diminished bone regeneration at the fracture site. In growing rats, DSF diminished growth plate height, primary and secondary spongiosa, mineralized osteoid and trabecular strength. Substantial decreased bone formation was also observed in the cortical site of these rats. We conclude that DSF has a strong osteopenia inducing effect by impairing osteoblast survival and differentiation due to the inhibition of ALDH2 function. PMID:24496638

  12. Low-level Ga-Al-As laser irradiation enhances osteoblast proliferation through activation of Hedgehog signaling pathway

    Science.gov (United States)

    Li, Qiushi; Qu, Zhou; Chen, Yingxin; Liu, Shujie; Zhou, Yanmin

    2014-12-01

    Low-level laser irradiation has been reported to promote bone formation, but the molecular mechanism is still unclear. Hedgehog signaling pathway has been reported to play an important role in promoting bone formation. The aim of the present study was to examine whether low-level Ga-Al-As laser (808 nm) irradiation could have an effect on Hedgehog signaling pathway during osteoblast proliferation in vitro. Mouse osteoblastic cell line MC3T3-E1 was cultured in vitro. The cultures after laser irradiation (3.75J/cm2) were treated with recombinant N-terminals Sonic Hedgehog (N-Shh)or Hedgehog inhibitor cyclopamine (cy). The experiment was divided into 4 group, group 1:laser irradiation, group 2: laser irradiation and N-Shh, group 3: laser irradiation and cy, group 4:control with no laser irradiation. On day 1,2 and 3,cell proliferation was determined by cell counting, Cell Counting Kit-8.On 12 h and 24 h, cell cycle was detected by flow cytometry. Proliferation activity of laser irradiation and N-Shh group was remarkably increased compared with those of laser irradiation group. Proliferation activity of laser irradiation and cy group was remarkably decreased compared with those of laser irradiation group, however proliferation activity of laser irradiation and cy group was remarkably increased compared with those of control group. These results suggest that low-level Ga-Al-As laser irradiation activate Hedgehog signaling pathway during osteoblast proliferation in vitro. Hedgehog signaling pathway is one of the signaling pathways by which low-level Ga-Al-As laser irradiation regulates osteoblast proliferation.

  13. Poly(lactic-co-glycolic Acid/Nanohydroxyapatite Scaffold Containing Chitosan Microspheres with Adrenomedullin Delivery for Modulation Activity of Osteoblasts and Vascular Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Lin Wang

    2013-01-01

    Full Text Available Adrenomedullin (ADM is a bioactive regulatory peptide that affects migration and proliferation of diverse cell types, including endothelial cells, smooth muscle cells, and osteoblast-like cells. This study investigated the effects of sustained release of ADM on the modulation activity of osteoblasts and vascular endothelial cells in vitro. Chitosan microspheres (CMs were developed for ADM delivery. Poly(lactic-co-glycolic acid and nano-hydroxyapatite were used to prepare scaffolds containing microspheres with ADM. The CMs showed rough surface morphology and high porosity, and they were well-distributed. The scaffolds exhibited relatively uniform pore sizes with interconnected pores. The addition of CMs improved the mechanical properties of the scaffolds without affecting their high porosity. In vitro degradation tests indicated that the addition of CMs increased the water absorption of the scaffolds and inhibited pH decline of phosphate-buffered saline medium. The expression levels of osteogenic-related and angiogenic-related genes were determined in MG63 cells and in human umbilical vein endothelial cells cultured on the scaffolds, respectively. The expression levels of osteogenic-related and angiogenic-related proteins were also detected by western blot analysis. Their expression levels in cells were improved on the ADM delivery scaffolds at a certain time point. The in vitro evaluation suggests that the microsphere-scaffold system is suitable as a model for bone tissue engineering.

  14. Tributyltin-binding protein type 1, a lipocalin, prevents inhibition of osteoblastic activity by tributyltin in fish scales.

    Science.gov (United States)

    Satone, Hina; Lee, Jae Man; Oba, Yumi; Kusakabe, Takahiro; Akahoshi, Eriko; Miki, Shizuho; Suzuki, Nobuo; Sasayama, Yuichi; Nassef, Mohamed; Shimasaki, Yohei; Kawabata, Shun-Ichiro; Honjo, Tsuneo; Oshima, Yuji

    2011-05-01

    Tributyltin-binding protein type 1 (TBT-bp1) is a member of the lipocalin family of proteins which bind to small hydrophobic molecules. In this study, we expressed a recombinant TBT-bp1 (rTBT-bp1, ca. 35kDa) in a baculovirus expression system and purified the protein from the hemolymph of silkworm larvae injected with recombinant baculovirus. After incubation of a mixture of rTBT-bp1 and TBT and its fractionation by means of gel filtration chromatography, TBT was detected in the elution peak of rTBT-bp1, confirming the binding potential of rTBT-bp1 for TBT. An assay of the ability of rTBT-bp1 or native TBT-bp1 (nTBT-bp1) to restore osteoblastic activity inhibited by TBT showed that co-treatment of the scales with rTBT-bp1 or nTBT-bp1 in combination with TBT restored osteoblastic activity in goldfish scales, whereas treatment with TBT alone significantly inhibited osteoblastic activity. These results suggest that TBT-bp1 as a lipocalin member might function to decrease the toxicity of TBT by binding to TBT. PMID:21396342

  15. MEK5 suppresses osteoblastic differentiation

    International Nuclear Information System (INIS)

    Extracellular signal-regulated kinase 5 (ERK5) is a member of the mitogen-activated protein kinase (MAPK) family and is activated by its upstream kinase, MAPK kinase 5 (MEK5), which is a member of the MEK family. Although the role of MEK5 has been investigated in several fields, little is known about its role in osteoblastic differentiation. In this study, we have demonstrated the role of MEK5 in osteoblastic differentiation in mouse preosteoblastic MC3T3-E1 cells and bone marrow stromal ST2 cells. We found that treatment with BIX02189, an inhibitor of MEK5, increased alkaline phosphatase (ALP) activity and the gene expression of ALP, osteocalcin (OCN) and osterix, as well as it enhanced the calcification of the extracellular matrix. Moreover, osteoblastic cell proliferation decreased at a concentration of greater than 0.5 μM. In addition, knockdown of MEK5 using siRNA induced an increase in ALP activity and in the gene expression of ALP, OCN, and osterix. In contrast, overexpression of wild-type MEK5 decreased ALP activity and attenuated osteoblastic differentiation markers including ALP, OCN and osterix, but promoted cell proliferation. In summary, our results indicated that MEK5 suppressed the osteoblastic differentiation, but promoted osteoblastic cell proliferation. These results implied that MEK5 may play a pivotal role in cell signaling to modulate the differentiation and proliferation of osteoblasts. Thus, inhibition of MEK5 signaling in osteoblasts may be of potential use in the treatment of osteoporosis. - Highlights: • MEK5 inhibitor BIX02189 suppresses proliferation of osteoblasts. • MEK5 knockdown and MEK5 inhibitor promote differentiation of osteoblasts. • MEK5 overexpression inhibits differentiation of osteoblasts

  16. MEK5 suppresses osteoblastic differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Kaneshiro, Shoichi [Department of Orthopaedic Surgery, Japan Community Health Care Organization Osaka Hospital, 4-2-78 Fukushima, Fukushima Ward, Osaka City, Osaka 553-0003 (Japan); Department of Orthopaedic Surgery, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Otsuki, Dai; Yoshida, Kiyoshi; Yoshikawa, Hideki [Department of Orthopaedic Surgery, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Higuchi, Chikahisa, E-mail: c-higuchi@umin.ac.jp [Department of Orthopaedic Surgery, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871 (Japan)

    2015-07-31

    Extracellular signal-regulated kinase 5 (ERK5) is a member of the mitogen-activated protein kinase (MAPK) family and is activated by its upstream kinase, MAPK kinase 5 (MEK5), which is a member of the MEK family. Although the role of MEK5 has been investigated in several fields, little is known about its role in osteoblastic differentiation. In this study, we have demonstrated the role of MEK5 in osteoblastic differentiation in mouse preosteoblastic MC3T3-E1 cells and bone marrow stromal ST2 cells. We found that treatment with BIX02189, an inhibitor of MEK5, increased alkaline phosphatase (ALP) activity and the gene expression of ALP, osteocalcin (OCN) and osterix, as well as it enhanced the calcification of the extracellular matrix. Moreover, osteoblastic cell proliferation decreased at a concentration of greater than 0.5 μM. In addition, knockdown of MEK5 using siRNA induced an increase in ALP activity and in the gene expression of ALP, OCN, and osterix. In contrast, overexpression of wild-type MEK5 decreased ALP activity and attenuated osteoblastic differentiation markers including ALP, OCN and osterix, but promoted cell proliferation. In summary, our results indicated that MEK5 suppressed the osteoblastic differentiation, but promoted osteoblastic cell proliferation. These results implied that MEK5 may play a pivotal role in cell signaling to modulate the differentiation and proliferation of osteoblasts. Thus, inhibition of MEK5 signaling in osteoblasts may be of potential use in the treatment of osteoporosis. - Highlights: • MEK5 inhibitor BIX02189 suppresses proliferation of osteoblasts. • MEK5 knockdown and MEK5 inhibitor promote differentiation of osteoblasts. • MEK5 overexpression inhibits differentiation of osteoblasts.

  17. Bio-functionalization of polycaprolactone infiltrated BCP scaffold with silicon and fibronectin enhances osteoblast activity in vitro

    Science.gov (United States)

    Kwak, Kyung-A.; Kim, Young-Hee; Kim, Minsung; Lee, Byong-Taek; Song, Ho-Yeon

    2013-08-01

    The surface property of a biomaterial plays a fundamental role in cell attachment, proliferation, differentiation, resorption, and biomolecular expression. In this study, the surface of a polycaprolactone-infiltrated biphasic calcium phosphate scaffold was biofunctionalized by silicon (Si) and fibronectin (FN) coating to evaluate the osteoblast-like cells activity in vitro. The surfaces of all scaffolds were characterized by scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS), whereas the presence of the functional group was determined by Fourier-transform infrared spectroscopy (FT-IR). Coomassie brilliant blue staining was applied to confirm the presence of FN on the scaffold surface. The in vitro bioactivity of the osteoblast-like cells was determined by one cell morphology and proliferation assay at 3, 7, and 14 days by SEM. Cell viability assay by MTT showed higher cell viability rate on coated scaffolds than in those coated with Si only or non-coated surfaces. The mRNA expressions of alkaline phosphatase (ALP) and osteocalcin (OC) were determined using RT-PCR and the expressions of osteopontin (OPN), type I collagen, and osteocalcin (OC) proteins were determined using Western blot. Thus the expression of genes and proteins further confirmed both early and intermediate phases of osteoblast-like cell activity which was found increased by Si-and Fn coating on PCL infiltrated BCP surfaces.

  18. Bio-functionalization of polycaprolactone infiltrated BCP scaffold with silicon and fibronectin enhances osteoblast activity in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Kwak, Kyung-A.; Kim, Young-Hee [Department of Microbiology and Immunology, School of Medicine, Soonchunhyang University, Cheonan, Chungnum 330-090 (Korea, Republic of); Kim, Minsung; Lee, Byong-Taek [Department of Biomedical Engineering and Materials, School of Medicine, Soonchunhyang University, Cheonan, Chungnum 330-090 (Korea, Republic of); Song, Ho-Yeon, E-mail: songmic@sch.ac.kr [Department of Microbiology and Immunology, School of Medicine, Soonchunhyang University, Cheonan, Chungnum 330-090 (Korea, Republic of)

    2013-08-15

    The surface property of a biomaterial plays a fundamental role in cell attachment, proliferation, differentiation, resorption, and biomolecular expression. In this study, the surface of a polycaprolactone-infiltrated biphasic calcium phosphate scaffold was biofunctionalized by silicon (Si) and fibronectin (FN) coating to evaluate the osteoblast-like cells activity in vitro. The surfaces of all scaffolds were characterized by scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS), whereas the presence of the functional group was determined by Fourier-transform infrared spectroscopy (FT-IR). Coomassie brilliant blue staining was applied to confirm the presence of FN on the scaffold surface. The in vitro bioactivity of the osteoblast-like cells was determined by one cell morphology and proliferation assay at 3, 7, and 14 days by SEM. Cell viability assay by MTT showed higher cell viability rate on coated scaffolds than in those coated with Si only or non-coated surfaces. The mRNA expressions of alkaline phosphatase (ALP) and osteocalcin (OC) were determined using RT-PCR and the expressions of osteopontin (OPN), type I collagen, and osteocalcin (OC) proteins were determined using Western blot. Thus the expression of genes and proteins further confirmed both early and intermediate phases of osteoblast-like cell activity which was found increased by Si-and Fn coating on PCL infiltrated BCP surfaces.

  19. Proteinase-activated receptor-2 is required for normal osteoblast and osteoclast differentiation during skeletal growth and repair.

    Science.gov (United States)

    Georgy, S R; Pagel, C N; Ghasem-Zadeh, A; Zebaze, R M D; Pike, R N; Sims, N A; Mackie, E J

    2012-03-01

    Proteinase-activated receptor-2 (PAR(2)) is a G-protein coupled receptor expressed by osteoblasts and monocytes. PAR(2) is activated by a number of proteinases including coagulation factors and proteinases released by inflammatory cells. The aim of the current study was to investigate the role of PAR(2) in skeletal growth and repair using wild type (WT) and PAR(2) knockout (KO) mice. Micro computed tomography and histomorphometry were used to examine the structure of tibias isolated from uninjured mice at 50 and 90 days of age, and from 98-day-old mice in a bone repair model in which a hole had been drilled through the tibias. Bone marrow was cultured and investigated for the presence of osteoblast precursors (alkaline phosphatase-positive fibroblastic colonies), and osteoclasts were counted in cultures treated with M-CSF and RANKL. Polymerase chain reaction (PCR) was used to determine which proteinases that activate PAR(2) are expressed in bone marrow. Regulation of PAR(2) expression in primary calvarial osteoblasts from WT mice was investigated by quantitative PCR. Cortical and trabecular bone volumes were significantly greater in the tibias of PAR(2) KO mice than in those of WT mice at 50 days of age. In trabecular bone, osteoclast surface, osteoblast surface and osteoid volume were significantly lower in KO than in WT mice. Bone marrow cultures from KO mice showed significantly fewer alkaline phosphatase-positive colony-forming units and osteoclasts compared to cultures from WT mice. Significantly less new bone and significantly fewer osteoclasts were observed in the drill sites of PAR(2) KO mice compared to WT mice 7 days post-surgery. A number of activators of PAR(2), including matriptase and kallikrein 4, were found to be expressed by normal bone marrow. Parathyroid hormone, 1,25 dihydroxyvitamin D(3), or interleukin-6 in combination with its soluble receptor down-regulated PAR(2) mRNA expression, and fibroblast growth factor-2 or thrombin stimulated PAR(2

  20. Biglycan deficiency increases osteoclast differentiation and activity due to defective osteoblasts

    DEFF Research Database (Denmark)

    Bi, Yanming; Nielsen, Karina L; Kilts, Tina M;

    2006-01-01

    effects of Bgn on 1alpha, 25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3))-induced osteoclast differentiation and bone resorption in an co-culture of calvariae-derived pre-osteoblasts and osteoclast precursors derived from spleen or bone marrow. Time course and dose response experiments showed that tartrate...... osteogenic cells. We have previously shown that the extracellular matrix protein, biglycan (Bgn), plays an important role in the differentiation of osteoblast precursors. In this paper, we showed that Bgn is involved in regulating osteoclast differentiation through its effect on osteoblasts and their...... precursors using both in vivo and in vitro experiments. The in vivo osteolysis experiment showed that LPS (lipopolisaccharide)-induced osteolysis occurred more rapidly and extensively in bgn deficient mice compared to wild type (WT) mice. To further understand the mechanism of action, we determined the...

  1. Quercetin Protects Primary Human Osteoblasts Exposed to Cigarette Smoke through Activation of the Antioxidative Enzymes HO-1 and SOD-1

    Directory of Open Access Journals (Sweden)

    Karl F. Braun

    2011-01-01

    Full Text Available Smokers frequently suffer from impaired fracture healing often due to poor bone quality and stability. Cigarette smoking harms bone cells and their homeostasis by increased formation of reactive oxygen species (ROS. The aim of this study was to investigate whether Quercetin, a naturally occurring antioxidant, can protect osteoblasts from the toxic effects of smoking. Human osteoblasts exposed to cigarette smoke medium (CSM rapidly produced ROS and their viability decreased concentration- and time-dependently. Co-, pre- and postincubation with Quercetin dose-dependently improved their viability. Quercetin increased the expression of the anti-oxidative enzymes heme-oxygenase- (HO- 1 and superoxide-dismutase- (SOD- 1. Inhibiting HO-1 activity abolished the protective effect of Quercetin. Our results demonstrate that CSM damages human osteoblasts by accumulation of ROS. Quercetin can diminish this damage by scavenging the radicals and by upregulating the expression of HO-1 and SOD-1. Thus, a dietary supplementation with Quercetin could improve bone matter, stability and even fracture healing in smokers.

  2. Endothelin‑1 induces oncostatin M expression in osteoarthritis osteoblasts by trans‑activating the oncostatin M gene promoter via Ets‑1.

    Science.gov (United States)

    Wu, Ren; Wang, Wanchun; Huang, Guoliang; Mao, Xinzhan; Chen, You; Tang, Qi; Liao, Lele

    2016-04-01

    Oncostatin M (OSM) contributes to cartilage degeneration in osteoarthritis (OA) and was demonstrated to be expressed in OA osteoblasts. Endothelin‑1 (ET‑1) is implicated in the degradation of OA articular cartilage, and osteoblast proliferation and bone development. In the present study, the effects of ET‑1 on OSM expression in human OA osteoblasts were investigated, to the best of our knowledge, for the first time. Primary human OA osteoblasts were treated with ET‑1 (1, 5, 10, 20 and 30 nM) for 0.5, 1, 2, 3 and 4 h with or without the selective ETA receptor (ETAR) antagonist, BQ123, ETB receptor antagonist, BQ788 or the phosphatidylinositol 3‑kinase (PI3K) inhibitor, BKM120. ET‑1 treatment induced OSM mRNA expression, and the intracellular and secreted protein levels of OA osteoblasts in a dose‑dependent manner. This effect was suppressed by BQ123 and BKM120, but not BQ788 administration. In combination with electrophoretic mobility shift assays, deletional and mutational analyses on the activity of a human OSM promoter/luciferase reporter demonstrated that ET‑1 induced OSM expression in OA osteoblasts by trans‑activating the OSM gene promoter through specific binding of Ets‑1 to an Ets‑1 binding site in the OSM promoter in an ETAR‑ and PI3K‑dependent manner. Furthermore, ET‑1 treatment increased the expression of Ets‑1 in a dose‑dependent manner, however the knockdown of Ets‑1 suppressed the ET1‑induced expression of OSM in OA osteoblasts. In conclusion, the present study demonstrated that ET‑1 induces the expression of OSM in OA osteoblasts by trans‑activating the OSM gene promoter primarily through increasing the expression level of Ets‑1 in an ETAR‑ and PI3K‑dependent manner. The current study suggested novel insights into the mechanistic role of ET‑1 in the pathophysiology of OA. PMID:26934912

  3. DHEA promotes osteoblast differentiation by regulating the expression of osteoblast-related genes and Foxp3(+) regulatory T cells.

    Science.gov (United States)

    Qiu, Xuemin; Gui, Yuyan; Xu, Yingping; Li, Dajin; Wang, Ling

    2015-10-01

    Several studies have reported that dehydroepiandrosterone (DHEA) promotes osteoblast proliferation and inhibits osteoblast apoptosis and that DHEA inhibits osteoclast maturation. However, whether DHEA regulates osteoblast differentiation remains unclear. The present study first examined the effect of DHEA on bone morphology in vivo. DHEA was found to increase bone volume (BV), bone mineral density (BMD), and the number of trabeculae in bone (Th.N) and it was found to decrease trabecular spacing in bone (Th.sp) in ovariectomized (OVX) mice. Next, the effect of DHEA on osteoblast differentiation was examined in vitro and osteoblastogenesis-related marker genes, such as Runx2, Osterix, Collagen1, and Osteocalcin, were also detected. DHEA increased osteoblast production in mesenchymal stem cells (MSCs) cultured in osteoblastogenic medium, and DHEA increased the expression of Runx2 and osterix, thereby increasing the expression of osteocalcin and collagen1. Immune cells and bone interact, so changes in immune cells were detected in vivo. DHEA increased the number of Foxp3(+) regulatory T cells (Tregs) in the spleen but it did not affect CTLA-4 or IL-10. When MSCs were treated with DHEA in the presence of Tregs, alkaline phosphatase (ALP) activity increased. Osteoblasts and adipocytes are both generated by MSCs. If osteoblast differentiation increases, adipocyte differentiation will decrease, and the reverse also holds true. DHEA was found to increase the number of adipocytes in osteoblastogenic medium but it had no effect on the number of adipocytes and expression of PPARγ mRNA in adipogenic medium. This finding suggests that osteoblasts may be involved in adipocyte production. In conclusion, the current results suggest that DHEA can improve postmenopausal osteoporosis (PMO) by up-regulating osteoblast differentiation via the up-regulation of the expression of osteoblastogenesis-related genes and via an increase in Foxp3(+) Tregs. PMID:26559023

  4. DNA–PKcs–SIN1 complexation mediates low-dose X-ray irradiation (LDI)-induced Akt activation and osteoblast differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Yong; Fang, Shi-ji [The Department of Orthopedics, The Second Affiliated Hospital of Soochow University, Suzhou 215000 (China); Zhu, Li-juan [Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and Institute of Neuroscience, Soochow University, Suzhou, Jiangsu 215021 (China); Zhu, Lun-qing, E-mail: xiaodongwangsz@163.com [The Center of Diagnosis and Treatment for Children’s Bone Diseases, The Children’s Hospital Affiliated to Soochow University, Suzhou, Jiangsu 215000 (China); Zhou, Xiao-zhong, E-mail: zhouxz@suda.edu.cn [The Department of Orthopedics, The Second Affiliated Hospital of Soochow University, Suzhou 215000 (China)

    2014-10-24

    Highlights: • LDI increases ALP activity, promotes type I collagen (Col I)/Runx2 mRNA expression. • LDI induces DNA–PKcs activation, which is required for osteoblast differentiation. • Akt activation mediates LDI-induced ALP activity and Col I/Runx2 mRNA increase. • DNA–PKcs–SIN1 complexation mediates LDI-induced Akt Ser-473 phosphorylation. • DNA–PKcs–SIN1 complexation is important for osteoblast differentiation. - Abstract: Low-dose irradiation (LDI) induces osteoblast differentiation, however the underlying mechanisms are not fully understood. In this study, we explored the potential role of DNA-dependent protein kinase catalytic subunit (DNA–PKcs)–Akt signaling in LDI-induced osteoblast differentiation. We confirmed that LDI promoted mouse calvarial osteoblast differentiation, which was detected by increased alkaline phosphatase (ALP) activity as well as mRNA expression of type I collagen (Col I) and runt-related transcription factor 2 (Runx2). In mouse osteoblasts, LDI (1 Gy) induced phosphorylation of DNA–PKcs and Akt (mainly at Ser-473). The kinase inhibitors against DNA–PKcs (NU-7026 and NU-7441) or Akt (LY294002, perifosine and MK-2206), as well as partial depletion of DNA–PKcs or Akt1 by targeted-shRNA, dramatically inhibited LDI-induced Akt activation and mouse osteoblast differentiation. Further, siRNA-knockdown of SIN1, a key component of mTOR complex 2 (mTORC2), also inhibited LDI-induced Akt Ser-473 phosphorylation as well as ALP activity increase and Col I/Runx2 expression in mouse osteoblasts. Co-immunoprecipitation (Co-IP) assay results demonstrated that LDI-induced DNA–PKcs–SIN1 complexation, which was inhibited by NU-7441 or SIN1 siRNA-knockdown in mouse osteoblasts. In summary, our data suggest that DNA–PKcs–SIN1 complexation-mediated Akt activation (Ser-473 phosphorylation) is required for mouse osteoblast differentiation.

  5. Effects of activated and nonactivated platelet-rich plasma on proliferation of human osteoblasts in vitro

    Czech Academy of Sciences Publication Activity Database

    Slapnička, J.; Fassmann, A.; Strašák, Luděk; Augustin, P.; Vaněk, J.

    2008-01-01

    Roč. 66, č. 2 (2008), s. 297-301. ISSN 0278-2391 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : platelet - rich plasma * osteoblast * proliferation Subject RIV: BO - Biophysics Impact factor: 1.241, year: 2008

  6. Dual reporter transgene driven by 2.3Col1a1 promoter is active in differentiated osteoblasts

    Science.gov (United States)

    Marijanovic, Inga; Jiang, Xi; Kronenberg, Mark S.; Stover, Mary Louise; Erceg, Ivana; Lichtler, Alexander C.; Rowe, David W.

    2003-01-01

    AIM: As quantitative and spatial analyses of promoter reporter constructs are not easily performed in intact bone, we designed a reporter gene specific to bone, which could be analyzed both visually and quantitatively by using chloramphenicol acetyltransferase (CAT) and a cyan version of green fluorescent protein (GFPcyan), driven by a 2.3-kb fragment of the rat collagen promoter (Col2.3). METHODS: The construct Col2.3CATiresGFPcyan was used for generating transgenic mice. Quantitative measurement of promoter activity was performed by CAT analysis of different tissues derived from transgenic animals; localization was performed by visualized GFP in frozen bone sections. To assess transgene expression during in vitro differentiation, marrow stromal cell and neonatal calvarial osteoblast cultures were analyzed for CAT and GFP activity. RESULTS: In mice, CAT activity was detected in the calvaria, long bone, teeth, and tendon, whereas histology showed that GFP expression was limited to osteoblasts and osteocytes. In cell culture, increased activity of CAT correlated with increased differentiation, and GFP activity was restricted to mineralized nodules. CONCLUSION: The concept of a dual reporter allows a simultaneous visual and quantitative analysis of transgene activity in bone.

  7. Adiponectin and AMP kinase activator stimulate proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells

    Directory of Open Access Journals (Sweden)

    Yamauchi Mika

    2007-11-01

    Full Text Available Abstract Background Adiponectin is a key mediator of the metabolic syndrome that is caused by visceral fat accumulation. Adiponectin and its receptors are known to be expressed in osteoblasts, but their actions with regard to bone metabolism are still unclear. In this study, we investigated the effects of adiponectin on the proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells. Results Adiponectin receptor type 1 (AdipoR1 mRNA was detected in the cells by RT-PCR. The adenosine monophosphate-activated protein kinase (AMP kinase was phosphorylated by both adiponectin and a pharmacological AMP kinase activator, 5-amino-imidazole-4-carboxamide-riboside (AICAR, in the cells. AdipoR1 small interfering RNA (siRNA transfection potently knocked down the receptor mRNA, and the effect of this knockdown persisted for as long as 10 days after the transfection. The transfected cells showed decreased expressions of type I collagen and osteocalcin mRNA, as determined by real-time PCR, and reduced ALP activity and mineralization, as determined by von Kossa and Alizarin red stainings. In contrast, AMP kinase activation by AICAR (0.01–0.5 mM in wild-type MC3T3-E1 cells augmented their proliferation, differentiation, and mineralization. BrdU assay showed that the addition of adiponectin (0.01–1.0 μg/ml also promoted their proliferation. Osterix, but not Runx-2, appeared to be involved in these processes because AdipoR1 siRNA transfection and AICAR treatments suppressed and enhanced osterix mRNA expression, respectively. Conclusion Taken together, this study suggests that adiponectin stimulates the proliferation, differentiation, and mineralization of osteoblasts via the AdipoR1 and AMP kinase signaling pathways in autocrine and/or paracrine fashions.

  8. Mechanisms regulating osteoblast response to surface microtopography and vitamin D

    Science.gov (United States)

    Bell, Bryan Frederick, Jr.

    (OH) 2D3. Silencing of the beta1 integrin in osteoblast-like MG63 cells significantly reduced osteogenic response to surface topography and 1alpha,25(OH)2D3. Silencing of the alpha 5 subunit did not alter the response of MG63 cells to changing surface roughness or chemistry, although future work must confirm these results given similar cell surface alpha5 integrin expression observed in control and alpha5-silenced cells. Multifunctional RGD, KRSR, and KSSR coated surfaces show that RGD increased osteoblast proliferation and reduced differentiation, KRSR had no affect on osteoblast phenotype, and KSSR increased osteoblast differentiation. These results suggest that titanium surfaces can be modified to manipulate proliferation and differentiation and that RGD/KSSR functionalized surfaces could be further investigated for use as osteointegrative surfaces. The results using VDR deficient osteoblasts demonstrate that 1alpha,25(OH)2D3 acts via VDR-dependent mechanisms in cells cultured on titanium surfaces that support terminal differentiation. In caveolae deficient osteoblasts, 1alpha,25(OH)2D3 affected cell number, alkaline phosphatase activity, and TGF-beta1 levels, although levels of osteocalcin and PGE2 were not affected. These results are consistent with the hypothesis that VDR is required for the actions of 1alpha,25(OH)2D3, but that caveolae-dependent membrane 1alpha,25(OH)2D3 signaling modulates traditional VDR signaling. The exact mechanisms for this interaction remain to be shown. Overall, these results are important in better understanding the role of beta 1 integrin partners in mediating osteoblast response to implant surfaces and in understanding how integrin signaling can alter osteoblast differentiation and responsiveness to 1alpha,25(OH)2D3 via genomic and non-genomic pathways.

  9. Autocrine stimulation of osteoblast activity by Wnt5a in response to TNF-α in human mesenchymal stem cells

    International Nuclear Information System (INIS)

    Highlights: ► Ankylosing spondylitis (AS) leads to bone fusions and ankylosis. ► TNF-α stimulates osteoblasts through growth factors in AS. ► We compare the involvement of canonical vs non-canonical Wnt signaling. ► Canonical Wnt signaling is not involved in TNF-α effects in differentiating hMSCs. ► TNF-α stimulates osteoblasts through Wnt5a autocrine secretion in hMSCs. -- Abstract: Although anti-tumor necrosis factor (TNF)-α treatments efficiently block inflammation in ankylosing spondylitis (AS), they are inefficient to prevent excessive bone formation. In AS, ossification seems more prone to develop in sites where inflammation has resolved following anti-TNF therapy, suggesting that TNF-α indirectly stimulates ossification. In this context, our objectives were to determine and compare the involvement of Wnt proteins, which are potent growth factors of bone formation, in the effects of TNF-α on osteoblast function. In human mesenchymal stem cells (MSCs), TNF-α significantly increased the levels of Wnt10b and Wnt5a. Associated with this effect, TNF-α stimulated tissue-non specific alkaline phosphatase (TNAP) and mineralization. This effect was mimicked by activation of the canonical β-catenin pathway with either anti-Dkk1 antibodies, lithium chloride (LiCl) or SB216763. TNF-α reduced, and activation of β-catenin had little effect on expression of osteocalcin, a late marker of osteoblast differentiation. Surprisingly, TNF-α failed to stabilize β-catenin and Dkk1 did not inhibit TNF-α effects. In fact, Dkk1 expression was also enhanced in response to TNF-α, perhaps explaining why canonical signaling by Wnt10b was not activated by TNF-α. However, we found that Wnt5a also stimulated TNAP in MSCs cultured in osteogenic conditions, and increased the levels of inflammatory markers such as COX-2. Interestingly, treatment with anti-Wnt5a antibodies reduced endogenous TNAP expression and activity. Collectively, these data suggest that increased

  10. Autocrine stimulation of osteoblast activity by Wnt5a in response to TNF-α in human mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Briolay, A. [ICBMS, UMR CNRS 5246, University of Lyon 1, Bâtiment Raulin, 43 Bd du 11 novembre 1918, 69622 Villeurbanne Cedex (France); Lencel, P. [Physiopathology of Inflammatory Bone Diseases, EA4490, ULCO. Quai Masset, Bassin Napoléon BP120, 62327 Boulogne/Mer (France); Bessueille, L. [ICBMS, UMR CNRS 5246, University of Lyon 1, Bâtiment Raulin, 43 Bd du 11 novembre 1918, 69622 Villeurbanne Cedex (France); Caverzasio, J. [Service of Bone Diseases, Department of Internal Medicine Specialties, University Hospital of Geneva, CH-1211 Geneva 14 (Switzerland); Buchet, R. [ICBMS, UMR CNRS 5246, University of Lyon 1, Bâtiment Raulin, 43 Bd du 11 novembre 1918, 69622 Villeurbanne Cedex (France); Magne, D., E-mail: david.magne@univ-lyon1.fr [ICBMS, UMR CNRS 5246, University of Lyon 1, Bâtiment Raulin, 43 Bd du 11 novembre 1918, 69622 Villeurbanne Cedex (France)

    2013-01-18

    Highlights: ► Ankylosing spondylitis (AS) leads to bone fusions and ankylosis. ► TNF-α stimulates osteoblasts through growth factors in AS. ► We compare the involvement of canonical vs non-canonical Wnt signaling. ► Canonical Wnt signaling is not involved in TNF-α effects in differentiating hMSCs. ► TNF-α stimulates osteoblasts through Wnt5a autocrine secretion in hMSCs. -- Abstract: Although anti-tumor necrosis factor (TNF)-α treatments efficiently block inflammation in ankylosing spondylitis (AS), they are inefficient to prevent excessive bone formation. In AS, ossification seems more prone to develop in sites where inflammation has resolved following anti-TNF therapy, suggesting that TNF-α indirectly stimulates ossification. In this context, our objectives were to determine and compare the involvement of Wnt proteins, which are potent growth factors of bone formation, in the effects of TNF-α on osteoblast function. In human mesenchymal stem cells (MSCs), TNF-α significantly increased the levels of Wnt10b and Wnt5a. Associated with this effect, TNF-α stimulated tissue-non specific alkaline phosphatase (TNAP) and mineralization. This effect was mimicked by activation of the canonical β-catenin pathway with either anti-Dkk1 antibodies, lithium chloride (LiCl) or SB216763. TNF-α reduced, and activation of β-catenin had little effect on expression of osteocalcin, a late marker of osteoblast differentiation. Surprisingly, TNF-α failed to stabilize β-catenin and Dkk1 did not inhibit TNF-α effects. In fact, Dkk1 expression was also enhanced in response to TNF-α, perhaps explaining why canonical signaling by Wnt10b was not activated by TNF-α. However, we found that Wnt5a also stimulated TNAP in MSCs cultured in osteogenic conditions, and increased the levels of inflammatory markers such as COX-2. Interestingly, treatment with anti-Wnt5a antibodies reduced endogenous TNAP expression and activity. Collectively, these data suggest that increased

  11. The role of osteoblasts in peri-prosthetic osteolysis.

    LENUS (Irish Health Repository)

    O'Neill, S C

    2013-08-01

    Peri-prosthetic osteolysis and subsequent aseptic loosening is the most common reason for revising total hip replacements. Wear particles originating from the prosthetic components interact with multiple cell types in the peri-prosthetic region resulting in an inflammatory process that ultimately leads to peri-prosthetic bone loss. These cells include macrophages, osteoclasts, osteoblasts and fibroblasts. The majority of research in peri-prosthetic osteolysis has concentrated on the role played by osteoclasts and macrophages. The purpose of this review is to assess the role of the osteoblast in peri-prosthetic osteolysis. In peri-prosthetic osteolysis, wear particles may affect osteoblasts and contribute to the osteolytic process by two mechanisms. First, particles and metallic ions have been shown to inhibit the osteoblast in terms of its ability to secrete mineralised bone matrix, by reducing calcium deposition, alkaline phosphatase activity and its ability to proliferate. Secondly, particles and metallic ions have been shown to stimulate osteoblasts to produce pro inflammatory mediators in vitro. In vivo, these mediators have the potential to attract pro-inflammatory cells to the peri-prosthetic area and stimulate osteoclasts to absorb bone. Further research is needed to fully define the role of the osteoblast in peri-prosthetic osteolysis and to explore its potential role as a therapeutic target in this condition.

  12. Osteocytes subjected to pulsating fluid flow regulate osteoblast proliferation and differentiation

    International Nuclear Information System (INIS)

    Osteocytes are thought to orchestrate bone remodeling, but it is unclear exactly how osteocytes influence neighboring bone cells. Here, we tested whether osteocytes, osteoblasts, and periosteal fibroblasts subjected to pulsating fluid flow (PFF) produce soluble factors that modulate the proliferation and differentiation of cultured osteoblasts and periosteal fibroblasts. We found that osteocyte PFF conditioned medium (CM) inhibited bone cell proliferation, and osteocytes produced the strongest inhibition of proliferation compared to osteoblasts and periosteal fibroblasts. The nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) attenuated the inhibitory effects of osteocyte PFF CM, suggesting that a change in NO release is at least partially responsible for the inhibitory effects of osteocyte PFF CM. Furthermore, osteocyte PFF CM stimulated osteoblast differentiation measured as increased alkaline phosphatase activity, and L-NAME decreased the stimulatory effects of osteocyte PFF CM on osteoblast differentiation. We conclude that osteocytes subjected to PFF inhibit proliferation but stimulate differentiation of osteoblasts in vitro via soluble factors and that the release of these soluble factors was at least partially dependent on the activation of a NO pathway in osteocytes in response to PFF. Thus, the osteocyte appears to be more responsive to PFF than the osteoblast or periosteal fibroblast with respect to the production of soluble signaling molecules affecting osteoblast proliferation and differentiation

  13. Neuropeptide Y, substance P, and human bone morphogenetic protein 2 stimulate human osteoblast osteogenic activity by enhancing gap junction intercellular communication

    Energy Technology Data Exchange (ETDEWEB)

    Ma, W.H.; Liu, Y.J.; Wang, W.; Zhang, Y.Z. [The Third Hospital of Hebei Medical University, The Provincial Key Laboratory for Orthopedic Biomechanics of Hebei, Shijiazhuang, Hebei Province (China)

    2015-02-13

    Bone homeostasis seems to be controlled by delicate and subtle “cross talk” between the nervous system and “osteo-neuromediators” that control bone remodeling. The purpose of this study was to evaluate the effect of interactions between neuropeptides and human bone morphogenetic protein 2 (hBMP2) on human osteoblasts. We also investigated the effects of neuropeptides and hBMP2 on gap junction intercellular communication (GJIC). Osteoblasts were treated with neuropeptide Y (NPY), substance P (SP), or hBMP2 at three concentrations. At various intervals after treatment, cell viability was measured by the MTT assay. In addition, cellular alkaline phosphatase (ALP) activity and osteocalcin were determined by colorimetric assay and radioimmunoassay, respectively. The effects of NPY, SP and hBMP on GJIC were determined by laser scanning confocal microscopy. The viability of cells treated with neuropeptides and hBMP2 increased significantly in a time-dependent manner, but was inversely associated with the concentration of the treatments. ALP activity and osteocalcin were both reduced in osteoblasts exposed to the combination of neuropeptides and hBMP2. The GJIC of osteoblasts was significantly increased by the neuropeptides and hBMP2. These results suggest that osteoblast activity is increased by neuropeptides and hBMP2 through increased GJIC. Identification of the GJIC-mediated signal transduction capable of modulating the cellular activities of bone cells represents a novel approach to studying the biology of skeletal innervation.

  14. Neuropeptide Y, substance P, and human bone morphogenetic protein 2 stimulate human osteoblast osteogenic activity by enhancing gap junction intercellular communication

    International Nuclear Information System (INIS)

    Bone homeostasis seems to be controlled by delicate and subtle “cross talk” between the nervous system and “osteo-neuromediators” that control bone remodeling. The purpose of this study was to evaluate the effect of interactions between neuropeptides and human bone morphogenetic protein 2 (hBMP2) on human osteoblasts. We also investigated the effects of neuropeptides and hBMP2 on gap junction intercellular communication (GJIC). Osteoblasts were treated with neuropeptide Y (NPY), substance P (SP), or hBMP2 at three concentrations. At various intervals after treatment, cell viability was measured by the MTT assay. In addition, cellular alkaline phosphatase (ALP) activity and osteocalcin were determined by colorimetric assay and radioimmunoassay, respectively. The effects of NPY, SP and hBMP on GJIC were determined by laser scanning confocal microscopy. The viability of cells treated with neuropeptides and hBMP2 increased significantly in a time-dependent manner, but was inversely associated with the concentration of the treatments. ALP activity and osteocalcin were both reduced in osteoblasts exposed to the combination of neuropeptides and hBMP2. The GJIC of osteoblasts was significantly increased by the neuropeptides and hBMP2. These results suggest that osteoblast activity is increased by neuropeptides and hBMP2 through increased GJIC. Identification of the GJIC-mediated signal transduction capable of modulating the cellular activities of bone cells represents a novel approach to studying the biology of skeletal innervation

  15. UV-activated 7-dehydrocholesterol-coated titanium implants promote differentiation of human umbilical cord mesenchymal stem cells into osteoblasts.

    Science.gov (United States)

    Satué, María; Ramis, Joana M; Monjo, Marta

    2016-01-01

    Vitamin D metabolites are essential for bone regeneration and mineral homeostasis. The vitamin D precursor 7-dehydrocholesterol can be used after UV irradiation to locally produce active vitamin D by osteoblastic cells. Furthermore, UV-irradiated 7-dehydrocholesterol is a biocompatible coating for titanium implants with positive effects on osteoblast differentiation. In this study, we examined the impact of titanium implants surfaces coated with UV-irradiated 7-dehydrocholesterol on the osteogenic differentiation of human umbilical cord mesenchymal stem cells. First, the synthesis of cholecalciferol (D3) was achieved through the incubation of the UV-activated 7-dehydrocholesterol coating for 48 h at 23℃. Further, we investigated in vitro the biocompatibility of this coating in human umbilical cord mesenchymal stem cells and its potential to enhance their differentiation towards the osteogenic lineage. Human umbilical cord mesenchymal stem cells cultured onto UV-irradiated 7-dehydrocholesterol-coated titanium implants surfaces, combined with osteogenic supplements, upregulated the gene expression of several osteogenic markers and showed higher alkaline phosphatase activity and calcein blue staining, suggesting increased mineralization. Thus, our results show that the use of UV irradiation on 7-dehydrocholesterol -treated titanium implants surfaces generates a bioactive coating that promotes the osteogenic differentiation of human umbilical cord mesenchymal stem cells, with regenerative potential for improving osseointegration in titanium-based bone anchored implants. PMID:25899927

  16. FHL2 mediates dexamethasone-induced mesenchymal cell differentiation into osteoblasts by activating Wnt/beta-catenin signaling-dependent Runx2 expression.

    Science.gov (United States)

    Hamidouche, Zahia; Haÿ, Eric; Vaudin, Pascal; Charbord, Pierre; Schüle, Roland; Marie, Pierre J; Fromigué, Olivia

    2008-11-01

    The differentiation of bone marrow mesenchymal stem cells (MSCs) into osteoblasts is a crucial step in bone formation. However, the mechanisms involved in the early stages of osteogenic differentiation are not well understood. In this study, we identified FHL2, a member of the LIM-only subclass of the LIM protein superfamily, that is up-regulated during early osteoblast differentiation induced by dexamethasone in murine and human MSCs. Gain-of-function studies showed that FHL2 promotes the expression of the osteoblast transcription factor Runx2, alkaline phosphatase, type I collagen, as well as in vitro extracellular matrix mineralization in murine and human mesenchymal cells. Knocking down FHL2 using sh-RNA reduces basal and dexamethasone-induced osteoblast marker gene expression in MSCs. We demonstrate that FHL2 interacts with beta-catenin, a key player involved in bone formation induced by Wnt signaling. FHL2-beta-catenin interaction potentiates beta-catenin nuclear translocation and TCF/LEF transcription, resulting in increased Runx2 and alkaline phosphatase expression, which was inhibited by the Wnt inhibitor DKK1. Reduction of Runx2 transcriptional activity using a mutant Runx2 results in inhibition of FHL2-induced alkaline phosphatase expression in MSCs. These findings reveal that FHL2 acts as an endogenous activator of mesenchymal cell differentiation into osteoblasts and mediates osteogenic differentiation induced by dexamethasone in MSCs through activation of Wnt/beta-catenin signaling- dependent Runx2 expression. PMID:18653765

  17. Effect of water soluble extract of nacre (Pinctada maxima) on alkaline phosphatase activity and Bcl-2 expression in primary cultured osteoblasts from neonatal rat calvaria.

    Science.gov (United States)

    Moutahir-Belqasmi, F; Balmain, N; Lieberrher, M; Borzeix, S; Berland, S; Barthelemy, M; Peduzzi, J; Milet, C; Lopez, E

    2001-01-01

    The nacre (mother of pearl) layer of the oyster Pinctada maxima shell can initiate bone formation by human osteoblasts in vivo and in vitro and is a new biomaterial that induces osteogenesis. This activity of nacre could be due to its water-soluble matrix. We examined the action of a water-soluble extract of nacre on the osteoblast phenotype of cells isolated from rat neonatal calvaria by measuring alkaline phosphatase (ALP) activity and by localization of the anti-apoptotic protein Bcl-2 by immunocytochemistry. ALP activity was increased 7% (pproduction in osteoblasts, that is correlated with the cell cycle. Bcl-2 was also abundant in the nucleoli of extract-treated cells. Thus, the concentration and subcellular distribution of Bcl-2 in osteoblasts in primary cultures is influenced by nacre extract, and related to the cell cycle and the regulation of gene expression. Hence, knowledge of how water-soluble extracts of Pinctada maxima nacre act on osteoblasts in vitro may reveal the mechanisms involved in its action in vivo on bone cells and bone regeneration. PMID:15348370

  18. Activation of β–catenin Signaling in MLO-Y4 Osteocytic Cells versus 2T3 Osteoblastic Cells by Fluid Flow Shear Stress and PGE2: Implications for the Study of Mechanosensation in Bone

    OpenAIRE

    Kamel, Mohamed A; Picconi, Jason L; Lara-Castillo, Nuria; Johnson, Mark L.

    2010-01-01

    The osteocyte is hypothesized to be the mechanosensory cell in bone. However, osteoblastic cell models have been most commonly used to investigate mechanisms of mechanosensation in bone. Therefore, we sought to determine if differences might exist between osteocytic and osteoblastic cell models relative to the activation of β-catenin signaling in MLO-Y4 osteocytic, 2T3 osteoblastic and primary neonatal calvarial cells (NCCs) in response to pulsatile fluid flow shear stress (PFFSS). β–catenin ...

  19. Cadmium-induced apoptosis and necrosis in human osteoblasts: role of caspases and mitogen-activated protein kinases pathways.

    Science.gov (United States)

    Brama, M; Politi, L; Santini, P; Migliaccio, S; Scandurra, R

    2012-02-01

    Cadmium is a widespread environmental pollutant which induces severe toxic alterations, including osteomalacia and osteoporosis, likely by estrogen receptor-dependent mechanisms. Indeed, cadmium has been described to act as an endocrine disruptor and its toxicity is exerted both in vivo and in vitro through induction of apoptosis and/or necrosis by not fully clarified intracellular mechanism(s) of action. Aim of the present study was to further investigate the molecular mechanism by which cadmium might alter homeostasis of estrogen target cells, such as osteoblast homeostasis, inducing cell apoptosis and/or necrosis. Human osteoblastic cells (hFOB 1.19) in culture were used as an in vitro model to characterize the intracellular mechanisms induced by this heavy metal. Cells were incubated in the presence/ absence of 10-50 μM cadmium chloride at different times and DNA fragmentation and activation of procaspases- 8 and -3 were induced upon CdCl(2) treatment triggering apoptotic and necrotic pathways. Addition of caspase-8 and -3 inhibitors (Z-IETD-FMK and Z-DQMD-FMK) partially blocked these effects. No activation of procaspase-9 was observed. To determine the role of mitogen-activated protein kinases (MAPK) in these events, we investigated c-jun N-terminal kinase (JNK), p38 and extracellular signal-regulated protein kinase (ERK1/2) phosphorylation which were activated by 10 μM CdCl(2). Chemical inhibitors of JNK, p38, and ERK1/2, SP600125, SB202190, and PD98059, significantly reduced the phosphorylation of the kinases and blunted apoptosis. In contrast, caspase inhibitors did not reduce the cadmium-induced MAPK phosphorylation, suggesting an independent activation of these pathways. In conclusion, at least 2 pathways appear activated by cadmium in osteoblasts: a direct induction of caspase-8 followed by activation of caspase-3 and an indirect induction by phosphorylation of ERK1/2, p38, and JNK MAPK triggering activation of caspase-8 and -3. PMID:21697648

  20. Osteoblasts in Bone Physiology—Mini Review

    Directory of Open Access Journals (Sweden)

    Orit Rosenberg

    2012-04-01

    Full Text Available Bone structural integrity and shape are maintained by removal of old matrix by osteoclasts and in-situ synthesis of new bone by osteoblasts. These cells comprise the basic multicellular unit (BMU. Bone mass maintenance is determined by the net anabolic activity of the BMU, when the matrix elaboration of the osteoblasts equals or exceeds the bone resorption by the osteoclasts. The normal function of the BMU causes a continuous remodeling process of the bone, with deposition of bony matrix (osteoid along the vectors of the generated force by gravity and attached muscle activity. The osteoblasts are derived from mesenchymal stem cells (MSCs. Circulating hormones and locally produced cytokines and growth factors modulate the replication and differentiation of osteoclast and osteoblast progenitors. The appropriate number of the osteoblasts in the BMU is determined by the differentiation of the precursor bone-marrow stem cells into mature osteoblasts, their proliferation with subsequent maturation into metabolically active osteocytes, and osteoblast degradation by apoptosis. Thus, the two crucial points to target when planning to control the osteoblast population are the processes of cell proliferation and apoptosis, which are regulated by cellular hedgehog and Wnt pathways that involve humoral and mechanical stimulations. Osteoblasts regulate both bone matrix synthesis and mineralization directly by their own synthetic activities, and bone resorption indirectly by its paracrinic effects on osteoclasts. The overall synthetic and regulatory activities of osteoblasts govern bone tissue integrity and shape.

  1. Saponins from the roots of Platycodon grandiflorum stimulate osteoblast differentiation via p38 MAPK- and ERK-dependent RUNX2 activation.

    Science.gov (United States)

    Jeong, Hyung Min; Han, Eun Hee; Jin, Yun Hey; Hwang, Yong Pil; Kim, Hyung Gyun; Park, Bong Hwan; Kim, Jin Young; Chung, Young Chul; Lee, Kwang Youl; Jeong, Hye Gwang

    2010-12-01

    Changkil (CK), the aqueous extract of the roots of Platycodon grandiflorum, has been used as a traditional oriental medicine for the treatment of chronic adult diseases. Although a saponin fraction derived from CK (CKS) has been suggested to have a variety of functional effects, its effect on bone is unknown. In the present study, the effects of CKS on osteoblast differentiation and function were determined by analyzing the activity of alkaline phosphatase (ALP), an osteoblast marker, and the regulation of RUNX2, a master gene of osteoblast differentiation, in a mesenchymal stem cell line. CKS upregulated ALP activity and the expression of osteogenic marker genes in C2C12 cells. In addition, CKS increased the expression and transcriptional activity of RUNX2. To determine which signaling pathways are involved in the osteogenic effects of CKS, we tested the effect of inhibitors of kinases known to regulate RUNX2. CKS-induced enhancement of RUNX2 and ALP was inhibited by treatment with a p38 inhibitor (SB203580) and an ERK inhibitor (U0126). These findings suggest that CKS stimulates osteoblast differentiation by activation of RUNX2 via mechanisms related to the p38 MAPK and ERK signaling pathways. The regulation of RUNX2 activation by CKS may be an important therapeutic target for osteoporosis. PMID:20828597

  2. IL-6 alters osteocyte signaling toward osteoblasts but not osteoclasts.

    Science.gov (United States)

    Bakker, A D; Kulkarni, R N; Klein-Nulend, J; Lems, W F

    2014-04-01

    Mechanosensitive osteocytes regulate bone mass in adults. Interleukin 6 (IL-6), such as present during orthodontic tooth movement, also strongly affects bone mass, but little is known about the effect of IL-6 on osteocyte function. Therefore we aimed to determine in vitro whether IL-6 affects osteocyte mechanosensitivity, and osteocyte regulation of osteoclastogenesis and osteoblast differentiation. MLO-Y4 osteocytes were incubated with/without IL-6 (1 or 10 pg/mL) for 24 hr. Subsequently, osteocytes were subjected to mechanical loading by pulsating fluid flow (PFF) for 1 hr. Mouse osteoclast precursors were cultured for 7 days on top of IL-6-treated osteocytes. Conditioned medium from osteocytes treated with/without IL-6 was added to MC3T3-E1 pre-osteoblasts for 14 days. Exogenous IL-6 (10 pg/mL) did not alter the osteocyte response to PFF. PFF significantly enhanced IL-6 production by osteocytes. IL-6 enhanced Rankl expression but reduced caspase 3/7 activity by osteocytes, and therefore did not affect osteocyte-stimulated osteoclastogenesis. Conditioned medium from IL-6-treated osteocytes reduced alkaline phosphatase (ALP) activity and Runx2 expression in osteoblasts, but increased expression of the proliferation marker Ki67 and osteocalcin. Our results suggest that IL-6 is produced by shear-loaded osteocytes and that IL-6 may affect bone mass by modulating osteocyte communication toward osteoblasts. PMID:24492932

  3. Peroxisomes in Different Skeletal Cell Types during Intramembranous and Endochondral Ossification and Their Regulation during Osteoblast Differentiation by Distinct Peroxisome Proliferator-Activated Receptors.

    Directory of Open Access Journals (Sweden)

    Guofeng Qian

    Full Text Available Ossification defects leading to craniofacial dysmorphism or rhizomelia are typical phenotypes in patients and corresponding knockout mouse models with distinct peroxisomal disorders. Despite these obvious skeletal pathologies, to date no careful analysis exists on the distribution and function of peroxisomes in skeletal tissues and their alterations during ossification. Therefore, we analyzed the peroxisomal compartment in different cell types of mouse cartilage and bone as well as in primary cultures of calvarial osteoblasts. The peroxisome number and metabolism strongly increased in chondrocytes during endochondral ossification from the reserve to the hypertrophic zone, whereas in bone, metabolically active osteoblasts contained a higher numerical abundance of this organelle than osteocytes. The high abundance of peroxisomes in these skeletal cell types is reflected by high levels of Pex11β gene expression. During culture, calvarial pre-osteoblasts differentiated into secretory osteoblasts accompanied by peroxisome proliferation and increased levels of peroxisomal genes and proteins. Since many peroxisomal genes contain a PPAR-responsive element, we analyzed the gene expression of PPARɑ/ß/ɣ in calvarial osteoblasts and MC3T3-E1 cells, revealing higher levels for PPARß than for PPARɑ and PPARɣ. Treatment with different PPAR agonists and antagonists not only changed the peroxisomal compartment and associated gene expression, but also induced complex alterations of the gene expression patterns of the other PPAR family members. Studies in M3CT3-E1 cells showed that the PPARß agonist GW0742 activated the PPRE-mediated luciferase expression and up-regulated peroxisomal gene transcription (Pex11, Pex13, Pex14, Acox1 and Cat, whereas the PPARß antagonist GSK0660 led to repression of the PPRE and a decrease of the corresponding mRNA levels. In the same way, treatment of calvarial osteoblasts with GW0742 increased in peroxisome number and

  4. Harmine promotes osteoblast differentiation through bone morphogenetic protein signaling

    International Nuclear Information System (INIS)

    Highlights: → Harmine promotes the activity and mRNA expression of ALP. → Harmine enhances the expressions of osteocalcin mRNA and protein. → Harmine induces osteoblastic mineralization. → Harmine upregulates the mRNA expressions of BMPs, Runx2 and Osterix. → BMP signaling pathways are involved in the actions of harmine. -- Abstract: Bone mass is regulated by osteoblast-mediated bone formation and osteoclast-mediated bone resorption. We previously reported that harmine, a β-carboline alkaloid, inhibits osteoclast differentiation and bone resorption in vitro and in vivo. In this study, we investigated the effects of harmine on osteoblast proliferation, differentiation and mineralization. Harmine promoted alkaline phosphatase (ALP) activity in MC3T3-E1 cells without affecting their proliferation. Harmine also increased the mRNA expressions of the osteoblast marker genes ALP and Osteocalcin. Furthermore, the mineralization of MC3T3-E1 cells was enhanced by treatment with harmine. Harmine also induced osteoblast differentiation in primary calvarial osteoblasts and mesenchymal stem cell line C3H10T1/2 cells. Structure-activity relationship studies using harmine-related β-carboline alkaloids revealed that the C3-C4 double bond and 7-hydroxy or 7-methoxy group of harmine were important for its osteogenic activity. The bone morphogenetic protein (BMP) antagonist noggin and its receptor kinase inhibitors dorsomorphin and LDN-193189 attenuated harmine-promoted ALP activity. In addition, harmine increased the mRNA expressions of Bmp-2, Bmp-4, Bmp-6, Bmp-7 and its target gene Id1. Harmine also enhanced the mRNA expressions of Runx2 and Osterix, which are key transcription factors in osteoblast differentiation. Furthermore, BMP-responsive and Runx2-responsive reporters were activated by harmine treatment. Taken together, these results indicate that harmine enhances osteoblast differentiation probably by inducing the expressions of BMPs and activating BMP and Runx2

  5. Icariin Stimulates Differentiation and Suppresses Adipocytic Transdifferentiation of Primary Osteoblasts Through Estrogen Receptor-Mediated Pathway.

    Science.gov (United States)

    Zhang, Dawei; Fong, Chichun; Jia, Zhenbin; Cui, Liao; Yao, Xinsheng; Yang, Mengsu

    2016-08-01

    Icariin, the main constituent of Herba Epimedii, appears to be a promising alternative to classic drugs used to treat osteoporosis. However, the detailed molecular mechanisms of its action and the role of icariin in the cross-talk between osteoblasts and adipocytes remain unclear. The present study was designed to investigate the gene expression profile of primary osteoblasts in the presence of icariin, and the effects of icariin on the differentiation and adipogenic transdifferentiation of osteoblasts. Cellular and molecular markers expressed during osteoblastic differentiation were assessed by cytochemical analysis, real-time quantitative PCR, Western blotting, and cDNA microarray analysis. Results indicated that icariin up-regulated the expression of runt-related transcription factor 2 (Runx2), bone morphogenetic protein 2 (Bmp2), and collagen type 1 (Col1) genes, and down-regulated the expression of the peroxisome proliferator-activated receptor γ (Pparg) and CCAAT/enhancer-binding protein β (Cebpb) genes. These effects were blocked by ICI 182,780, suggesting that icariin may be acting via the estrogen receptor (ER). Results also demonstrated that the ratio of osteoprotegerin (Opg)/receptor activator of nuclear factor kappa B ligand (Rankl) expression was up-regulated following treatment with icariin. In total, osteoblastic gene expression profile analysis suggested that 33 genes were affected by icariin; these could be sub-divided into nine functional categories. It appears that icariin could stimulate the differentiation and mineralization of osteoblasts, regulate the differentiation of osteoclasts, and inhibit the adipogenic transdifferentiation of osteoblasts, therefore increasing the number of osteoblasts undergoing differentiation to mature osteoblasts, via an ER-mediated pathway. In summary, icariin may exhibit beneficial effects on bone health, especially for patients with osteoporosis and obesity. PMID:27061090

  6. Evaluation of antibacterial activity and osteoblast-like cell viability of TiN, ZrN and (Ti1-xZrx)N coating on titanium

    OpenAIRE

    Ji, Min-Kyung; Park, Sang-Won; Lee, Kwangmin; Kang, In-Chol; Yun, Kwi-Dug; Kim, Hyun-Seung; Lim, Hyun-Pil

    2015-01-01

    PURPOSE The aim of this study was to evaluate antibacterial activity and osteoblast-like cell viability according to the ratio of titanium nitride and zirconium nitride coating on commercially pure titanium using an arc ion plating system. MATERIALS AND METHODS Polished titanium surfaces were used as controls. Surface topography was observed by scanning electron microscopy, and surface roughness was measured using a two-dimensional contact stylus profilometer. Antibacterial activity was evalu...

  7. Small oscillatory accelerations, independent of matrix deformations, increase osteoblast activity and enhance bone morphology.

    Directory of Open Access Journals (Sweden)

    Russell Garman

    Full Text Available A range of tissues have the capacity to adapt to mechanical challenges, an attribute presumed to be regulated through deformation of the cell and/or surrounding matrix. In contrast, it is shown here that extremely small oscillatory accelerations, applied as unconstrained motion and inducing negligible deformation, serve as an anabolic stimulus to osteoblasts in vivo. Habitual background loading was removed from the tibiae of 18 female adult mice by hindlimb-unloading. For 20 min/d, 5 d/wk, the left tibia of each mouse was subjected to oscillatory 0.6 g accelerations at 45 Hz while the right tibia served as control. Sham-loaded (n = 9 and normal age-matched control (n = 18 mice provided additional comparisons. Oscillatory accelerations, applied in the absence of weight bearing, resulted in 70% greater bone formation rates in the trabeculae of the metaphysis, but similar levels of bone resorption, when compared to contralateral controls. Quantity and quality of trabecular bone also improved as a result of the acceleration stimulus, as evidenced by a significantly greater bone volume fraction (17% and connectivity density (33%, and significantly smaller trabecular spacing (-6% and structural model index (-11%. These in vivo data indicate that mechanosensory elements of resident bone cell populations can perceive and respond to acceleratory signals, and point to an efficient means of introducing intense physical signals into a biologic system without putting the matrix at risk of overloading. In retrospect, acceleration, as opposed to direct mechanical distortion, represents a more generic and safe, and perhaps more fundamental means of transducing physical challenges to the cells and tissues of an organism.

  8. Novel targets of vitamin D activity in bone: action of the vitamin D receptor in osteoblasts, osteocytes and osteoclasts.

    Science.gov (United States)

    Ryan, Jackson W; Reinke, Daniel; Kogawa, Masakazu; Turner, Andrew G; Atkins, Gerald J; Anderson, Paul H; Morris, Howard A

    2013-12-01

    The active form of vitamin D, 1,25-dihydroxyvitamin D3, carries out its diverse range of biological activities by binding to the nuclear vitamin D receptor, present in almost every cell of the body. It is well established that adequate serum 25-hydroxyvitamin D levels correlate with a reduction in the incidence of osteoporosis; however, the physiological basis for this relationship remains elusive. Although, the endocrine actions of vitamin D are thoroughly appreciated, the effect of vitamin D on bone tissue and bone cells is yet to be completely understood. There exists a wealth of literature that suggests the VDR within the three major bone cell types, osteoblasts, osteocytes and osteoclasts, is responsible for the regulation of bone homeostasis. The circumstances, under which the action of 1,25-dihydroxyvitamin D3 elicits an anabolic or catabolic role have not been elucidated. However, it would seem that vitamin D can evoke both of these effects and that this is partly mediated by calcium homeostasis. This raises the possibility that dietary calcium intake and vitamin D metabolism act concomitantly at the kidney, intestine and the bone in a coordinated response. Thus, to maintain adequate bone homeostasis and reduce the risk of metabolic bone disease via the diet, it is important to consider this duality of vitamin D action in relation to the overall calcium economy. PMID:24010964

  9. Effects of different magnitudes of mechanical strain on Osteoblasts in vitro

    International Nuclear Information System (INIS)

    In addition to systemic and local factors, mechanical strain plays a crucial role in bone remodeling during growth, development, and fracture healing, and especially in orthodontic tooth movement. Although many papers have been published on the effects of mechanical stress on osteoblasts or osteoblastic cells, little is known about the effects of different magnitudes of mechanical strain on such cells. In the present study, we investigated how different magnitudes of cyclic tensile strain affected osteoblasts. MC3T3-E1 osteoblastic cells were subjected to 0%, 6%, 12% or 18% elongation for 24 h using a Flexercell Strain Unit, and then the mRNA and protein expressions of osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) were examined. The results showed that cyclic tensile strain induced a magnitude-dependent increase (0%, 6%, 12%, and 18%) in OPG synthesis and a concomitant decrease in RANKL mRNA expression and sRANKL release from the osteoblasts. Furthermore, the induction of OPG mRNA expression by stretching was inhibited by indomethacin or genistein, and the stretch-induced reduction of RANKL mRNA was inhibited by PD098059. These results indicate that different magnitudes of cyclic tensile strain influence the biological behavior of osteoblasts, which profoundly affects bone remodeling

  10. Microgravity Induces Pelvic Bone Loss through Osteoclastic Activity, Osteocytic Osteolysis, and Osteoblastic Cell Cycle Inhibition by CDKN1a/p21

    OpenAIRE

    Blaber, Elizabeth A.; Dvorochkin, Natalya; Lee, Chialing; Alwood, Joshua S.; Yousuf, Rukhsana; Pianetta, Piero; Globus, Ruth K.; Burns, Brendan P.; Almeida, Eduardo A.C.

    2013-01-01

    Bone is a dynamically remodeled tissue that requires gravity-mediated mechanical stimulation for maintenance of mineral content and structure. Homeostasis in bone occurs through a balance in the activities and signaling of osteoclasts, osteoblasts, and osteocytes, as well as proliferation and differentiation of their stem cell progenitors. Microgravity and unloading are known to cause osteoclast-mediated bone resorption; however, we hypothesize that osteocytic osteolysis, and cell cycle arres...

  11. The shunt from the cyclooxygenase to lipoxygenase pathway in human osteoarthritic subchondral osteoblasts is linked with a variable expression of the 5-lipoxygenase-activating protein

    OpenAIRE

    Maxis, Kelitha; Delalandre, Aline; Martel-Pelletier, Johanne; Pelletier, Jean-Pierre; Duval, Nicolas; Lajeunesse, Daniel

    2006-01-01

    Osteoarthritis (OA) is characterized by articular cartilage degradation and hypertrophic bone changes with osteophyte formation and abnormal bone remodeling. Two groups of OA patients were identified via the production of variable and opposite levels of prostaglandin E2 (PGE2) or leukotriene B4 (LTB4) by subchondral osteoblasts, PGE2 levels discriminating between low and high subgroups. We studied whether the expression of 5-lipoxygenase (5-LO) or 5-LO-activating protein (FLAP) is responsible...

  12. Graphene oxide and titanium: synergistic effects on the biomineralization ability of osteoblast cultures.

    Science.gov (United States)

    Zancanela, Daniela C; Simão, Ana Maria S; Francisco, Camila G; de Faria, Amanda N; Ramos, Ana Paula; Gonçalves, Rogéria R; Matsubara, Elaine Y; Rosolen, José Maurício; Ciancaglini, Pietro

    2016-04-01

    Graphene oxide (GO) has attracted remarkable attention in recent years due to properties such as extremely large surface area, biocompatibility, biostability, and easy chemical functionalization. Osteoblasts underlie the deposition of hydroxyapatite crystals in the bone protein matrix during biomineralization; hydroxyapatite deposition involves extracellular matrix vesicles that are rich in alkaline phosphatase (ALP). Here, we have investigated how GO affects osteoblast viability, ALP activity, and mineralized matrix formation in osteoblast cultures in three different phases of cell growth, in the presence and in the absence of titanium (Ti). Scanning electron microscopy (SEM), Raman spectra, and energy dispersive spectroscopy aided GO characterization. The presence of GO increased the viability of osteoblast cells grown on a plastic surface. However, osteoblast viability on Ti discs was lower in the presence than in the absence of GO. ALP activity emerged at 14 days for the cell culture incubated with GO. The total protein concentration also increased at 21 days on both the Ti discs and plastic surface. Osteoblasts grown on Ti discs had increased mineralized matrix formation in the presence of GO as compared to the cells grown in the absence of GO. SEM images of the cell cultures on plastic surfaces in the presence of GO suggested delayed mineralized matrix formation. In conclusion, applications requiring the presence of Ti, such as prostheses and implants, should benefit from the use of GO, which may increase mineralized nodule formation, stimulate biomineralization, and accelerate bone regeneration. PMID:26886819

  13. 不同表面处理方法对钛表面成骨细胞膜片 ALP活性的影响%Active Changes of ALP of Osteoblasts Cell Sheet with Different Treated Titanium Surfaces

    Institute of Scientific and Technical Information of China (English)

    毛久凤; 夏茜; 吴镭; 杨红; 周成菊; 方艺; 董强

    2016-01-01

    目的:探讨不同表面处理方法对钛表面的成骨细胞膜片碱性磷酸酶( ALP)活性的影响。方法:原代培养大鼠成骨细胞,通过形态学及ALP鉴定成骨细胞;以机械抛光处理的钛片为对照组,以棕刚玉颗粒喷砂材料处理的钛片为喷砂酸蚀( SLA)组,分别构建成骨细胞膜片,膜片连续培养1周或2周时,测量成骨细胞膜片中反映成骨效应的ALP活性的改变。结果:原代培养细胞ALP阳性,经形态及特性鉴定为成骨细胞;连续培养1周或2周时,SLA组ALP活性均高于对照组,差异有统计学意义( P<0.05);各组膜片ALP活性第1周高于第2周,差异有统计学意义( P<0.05)。结论:钛表面性质能够影响成骨细胞膜片的成骨分化能力。%[ Abstract]Objective:To study the ontogenesis of osteoblasts cell sheets with different treated surface of titanium. Methods:Primary cultured rat osteoblasts were identified through morphology observa-tion,alkaline phosphatase staining. Mechanically polished titanium sheets served as control group. The cell sheets were treated with brown fused alumina material as SLA group. Constructing osteoblast cell sheets respectively,and cultured successively for 1 or 2 weeks. Then,measuring changes of ALP activity. Results:Primary cultured cell ALP was positive,the morphology of cells conformed to the characteristics of osteoblasts. The alkaline phosphatase activity of the SLA group was higher than that of control group when cultured successively for 1 or 2 weeks,differences were statistically significant ( P<0 . 05 ). The alkaline phosphatase activity of the cell sheets formed in 1 st week was higher than that of 2nd week,differences were statistically significant(P<0. 05). Conclusion:The surface prop-erties of titanium could affect the osteogenic capacity of cell sheet.

  14. Calcitonin gene-related peptide promotes the expression of osteoblastic genes and activates the WNT signal transduction pathway in bone marrow stromal stem cells.

    Science.gov (United States)

    Zhou, Ri; Yuan, Zhi; Liu, Jierong; Liu, Jian

    2016-06-01

    Calcitonin gene-related peptide (CGRP) is known to induce osteoblastic differentiation and alkaline phosphatase activity in bone marrow stromal stem cells (BMSCs). However, it has remained elusive whether this effect is mediated by CGRP receptors directly or whether other signaling pathways are involved. The present study assessed the possible involvement of the Wnt/β‑catenin signaling pathway in the activation of CGRP signaling during the differentiation of BMSCs. First, the differentiation of BMSCs was induced in vitro and the expression of CGRP receptors was examined by western blot analysis. The effects of exogenous CGRP and LiCl, a stimulator of the Wnt/β‑catenin signaling pathway, on the osteoblastic differentiation of BMSCs were assessed; furthermore, the expression of mRNA and proteins involved in the Wnt/β‑catenin signaling pathway was assessed using quantitative PCR and western blot analyses. The results revealed that CGRP receptors were expressed throughout the differentiation of BMSCs, at days 7 and 14. Incubation with CGRP and LiCl led to the upregulation of the expression of osteoblastic genes associated with the Wnt/β‑catenin pathway, including the mRNA of c‑myc, cyclin D1, Lef1, Tcf7 and β‑catenin as well as β‑catenin protein. However, the upregulation of these genes and β‑catenin protein was inhibited by CGRP receptor antagonist or secreted frizzled‑related protein, an antagonist of the Wnt/β‑catenin pathway. The results of the present study therefore suggested that the Wnt/β-catenin signaling pathway may be involved in CGRP‑ and LiCl-promoted osteoblastic differentiation of BMSCs. PMID:27082317

  15. Nicotine inhibits collagen synthesis and alkaline phosphatase activity, but stimulates DNA synthesis in osteoblast-like cells

    International Nuclear Information System (INIS)

    Use of smokeless tobacco is associated with various oral lesions including periodontal damage and alveolar bone loss. This study was performed to test the effects of nicotine on bone-forming cells at concentrations that occur in the saliva of smokeless tobacco users. Confluent cultures of osteoblast-like cells isolated from chick embryo calvariae were incubated for 2 days with nicotine added to the culture medium (25-600 micrograms/ml). Nicotine inhibited alkaline phosphatase in the cell layer and released to the medium, whereas glycolysis (as indexed by lactate production) was unaffected or slightly elevated. The effects on medium and cell layer alkaline phosphatase were concentration dependent with maximal inhibition occurring at 600 micrograms nicotine/ml. Nicotine essentially did not affect the noncollagenous protein content of the cell layer, but did inhibit collagen synthesis (hydroxylation of [3H]proline and collagenase-digestible protein) at 100, 300, and 600 micrograms/ml. Release of [3H]hydroxyproline to the medium was also decreased in a dose-dependent manner, as was the collagenase-digestible protein for both the medium and cell layer. In contrast, DNA synthesis (incorporation of [3H]thymidine) was more than doubled by the alkaloid, whereas total DNA content was slightly inhibited at 600 micrograms/ml, suggesting stimulated cell turnover. Morphologic changes occurred in nicotine-treated cells including rounding up, detachment, and the occurrence of numerous large vacuoles. These results suggest that steps to reduce the salivary concentration of nicotine in smokeless tobacco users might diminish damaging effects of this product on alveolar bone

  16. The Rho-GEF Kalirin regulates bone mass and the function of osteoblasts and osteoclasts.

    Science.gov (United States)

    Huang, Su; Eleniste, Pierre P; Wayakanon, Kornchanok; Mandela, Prashant; Eipper, Betty A; Mains, Richard E; Allen, Matthew R; Bruzzaniti, Angela

    2014-03-01

    Bone homeostasis is maintained by the balance between bone resorption by osteoclasts and bone formation by osteoblasts. Dysregulation in the activity of the bone cells can lead to osteoporosis, a disease characterized by low bone mass and an increase in bone fragility and risk of fracture. Kalirin is a novel GTP-exchange factor protein that has been shown to play a role in cytoskeletal remodeling and dendritic spine formation in neurons. We examined Kalirin expression in skeletal tissue and found that it was expressed in osteoclasts and osteoblasts. Furthermore, micro-CT analyses of the distal femur of global Kalirin knockout (Kal-KO) mice revealed significantly reduced trabecular and cortical bone parameters in Kal-KO mice, compared to WT mice, with significantly reduced bone mass in 8, 14 and 36week-old female Kal-KO mice. Male mice also exhibited a decrease in bone parameters but not to the level seen in female mice. Histomorphometric analyses also revealed decreased bone formation rate in 14week-old female Kal-KO mice, as well as decreased osteoblast number/bone surface and increased osteoclast surface/bone surface. Consistent with our in vivo findings, the bone resorbing activity and differentiation of Kal-KO osteoclasts was increased in vitro. Although alkaline phosphatase activity by Kal-KO osteoblasts was increased in vitro, Kal-KO osteoblasts showed decreased mineralizing activity, as well as decreased secretion of OPG, which was inversely correlated with ERK activity. Taken together, our findings suggest that deletion of Kalirin directly affects osteoclast and osteoblast activity, leading to decreased OPG secretion by osteoblasts which is likely to alter the RANKL/OPG ratio and promote osteoclastogenesis. Therefore, Kalirin may play a role in paracrine and/or endocrine signaling events that control skeletal bone remodeling and the maintenance of bone mass. PMID:24380811

  17. Human activities affecting trace gases and climate

    International Nuclear Information System (INIS)

    The Earth's climate has been in a constant state of change throughout geologic time due to natural perturbations in the global geobiosphere. However, various human activities have the potential to cause future global warming over a relatively short amount of time. These activities, which affect the Earth's climate by altering the concentrations of trace gases in the atmosphere, include energy consumption, particularly fossil-fuel consumption; industrial processes (production and use of chlorofluorocarbons, halons, and chlorocarbons, landfilling of wastes, and cement manufacture); changes in land use patterns, particularly deforestation and biomass burning; and agricultural practices (waste burning, fertilizer usage, rice production, and animal husbandry). Population growth is an important underlying factor affecting the level of growth in each activity. This paper describes how the human activities listed above contribute to atmospheric change, the current pattern of each activity, and how levels of each activity have changed since the early part of this century

  18. Artificial Extracellular Matrices with Oversulfated Glycosaminoglycan Derivatives Promote the Differentiation of Osteoblast-Precursor Cells and Premature Osteoblasts

    Directory of Open Access Journals (Sweden)

    Ute Hempel

    2014-01-01

    Full Text Available Sulfated glycosaminoglycans (GAG are components of the bone marrow stem cell niche and to a minor extent of mature bone tissue with important functions in regulating stem cell lineage commitment and differentiation. We anticipated that artificial extracellular matrices (aECM composed of collagen I and synthetically oversulfated GAG derivatives affect preferentially the differentiation of osteoblast-precursor cells and early osteoblasts. A set of gradually sulfated chondroitin sulfate and hyaluronan derivatives was used for the preparation of aECM. All these matrices were analysed with human bone marrow stromal cells to identify the most potent aECM and to determine the influence of the degree and position of sulfate groups and the kind of disaccharide units on the osteogenic differentiation. Oversulfated GAG derivatives with a sulfate group at the C-6 position of the N-acetylglycosamine revealed the most pronounced proosteogenic effect as determined by tissue nonspecific alkaline phosphatase activity and calcium deposition. A subset of the aECM was further analysed with different primary osteoblasts and cell lines reflecting different maturation stages to test whether the effect of sulfated GAG derivatives depends on the maturation status of the cells. It was shown that the proosteogenic effect of aECM was most prominent in early osteoblasts.

  19. Whence Induced Demand: How Access Affects Activity

    OpenAIRE

    Levinson, David; Kanchi, Seshasai

    2000-01-01

    Additional highway capacity, by increasing travel speed, affects the individual share of time within a 24-hour budget allocated to various activities (time spent at and traveling to home, shop, work and other), some activities will be undertaken more, others less. This paper extends previous research that identified and quantified induced demand in terms of vehicle miles traveled, by considering questions of what type of demand is induced and which activities are consequently reduced. This pa...

  20. An osteoblast-derived proteinase controls tumor cell survival via TGF-beta activation in the bone microenvironment.

    Directory of Open Access Journals (Sweden)

    Sophie Thiolloy

    Full Text Available Breast to bone metastases frequently induce a "vicious cycle" in which osteoclast mediated bone resorption and proteolysis results in the release of bone matrix sequestered factors that drive tumor growth. While osteoclasts express numerous proteinases, analysis of human breast to bone metastases unexpectedly revealed that bone forming osteoblasts were consistently positive for the proteinase, MMP-2. Given the role of MMP-2 in extracellular matrix degradation and growth factor/cytokine processing, we tested whether osteoblast derived MMP-2 contributed to the vicious cycle of tumor progression in the bone microenvironment.To test our hypothesis, we utilized murine models of the osteolytic tumor-bone microenvironment in immunocompetent wild type and MMP-2 null mice. In longitudinal studies, we found that host MMP-2 significantly contributed to tumor progression in bone by protecting against apoptosis and promoting cancer cell survival (caspase-3; immunohistochemistry. Our data also indicate that host MMP-2 contributes to tumor induced osteolysis (μCT, histomorphometry. Further ex vivo/in vitro experiments with wild type and MMP-2 null osteoclast and osteoblast cultures identified that 1 the absence of MMP-2 did not have a deleterious effect on osteoclast function (cd11B isolation, osteoclast differentiation, transwell migration and dentin resorption assay; and 2 that osteoblast derived MMP-2 promoted tumor survival by regulating the bioavailability of TGFβ, a factor critical for cell-cell communication in the bone (ELISA, immunoblot assay, clonal and soft agar assays.Collectively, these studies identify a novel "mini-vicious cycle" between the osteoblast and metastatic cancer cells that is key for initial tumor survival in the bone microenvironment. In conclusion, the findings of our study suggest that the targeted inhibition of MMP-2 and/or TGFβ would be beneficial for the treatment of bone metastases.

  1. Identification of Rorβ targets in cultured osteoblasts and in human bone

    International Nuclear Information System (INIS)

    Highlights: •We examine the gene expression patterns controlled by Rorβ in osteoblasts. •Genes involved in extracellular matrix regulation and proliferation are affected. •Rorβ mRNA levels increase in aged, human bone biopsies. •Rorβ may affect osteoblast activity by modulation of these pathways. -- Abstract: Control of osteoblastic bone formation involves the cumulative action of numerous transcription factors, including both activating and repressive functions that are important during specific stages of differentiation. The nuclear receptor retinoic acid receptor-related orphan receptor β (Rorβ) has been recently shown to suppress the osteogenic phenotype in cultured osteoblasts, and is highly upregulated in bone marrow-derived osteogenic precursors isolated from aged osteoporotic mice, suggesting Rorβ is an important regulator of osteoblast function. However the specific gene expression patterns elicited by Rorβ are unknown. Using microarray analysis, we identified 281 genes regulated by Rorβ in an MC3T3-E1 mouse osteoblast cell model (MC3T3-Rorβ-GFP). Pathway analysis revealed alterations in genes involved in MAPK signaling, genes involved in extracellular matrix (ECM) regulation, and cytokine-receptor interactions. Whereas the identified Rorβ-regulated ECM genes normally decline during osteoblastic differentiation, they were highly upregulated in this non-mineralizing MC3T3-Rorβ-GFP model system, suggesting that Rorβ may exert its anti-osteogenic effects through ECM disruption. Consistent with these in vitro findings, the expression of both RORβ and a subset of RORβ-regulated genes were increased in bone biopsies from postmenopausal women (73 ± 7 years old) compared to premenopausal women (30 ± 5 years old), suggesting a role for RORβ in human age-related bone loss. Collectively, these data demonstrate that Rorβ regulates known osteogenic pathways, and may represent a novel therapeutic target for age-associated bone loss

  2. Identification of Rorβ targets in cultured osteoblasts and in human bone

    Energy Technology Data Exchange (ETDEWEB)

    Roforth, Matthew M., E-mail: roforth.matthew@mayo.edu; Khosla, Sundeep, E-mail: khosla.sundeep@mayo.edu; Monroe, David G., E-mail: monroe.david@mayo.edu

    2013-11-01

    Highlights: •We examine the gene expression patterns controlled by Rorβ in osteoblasts. •Genes involved in extracellular matrix regulation and proliferation are affected. •Rorβ mRNA levels increase in aged, human bone biopsies. •Rorβ may affect osteoblast activity by modulation of these pathways. -- Abstract: Control of osteoblastic bone formation involves the cumulative action of numerous transcription factors, including both activating and repressive functions that are important during specific stages of differentiation. The nuclear receptor retinoic acid receptor-related orphan receptor β (Rorβ) has been recently shown to suppress the osteogenic phenotype in cultured osteoblasts, and is highly upregulated in bone marrow-derived osteogenic precursors isolated from aged osteoporotic mice, suggesting Rorβ is an important regulator of osteoblast function. However the specific gene expression patterns elicited by Rorβ are unknown. Using microarray analysis, we identified 281 genes regulated by Rorβ in an MC3T3-E1 mouse osteoblast cell model (MC3T3-Rorβ-GFP). Pathway analysis revealed alterations in genes involved in MAPK signaling, genes involved in extracellular matrix (ECM) regulation, and cytokine-receptor interactions. Whereas the identified Rorβ-regulated ECM genes normally decline during osteoblastic differentiation, they were highly upregulated in this non-mineralizing MC3T3-Rorβ-GFP model system, suggesting that Rorβ may exert its anti-osteogenic effects through ECM disruption. Consistent with these in vitro findings, the expression of both RORβ and a subset of RORβ-regulated genes were increased in bone biopsies from postmenopausal women (73 ± 7 years old) compared to premenopausal women (30 ± 5 years old), suggesting a role for RORβ in human age-related bone loss. Collectively, these data demonstrate that Rorβ regulates known osteogenic pathways, and may represent a novel therapeutic target for age-associated bone loss.

  3. Dexamethasone, BMP-2, and 1,25-dihydroxyvitamin D enhance a more differentiated osteoblast phenotype

    DEFF Research Database (Denmark)

    Jørgensen, Niklas Rye; Henriksen, Z; Sørensen, O H;

    2004-01-01

    D), 100 nM Dex, and/or 100 ng/ml BMP-2. The osteoblast phenotype was assessed as alkaline phosphatase (AP) activity/staining, production of osteocalcin and procollagen type 1 (P1NP), parathyroid hormone (PTH)-induced cyclic adenosine mono-phosphate (cAMP) production, and in vitro mineralization. AP...... activity was increased by Dex, but not by BMP-2 treatment. P1NP production was decreased after Dex treatment, while BMP-2 had no effect on P1NP levels. Osteocalcin production was low in cultures not stimulated with vitamin D. Dex or BMP-2 treatment alone did not affect the basic osteocalcin levels, but in...... osteoblastic cells with different phenotypic characteristics, and a selective activation of some of the most important genes and functions of the mature osteoblast can thus be performed in vitro....

  4. Biochemical markers of bone metabolism reflect osteoclastic and osteoblastic activity in multiple myeloma

    DEFF Research Database (Denmark)

    Abildgaard, N; Glerup, H; Rungby, Jørgen;

    2000-01-01

    histomorphometric findings. MARKERS OF BONE FORMATION: Serum C-terminal propeptide of procollagen I (PICP) and serum bone-specific alkaline phosphatase (bAP) showed significant correlations with the dynamic parameters of bone formation (r=0.57-0.58), whereas serum osteocalcin and serum total AP did not. CYTOKINES......: Highly significant correlations were observed between marrow IL-6 and rates of bone resorption and activation frequency (r=0.76-0.82) and with serum ICTP (r=0.63). Minor, but also significant correlations were observed between the resorptive indices and IL-6sR and IL-1beta. The data indicate that...

  5. Effect of La3+ on osteoblastic differentiation of rat bone marrow stromal cells

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    In the present work, the effect of La3+ on osteoblastic differentiation of primary rat bone marrow stromal cells (MSCs) as well as the related mechanisms are studied. Differentiation is monitored by detection of alkaline phosphatase (ALP) activity, osteocalcin secretion, the mRNA levels of Type I collagen and osteocalcin, and matrix mineralization. The results show that La3+ inhibits osteoblastic differentiation of MSCs in the early and middle stages of culture, as demonstrated by the decrease of ALP activity, osteocalcin secretion, and down-regulation of the mRNA level of osteocalcin. However, La3+ does not affect the matrix mineralization in advanced MSCs, because it up-regulates the mRNA levels of Type I collagen, and promotes ALP activity and osteocalcin secretion in MSCs in the late stage of culture. In addition, Western blot analysis exhibits that La3+ induces the phosphorylation and activation of mitogen-activated protein kinase (MAPK). Furthermore, MAPK kinase inhibitor PD98059 completely blocks the inhibitory effect of La3+ on ALP activity of MSCs in the middle stage of culture. These results suggest that La3+ affects MSCs osteoblastic differentiation depending on differentiation stages. La3+ inhibits osteoblastic differentiation of MSCs in the early and middle stages by a MAPK-dependent mechanism, but does not affect the matrix mineralization in advanced MSCs.

  6. The predominant mechanism of intercellular calcium wave propagation changes during long-term culture of human osteoblast-like cells

    DEFF Research Database (Denmark)

    Henriksen, Zanne; Hiken, Jeffrey F; Steinberg, Thomas H;

    2006-01-01

    cells still responded to addition of ATP, but P2Y desensitization did not inhibit ICW propagation. Our data indicate that the relative role of P2Y-mediated and gap junction-mediated ICW changes during osteoblast differentiation in vitro. In less differentiated cells, P2Y-mediated ICW predominate, but as......Intercellular calcium waves (ICW) are calcium transients that spread from cell to cell in response to different stimuli. We previously demonstrated that human osteoblast-like cells in culture propagate ICW in response to mechanical stimulation by two mechanisms. One mechanism involves autocrine...... activation of P2Y receptors, and the other requires gap junctional communication. In the current work we ask whether long-term culture of osteoblast-like cells affects the propagation of ICW by these two mechanisms. Human osteoblast-like cells were isolated from bone marrow. Mechanically induced ICW were...

  7. Tartrate-resistant acid phosphatase (TRAP) co-localizes with receptor activator of NF-KB ligand (RANKL) and osteoprotegerin (OPG) in lysosomal-associated membrane protein 1 (LAMP1)-positive vesicles in rat osteoblasts and osteocytes.

    Science.gov (United States)

    Solberg, L B; Stang, E; Brorson, S-H; Andersson, G; Reinholt, F P

    2015-02-01

    Tartrate-resistant acid phosphatase (TRAP) is well known as an osteoclast marker; however, a recent study from our group demonstrated enhanced number of TRAP + osteocytes as well as enhanced levels of TRAP located to intracellular vesicles in osteoblasts and osteocytes in experimental osteoporosis in rats. Such vesicles were especially abundant in osteoblasts and osteocytes in cancellous bone as well as close to bone surface and intracortical remodeling sites. To further investigate TRAP in osteoblasts and osteocytes, long bones from young, growing rats were examined. Immunofluorescence confocal microscopy displayed co-localization of TRAP with receptor activator of NF-KB ligand (RANKL) and osteoprotegerin (OPG) in hypertrophic chondrocytes and diaphyseal osteocytes with Pearson's correlation coefficient ≥0.8. Transmission electron microscopy showed co-localization of TRAP and RANKL in lysosomal-associated membrane protein 1 (LAMP1) + vesicles in osteoblasts and osteocytes supporting the results obtained by confocal microscopy. Recent in vitro data have demonstrated OPG as a traffic regulator for RANKL to LAMP1 + secretory lysosomes in osteoblasts and osteocytes, which seem to serve as temporary storage compartments for RANKL. Our in situ observations indicate that TRAP is located to RANKL-/OPG-positive secretory lysosomes in osteoblasts and osteocytes, which may have implications for osteocyte regulation of osteoclastogenesis. PMID:25201349

  8. Avenanthramides Prevent Osteoblast and Osteocyte Apoptosis and Induce Osteoclast Apoptosis in Vitro in an Nrf2-Independent Manner

    Directory of Open Access Journals (Sweden)

    Gretel G. Pellegrini

    2016-07-01

    Full Text Available Oats contain unique bioactive compounds known as avenanthramides (AVAs with antioxidant properties. AVAs might enhance the endogenous antioxidant cellular response by activation of the transcription factor Nrf2. Accumulation of reactive oxygen species plays a critical role in many chronic and degenerative diseases, including osteoporosis. In this disease, there is an imbalance between bone formation by osteoblasts and bone resorption by osteoclasts, which is accompanied by increased osteoblast/osteocyte apoptosis and decreased osteoclast apoptosis. We investigated the ability of the synthethic AVAs 2c, 2f and 2p, to 1-regulate gene expression in bone cells, 2-affect the viability of osteoblasts, osteocytes and osteoclasts, and the generation of osteoclasts from their precursors, and 3-examine the potential involvement of the transcription factor Nrf2 in these actions. All doses of AVA 2c and 1 and 5 µM dose of 2p up-regulated collagen 1A expression. Lower doses of AVAs up-regulated OPG (osteoprotegerin in OB-6 osteoblastic cells, whereas 100 μM dose of 2f and all concentrations of 2c down-regulated RANKL gene expression in MLO-Y4 osteocytic cells. AVAs did not affect apoptosis of OB-6 osteoblastic cells or MLO-Y4 osteocytic cells; however, they prevented apoptosis induced by the DNA topoisomerase inhibitor etoposide, the glucocorticoid dexamethasone, and hydrogen peroxide. AVAs prevented apoptosis of both wild type (WT and Nrf2 Knockout (KO osteoblasts, demonstrating that AVAs-induced survival does not require Nrf2 expression. Further, KO osteoclast precursors produced more mature osteoclasts than WT; and KO cultures exhibited less apoptotic osteoclasts than WT cultures. Although AVAs did not affect WT osteoclasts, AVA 2p reversed the low apoptosis of KO osteoclasts. These in vitro results demonstrate that AVAs regulate, in part, the function of osteoblasts and osteocytes and prevent osteoblast/osteocyte apoptosis and increase osteoclast

  9. Avenanthramides Prevent Osteoblast and Osteocyte Apoptosis and Induce Osteoclast Apoptosis in Vitro in an Nrf2-Independent Manner.

    Science.gov (United States)

    Pellegrini, Gretel G; Morales, Cynthya C; Wallace, Taylor C; Plotkin, Lilian I; Bellido, Teresita

    2016-01-01

    Oats contain unique bioactive compounds known as avenanthramides (AVAs) with antioxidant properties. AVAs might enhance the endogenous antioxidant cellular response by activation of the transcription factor Nrf2. Accumulation of reactive oxygen species plays a critical role in many chronic and degenerative diseases, including osteoporosis. In this disease, there is an imbalance between bone formation by osteoblasts and bone resorption by osteoclasts, which is accompanied by increased osteoblast/osteocyte apoptosis and decreased osteoclast apoptosis. We investigated the ability of the synthethic AVAs 2c, 2f and 2p, to 1-regulate gene expression in bone cells, 2-affect the viability of osteoblasts, osteocytes and osteoclasts, and the generation of osteoclasts from their precursors, and 3-examine the potential involvement of the transcription factor Nrf2 in these actions. All doses of AVA 2c and 1 and 5 µM dose of 2p up-regulated collagen 1A expression. Lower doses of AVAs up-regulated OPG (osteoprotegerin) in OB-6 osteoblastic cells, whereas 100 μM dose of 2f and all concentrations of 2c down-regulated RANKL gene expression in MLO-Y4 osteocytic cells. AVAs did not affect apoptosis of OB-6 osteoblastic cells or MLO-Y4 osteocytic cells; however, they prevented apoptosis induced by the DNA topoisomerase inhibitor etoposide, the glucocorticoid dexamethasone, and hydrogen peroxide. AVAs prevented apoptosis of both wild type (WT) and Nrf2 Knockout (KO) osteoblasts, demonstrating that AVAs-induced survival does not require Nrf2 expression. Further, KO osteoclast precursors produced more mature osteoclasts than WT; and KO cultures exhibited less apoptotic osteoclasts than WT cultures. Although AVAs did not affect WT osteoclasts, AVA 2p reversed the low apoptosis of KO osteoclasts. These in vitro results demonstrate that AVAs regulate, in part, the function of osteoblasts and osteocytes and prevent osteoblast/osteocyte apoptosis and increase osteoclast apoptosis; further

  10. Methylsulfonylmethane enhances BMP‑2‑induced osteoblast differentiation in mesenchymal stem cells.

    Science.gov (United States)

    Kim, Don Nam; Joung, Youn Hee; Darvin, Pramod; Kang, Dong Young; Sp, Nipin; Byun, Hyo Joo; Cho, Kwang Hyun; Park, Kyung Do; Lee, Hak Kyo; Yang, Young Mok

    2016-07-01

    As human lifespans have increased, the incidence of osteoporosis has also increased. Methylsulfonylmethane (MSM) affects the process of mesenchymal stem cell (MSC) differentiation into osteoblasts via the Janus kinase 2 (Jak2)/signal transducer and activator of transcription (STAT)5b signaling pathway, and bone morphogenetic protein 2 (BMP‑2) is also known to significantly affect bone health. In addition, the phosphorylation of small mothers against decapentaplegic (Smad)1/5/8 regulates the Runt‑related transcription factor 2 (Runx2) gene, which encodes a transcription factor for osteoblast differentiation markers. In the present study, the differentiation of MSCs treated with MSM, BMP‑2, and their combination were examined. The differentiation of osteoblasts was demonstrated through observation of morphological changes and mineralization, using alizarin red and Von Kossa staining. Western blotting analysis demonstrated that the combination of MSM and BMP-2 increased the phosphorylation of the BMP signaling-associated protein, Smad1/5/8. Combination of MSM and BMP-2 significantly increased osteogenic differentiation and mineralization of the MSCs compared with either MSM or BMP-2 alone. Additionally, reverse transcription-polymerase chain reaction analysis demonstrated that combination of MSM and BMP-2 increased the expression level of the Runx2 gene and the osteoblast differentiation marker genes, alkaline phosphatase, bone sialoprotein and osteocalcin, in MSCs compared with controls. Thus, the combination of MSM and BMP-2 may promote the differentiation of MSCs into osteoblasts. PMID:27175741

  11. Photothermal effects of laser-activated surface plasmonic gold nanoparticles on the apoptosis and osteogenesis of osteoblast-like cells

    Science.gov (United States)

    Rau, Lih-Rou; Huang, Wan-Yu; Liaw, Jiunn-Woei; Tsai, Shiao-Wen

    2016-01-01

    The specific properties of gold nanoparticles (AuNPs) make them a novel class of photothermal agents that can induce cancer cell damage and even death through the conversion of optical energy to thermal energy. Most relevant studies have focused on increasing the precision of cell targeting, improving the efficacy of energy transfer, and exploring additional functions. Nevertheless, most cells can uptake nanosized particles through nonspecific endocytosis; therefore, before hyperthermia via AuNPs can be applied for clinical use, it is important to understand the adverse optical–thermal effects of AuNPs on nontargeted cells. However, few studies have investigated the thermal effects induced by pulsed laser-activated AuNPs on nearby healthy cells due to nonspecific treatment. The aim of this study is to evaluate the photothermal effects induced by AuNPs plus a pulsed laser on MG63, an osteoblast-like cell line, specifically examining the effects on cell morphology, viability, death program, and differentiation. The cells were treated with media containing 50 nm AuNPs at a concentration of 5 ppm for 1 hour. Cultured cells were then exposed to irradiation at 60 mW/cm2 and 80 mW/cm2 by a Nd:YAG laser (532 nm wavelength). We observed that the cytoskeletons of MG63 cells treated with bare AuNPs followed by pulsed laser irradiation were damaged, and these cells had few bubbles on the cell membrane compared with those that were not treated (control) or were treated with AuNPs or the laser alone. There were no significant differences between the AuNPs plus laser treatment group and the other groups in terms of cell viability, death program analysis results, or alkaline phosphatase and calcium accumulation during culture for up to 21 days. However, the calcium deposit areas in the cells treated with AuNPs plus laser were larger than those in other groups during the early culture period. PMID:27555768

  12. Photothermal effects of laser-activated surface plasmonic gold nanoparticles on the apoptosis and osteogenesis of osteoblast-like cells.

    Science.gov (United States)

    Rau, Lih-Rou; Huang, Wan-Yu; Liaw, Jiunn-Woei; Tsai, Shiao-Wen

    2016-01-01

    The specific properties of gold nanoparticles (AuNPs) make them a novel class of photothermal agents that can induce cancer cell damage and even death through the conversion of optical energy to thermal energy. Most relevant studies have focused on increasing the precision of cell targeting, improving the efficacy of energy transfer, and exploring additional functions. Nevertheless, most cells can uptake nanosized particles through nonspecific endocytosis; therefore, before hyperthermia via AuNPs can be applied for clinical use, it is important to understand the adverse optical-thermal effects of AuNPs on nontargeted cells. However, few studies have investigated the thermal effects induced by pulsed laser-activated AuNPs on nearby healthy cells due to nonspecific treatment. The aim of this study is to evaluate the photothermal effects induced by AuNPs plus a pulsed laser on MG63, an osteoblast-like cell line, specifically examining the effects on cell morphology, viability, death program, and differentiation. The cells were treated with media containing 50 nm AuNPs at a concentration of 5 ppm for 1 hour. Cultured cells were then exposed to irradiation at 60 mW/cm(2) and 80 mW/cm(2) by a Nd:YAG laser (532 nm wavelength). We observed that the cytoskeletons of MG63 cells treated with bare AuNPs followed by pulsed laser irradiation were damaged, and these cells had few bubbles on the cell membrane compared with those that were not treated (control) or were treated with AuNPs or the laser alone. There were no significant differences between the AuNPs plus laser treatment group and the other groups in terms of cell viability, death program analysis results, or alkaline phosphatase and calcium accumulation during culture for up to 21 days. However, the calcium deposit areas in the cells treated with AuNPs plus laser were larger than those in other groups during the early culture period. PMID:27555768

  13. TNF-alpha-dependent activation of NF-kappa B in human osteoblastic HOS-TE85 cells is repressed in vector-averaged gravity using clinostat rotation.

    Science.gov (United States)

    Kobayashi, K; Kambe, F; Kurokouchi, K; Sakai, T; Ishiguro, N; Iwata, H; Koga, K; Gruener, R; Seo, H

    2000-12-01

    Effects of vector-averaged gravity on tumor necrosis factor (TNF)-alpha-dependent activation of nuclear factor kappa B (NF-kappa B) in human osteoblastic HOS-TE85 cells were investigated by culturing the cells using clinostat rotation (clinorotation). Cell cultures were rotated for 72 h at 40 rpm in a clinostat. At the end of clinorotation, the cells were treated with TNF-alpha for 30 min under stationary conditions. Electrophoretic mobility shift assays revealed that TNF-alpha-dependent activation of NF-kappa B was markedly reduced in the clinorotated cells when compared with the cells in control stationary cultures or after horizontal rotation (motional controls). The NF-kappa B-dependent transactivation was also impaired in the clinorotated cells, as evidenced by a transient transfection assay with a reporter plasmid containing multimerized NF-kappa B sites. Consistent with these findings, the TNF-alpha-dependent induction of endogenous NF-kappa B-responsive genes p105, I kappa B-alpha, and IL-8, was significantly attenuate in clinorotated cells. These results demonstrate that vector-averaged gravity inhibits the responsiveness of osteoblasts to TNF-alpha by repressing NF-kappa B activation. PMID:11112449

  14. Do recreational activities affect coastal biodiversity?

    Science.gov (United States)

    Riera, Rodrigo; Menci, Cristiano; Sanabria-Fernández, José Antonio; Becerro, Mikel A.

    2016-09-01

    Human activities are largely affecting coastal communities worldwide. Recreational perturbations have been overlooked in comparison to other perturbations, yet they are potential threats to marine biodiversity. They affect coastal communities in different ways, underpinning consistent shifts in fish and invertebrates assemblages. Several sites were sampled subjected to varying effects by recreational fishermen (low and high pressure) and scuba divers (low and high) in an overpopulated Atlantic island. Non-consistent differences in ecological, trophic and functional diversity were found in coastal communities, considering both factors ("diving" and "fishing"). Multivariate analyses only showed significant differences in benthic invertebrates between intensively-dived and non-dived sites. The lack of clear trends may be explained by the depletion of coastal resources in the study area, an extensively-affected island by overfishing.

  15. Prunetin signals via G-protein-coupled receptor, GPR30(GPER1): Stimulation of adenylyl cyclase and cAMP-mediated activation of MAPK signaling induces Runx2 expression in osteoblasts to promote bone regeneration.

    Science.gov (United States)

    Khan, Kainat; Pal, Subhashis; Yadav, Manisha; Maurya, Rakesh; Trivedi, Arun Kumar; Sanyal, Sabyasachi; Chattopadhyay, Naibedya

    2015-12-01

    Prunetin is found in red clover and fruit of Prunus avium (red cherry). The effect of prunetin on osteoblast function, its mode of action and bone regeneration in vivo were investigated. Cultures of primary osteoblasts, osteoblastic cell line and HEK293T cells were used for various in vitro studies. Adult female rats received drill-hole injury at the femur diaphysis to assess the bone regenerative effect of prunetin. Prunetin at 10nM significantly (a) increased proliferation and differentiation of primary cultures of osteoblasts harvested from rats and (b) promoted formation of mineralized nodules by bone marrow stromal/osteoprogenitor cells. At this concentration, prunetin did not activate any of the two nuclear estrogen receptors (α and β). However, prunetin triggered signaling via a G-protein-coupled receptor, GPR30/GPER1, and enhanced cAMP levels in osteoblasts. G15, a selective GPR30 antagonist, abolished prunetin-induced increases in osteoblast proliferation, differentiation and intracellular cAMP. In osteoblasts, prunetin up-regulated runt-related transcription factor 2 (Runx2) protein through cAMP-dependent Erk/MAP kinase activation that ultimately resulted in the up-regulation of GPR30. Administration of prunetin at 0.25mg/kg given to rats stimulated bone regeneration at the site of drill hole and up-regulated Runx2 expression in the fractured callus and the effect was comparable to human parathyroid hormone, the only clinically used osteogenic therapy. We conclude that prunetin promotes osteoinduction in vivo and the mechanism is defined by signaling through GPR30 resulting in the up-regulation of the key osteogenic gene Runx2 that in turn up-regulates GPR30. PMID:26345541

  16. Promoter-dependent and -independent activation of insulin-like growth factor binding protein-5 gene expression by prostaglandin E2 in primary rat osteoblasts

    Science.gov (United States)

    McCarthy, T. L.; Casinghino, S.; Mittanck, D. W.; Ji, C. H.; Centrella, M.; Rotwein, P.

    1996-01-01

    Insulin-like growth factor (IGF) action is mediated by high affinity cell surface IGF receptors and modulated by a family of secreted IGF binding proteins (IGFBPs). IGFBP-5, the most conserved of six IGFBPs characterized to date, uniquely potentiates the anabolic actions of IGF-I for skeletal cells. In osteoblasts, IGFBP-5 production is stimulated by prostaglandin E2 (PGE2), a local factor that mediates certain effects induced by parathyroid hormone, cytokines such as interleukin-1 and transforming growth factor-beta, and mechanical strain. In this study, we show that transcriptional and post-transcriptional events initiated by PGE2 collaborate to enhance IGFBP-5 gene expression in primary fetal rat osteoblast cultures. PGE2 treatment stimulated up to a 7-fold rise in steady-state levels of IGFBP-5 mRNA throughout 32 h of incubation. Analysis of nascent IGFBP-5 mRNA suggested that PGE2 had only a modest stimulatory effect on IGFBP-5 gene transcription, and transient transfection studies with IGFBP-5 promoter-reporter genes confirmed that PGE2 enhanced promoter activity by approximately 2-fold. Similar stimulatory effects were seen with forskolin. A DNA fragment with only 51 base pairs of the 5'-flanking sequence retained hormonal responsiveness, which may be mediated by a binding site for transcription factor AP-2 located at positions -44 to -36 in the proximal IGFBP-5 promoter. Incubation of osteoblasts with the mRNA transcriptional inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole demonstrated that PGE2 enhanced IGFBP-5 mRNA stability by 2-fold, increasing the t1/2 from 9 to 18 h. The effects of PGE2 on steady-state IGFBP-5 transcripts were abrogated by preincubating cells with cycloheximide, indicating that the effects of PGE2 on both gene transcription and mRNA stability required ongoing protein synthesis. Therefore, both promoter-dependent and -independent pathways converge to enhance IGFBP-5 gene expression in response to PGE2 in osteoblasts.

  17. Electrical activity of ferroelectric biomaterials and its effects on the adhesion, growth and enzymatic activity of human osteoblast-like cells

    Science.gov (United States)

    Vaněk, P.; Kolská, Z.; Luxbacher, T.; García, J. A. L.; Lehocký, M.; Vandrovcová, M.; Bačáková, L.; Petzelt, J.

    2016-05-01

    Ferroelectrics have been, among others, studied as electroactive implant materials. Previous investigations have indicated that such implants induce improved bone formation. If a ferroelectric is immersed in a liquid, an electric double layer and a diffusion layer are formed at the interface. This is decisive for protein adsorption and bioactive behaviour, particularly for the adhesion and growth of cells. The charge distribution can be characterized, in a simplified way, by the zeta potential. We measured the zeta potential in dependence on the surface polarity on poled ferroelectric single crystalline LiNbO3 plates. Both our results and recent results of colloidal probe microscopy indicate that the charge distribution at the surface can be influenced by the surface polarity of ferroelectrics under certain ‘ideal’ conditions (low ionic strength, non-contaminated surface, very low roughness). However, suggested ferroelectric coatings on the surface of implants are far from ideal: they are rough, polycrystalline, and the body fluid is complex and has high ionic strength. In real cases, it can therefore be expected that there is rather low influence of the sign of the surface polarity on the electric diffusion layer and thus on the specific adsorption of proteins. This is supported by our results from studies of the adhesion, growth and the activity of alkaline phosphatase of human osteoblast-like Saos-2 cells on ferroelectric LiNbO3 plates in vitro.

  18. Approximating bone ECM: Crosslinking directs individual and coupled osteoblast/osteoclast behavior.

    Science.gov (United States)

    Hwang, Mintai P; Subbiah, Ramesh; Kim, In Gul; Lee, Kyung Eun; Park, Jimin; Kim, Sang Heon; Park, Kwideok

    2016-10-01

    Osteoblast and osteoclast communication (i.e. osteocoupling) is an intricate process, in which the biophysical profile of bone ECM is an aggregate product of their activities. While the effect of microenvironmental cues on osteoblast and osteoclast maturation has been resolved into individual variables (e.g. stiffness or topography), a single cue can be limited with regards to reflecting the full biophysical scope of natural bone ECM. Additionally, the natural modulation of bone ECM, which involves collagenous fibril and elastin crosslinking via lysyl oxidase, has yet to be reflected in current synthetic platforms. Here, we move beyond traditional substrates and use cell-derived ECM to examine individual and coupled osteoblast and osteoclast behavior on a physiological platform. Specifically, preosteoblast-derived ECM is crosslinked with genipin, a biocompatible crosslinker, to emulate physiological lysyl oxidase-mediated ECM crosslinking. We demonstrate that different concentrations of genipin yield changes to ECM density, stiffness, and roughness while retaining biocompatibility. By approximating various bone ECM profiles, we examine how individual and coupled osteoblast and osteoclast behavior are affected. Ultimately, we demonstrate an increase in osteoblast and osteoclast differentiation on compact and loose ECM, respectively, and identify ECM crosslinking density as an underlying force in osteocoupling behavior. PMID:27376556

  19. Effects of radiation on macrophage colony stimulating factor in primary osteoblasts

    International Nuclear Information System (INIS)

    Objective: To investigate the radiation effect on the expression of macrophage colony stimulating factor (M-CSF) in primary osteoblasts and the molecular mechanism of bone injury induced by radiation. Methods: Osteoblasts differentiated from bone marrow stromal cells (BMSCs) were analyzed by real-time PCR and Western blot after 2 Gy or 4 Gy 137Cs γ-irradiation. Results: M-CSF mRNA and protein expression level were up-regulated after 2 Gy and 4 Gy irradiation (t=-17.329, P<0.01; t=-3.841, P<0.05) in osteoblasts. 4 Gy irradiation increased M-CSF mRNA in osteoblast precursors (t=-4.478, P<0.05), but do not affect the protein expression level. Conclusions: These results indicated that up-regulated M-CSF in osteoblasts could enhance the function of osteoclasts' differentiation and maturation which induced by receptor activator of nuclear factor κB ligand after 2 Gy and 4 Gy irradiation. Further more,up-regulated M-CSF expression could enhance osteoclastic bone resorption of mature osteoclasts. (authors)

  20. Sp1/Sp3 and DNA-methylation contribute to basal transcriptional activation of human podoplanin in MG63 versus Saos-2 osteoblastic cells

    Directory of Open Access Journals (Sweden)

    Puri Christina

    2007-03-01

    Full Text Available Abstract Background Podoplanin is a membrane mucin that, among a series of tissues, is expressed on late osteoblasts and osteocytes. Since recent findings have focussed on podoplanin's potential role as a tumour progression factor, we aimed at identifying regulatory elements conferring PDPN promoter activity. Here, we characterized the molecular mechanism controlling basal PDPN transcription in human osteoblast-like MG63 versus Saos-2 cells. Results We cloned and sequenced 2056 nucleotides from the 5'-flanking region of the PDPN gene and a computational search revealed that the TATA and CAAT box-lacking promoter possesses features of a growth-related gene, such as a GC-rich 5' region and the presence of multiple putative Sp1, AP-4 and NF-1 sites. Reporter gene assays demonstrated a functional promoter in MG63 cells exhibiting 30-fold more activity than in Saos-2 cells. In vitro DNase I footprinting revealed eight protected regions flanked by DNaseI hypersensitive sites within the region bp -728 to -39 present in MG63, but not in Saos-2 cells. Among these regions, mutation and supershift electrophoretic mobility shift assays (EMSA identified four Sp1/Sp3 binding sites and two binding sites for yet unknown transcription factors. Deletion studies demonstrated the functional importance of two Sp1/Sp3 sites for PDPN promoter activity. Overexpression of Sp1 and Sp3 independently increased the stimulatory effect of the promoter and podoplanin mRNA levels in MG63 and Saos-2 cells. In SL2 cells, Sp3 functioned as a repressor, while Sp1 and Sp3 acted positively synergistic. Weak PDPN promoter activity of Saos-2 cells correlated with low Sp1/Sp3 nuclear levels, which was confirmed by Sp1/Sp3 chromatin immunoprecipitations in vivo. Moreover, methylation-sensitive Southern blot analyses and bisulfite sequencing detected strong methylation of CpG sites upstream of bp -464 in MG63 cells, but hypomethylation of these sites in Saos-2 cells. Concomitantly

  1. AMPK Activation Affects Glutamate Metabolism in Astrocytes

    DEFF Research Database (Denmark)

    Voss, Caroline Marie; Pajęcka, Kamilla; Stridh, Malin H;

    2015-01-01

    acid (TCA) cycle was studied using high-performance liquid chromatography analysis supplemented with gas chromatography-mass spectrometry technology. It was found that AMPK activation had profound effects on the pathways involved in glutamate metabolism since the entrance of the glutamate carbon...... affected by a reduction of the flux of glutamate derived carbon through the malic enzyme and pyruvate carboxylase catalyzed reactions. Finally, it was found that in the presence of glutamate as an additional substrate, glucose metabolism monitored by the use of tritiated deoxyglucose was unaffected by AMPK...

  2. Abdominal Fat and Sarcopenia in Women Significantly Alter Osteoblasts Homeostasis In Vitro by a WNT/β-Catenin Dependent Mechanism

    OpenAIRE

    Francesca Wannenes; Vincenza Papa; Greco, Emanuela A.; Rachele Fornari; Chiara Marocco; Carlo Baldari; Luigi Di Luigi; Gian Pietro Emerenziani; Eleonora Poggiogalle; Laura Guidetti; Donini, Lorenzo M.; Andrea Lenzi; Silvia Migliaccio

    2014-01-01

    Obesity and sarcopenia have been associated with mineral metabolism derangement and low bone mineral density (BMD). We investigated whether imbalance of serum factors in obese or obese sarcopenic patients could affect bone cell activity in vitro. To evaluate and characterize potential cellular and molecular changes of human osteoblasts, cells were exposed to sera of four groups of patients: (1) affected by obesity with normal BMD (O), (2) affected by obesity with low BMD (OO), (3) affected by...

  3. Morphological and proteomic analysis of early stage of osteoblast differentiation in osteoblastic progenitor cells

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Dun [Population Council, 1230 York Avenue, New York, NY 10065 (United States); Orthopedic Department, Taizhou Hospital, Wenzhou Medical College, Linhai, Zhejiang 317000 (China); Chen, Hai-Xiao, E-mail: Hxchen-1@163.net [Orthopedic Department, Taizhou Hospital, Wenzhou Medical College, Linhai, Zhejiang 317000 (China); Yu, Hai-Qiang [Proteomics Resource Center, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Liang, Yong; Wang, Carrie [Population Council, 1230 York Avenue, New York, NY 10065 (United States); Lian, Qing-Quan [The 2nd Affiliated Hospital, Wenzhou Medical College, Wenzhou, Zhejiang 325000 (China); Deng, Hai-Teng, E-mail: dengh@mail.rockefeller.edu [Proteomics Resource Center, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Ge, Ren-Shan, E-mail: rge@popcbr.rockefeller.edu [Population Council, 1230 York Avenue, New York, NY 10065 (United States); The 2nd Affiliated Hospital, Wenzhou Medical College, Wenzhou, Zhejiang 325000 (China)

    2010-08-15

    Bone remodeling relies on a dynamic balance between bone formation and resorption, mediated by osteoblasts and osteoclasts, respectively. Under certain stimuli, osteoprogenitor cells may differentiate into premature osteoblasts and further into mature osteoblasts. This process is marked by increased alkaline phosphatase (ALP) activity and mineralized nodule formation. In this study, we induced osteoblast differentiation in mouse osteoprogenitor MC3T3-E1 cells and divided the process into three stages. In the first stage (day 3), the MC3T3-E1 cell under osteoblast differentiation did not express ALP or deposit a mineralized nodule. In the second stage, the MC3T3-E1 cell expressed ALP but did not form a mineralized nodule. In the third stage, the MC3T3-E1 cell had ALP activity and formed mineralized nodules. In the present study, we focused on morphological and proteomic changes of MC3T3-E1 cells in the early stage of osteoblast differentiation - a period when premature osteoblasts transform into mature osteoblasts. We found that mean cell area and mean stress fiber density were increased in this stage due to enhanced cell spreading and decreased cell proliferation. We further analyzed the proteins in the signaling pathway of regulation of the cytoskeleton using a proteomic approach and found upregulation of IQGAP1, gelsolin, moesin, radixin, and Cfl1. After analyzing the focal adhesion signaling pathway, we found the upregulation of FLNA, LAMA1, LAMA5, COL1A1, COL3A1, COL4A6, and COL5A2 as well as the downregulation of COL4A1, COL4A2, and COL4A4. In conclusion, the signaling pathway of regulation of the cytoskeleton and focal adhesion play critical roles in regulating cell spreading and actin skeleton formation in the early stage of osteoblast differentiation.

  4. Osteoblast-secreted collagen upregulates paracrine Sonic hedgehog signaling by prostate cancer cells and enhances osteoblast differentiation

    Directory of Open Access Journals (Sweden)

    Zunich Samantha M

    2012-07-01

    Full Text Available Abstract Background Induction of osteoblast differentiation by paracrine Sonic hedgehog (Shh signaling may be a mechanism through which Shh-expressing prostate cancer cells initiate changes in the bone microenvironment and promote metastases. A hallmark of osteoblast differentiation is the formation of matrix whose predominant protein is type 1 collagen. We investigated the formation of a collagen matrix by osteoblasts cultured with prostate cancer cells, and its effects on interactions between prostate cancer cells and osteoblasts. Results In the presence of exogenous ascorbic acid (AA, a co-factor in collagen synthesis, mouse MC3T3 pre-osteoblasts in mixed cultures with human LNCaP prostate cancer cells or LNCaP cells modified to overexpress Shh (LNShh cells formed collagen matrix with distinct fibril ultrastructural characteristics. AA increased the activity of alkaline phosphatase and the expression of the alkaline phosphatase gene Akp2, markers of osteoblast differentiation, in MC3T3 pre-osteoblasts cultured with LNCaP or LNShh cells. However, the AA-stimulated increase in Akp2 expression in MC3T3 pre-osteoblasts cultured with LNShh cells far exceeded the levels observed in MC3T3 cells cultured with either LNCaP cells with AA or LNShh cells without AA. Therefore, AA and Shh exert a synergistic effect on osteoblast differentiation. We determined whether the effect of AA on LNShh cell-induced osteoblast differentiation was mediated by Shh signaling. AA increased the expression of Gli1 and Ptc1, target genes of the Shh pathway, in MC3T3 pre-osteoblasts cultured with LNShh cells to at least twice their levels without AA. The ability of AA to upregulate Shh signaling and enhance alkaline phosphatase activity was blocked in MC3T3 cells that expressed a dominant negative form of the transcription factor GLI1. The AA-stimulated increase in Shh signaling and Shh-induced osteoblast differentiation was also inhibited by the specific collagen synthesis

  5. The effect of a new direct Factor Xa inhibitor on human osteoblasts: an in-vitro study comparing the effect of rivaroxaban with enoxaparin

    LENUS (Irish Health Repository)

    Solayar, Gandhi N

    2011-10-28

    Abstract Background Current treatments for the prevention of thromboembolism include heparin and low-molecular weight heparins (LMWHs). A number of studies have suggested that long term administration of these drugs may adversely affect osteoblasts and therefore, bone metabolism. Xarelto™ (Rivaroxaban) is a new anti-thrombotic drug for the prevention of venous thromboembolism in adult patients undergoing elective hip and knee replacement surgery. The aim of this in vitro study was to investigate the possible effects of rivaroxaban on osteoblast viability, function and gene expression compared to enoxaparin, a commonly used LMWH. Methods Primary human osteoblast cultures were treated with varying concentrations of rivaroxaban (0.013, 0.13, 1.3 and 13 μg\\/ml) or enoxaparin (1, 10 and 100 μg\\/ml). The effect of each drug on osteoblast function was evaluated by measuring alkaline phosphatase activity. The MTS assay was used to assess the effect of drug treatments on cell proliferation. Changes in osteocalcin, Runx2 and BMP-2 messenger RNA (mRNA) expression following drug treatments were measured by real-time polymerase chain reaction (PCR). Results Rivaroxaban and enoxaparin treatment did not adversely affect osteoblast viability. However, both drugs caused a significant reduction in osteoblast function, as measured by alkaline phosphatase activity. This reduction in osteoblast function was associated with a reduction in the mRNA expression of the bone marker, osteocalcin, the transcription factor, Runx2, and the osteogenic factor, BMP-2. Conclusions These data show that rivaroxaban treatment may negatively affect bone through a reduction in osteoblast function.

  6. The effect of a new direct Factor Xa inhibitor on human osteoblasts: an in-vitro study comparing the effect of rivaroxaban with enoxaparin

    Directory of Open Access Journals (Sweden)

    Mulhall Kevin J

    2011-10-01

    Full Text Available Abstract Background Current treatments for the prevention of thromboembolism include heparin and low-molecular weight heparins (LMWHs. A number of studies have suggested that long term administration of these drugs may adversely affect osteoblasts and therefore, bone metabolism. Xarelto™ (Rivaroxaban is a new anti-thrombotic drug for the prevention of venous thromboembolism in adult patients undergoing elective hip and knee replacement surgery. The aim of this in vitro study was to investigate the possible effects of rivaroxaban on osteoblast viability, function and gene expression compared to enoxaparin, a commonly used LMWH. Methods Primary human osteoblast cultures were treated with varying concentrations of rivaroxaban (0.013, 0.13, 1.3 and 13 μg/ml or enoxaparin (1, 10 and 100 μg/ml. The effect of each drug on osteoblast function was evaluated by measuring alkaline phosphatase activity. The MTS assay was used to assess the effect of drug treatments on cell proliferation. Changes in osteocalcin, Runx2 and BMP-2 messenger RNA (mRNA expression following drug treatments were measured by real-time polymerase chain reaction (PCR. Results Rivaroxaban and enoxaparin treatment did not adversely affect osteoblast viability. However, both drugs caused a significant reduction in osteoblast function, as measured by alkaline phosphatase activity. This reduction in osteoblast function was associated with a reduction in the mRNA expression of the bone marker, osteocalcin, the transcription factor, Runx2, and the osteogenic factor, BMP-2. Conclusions These data show that rivaroxaban treatment may negatively affect bone through a reduction in osteoblast function.

  7. Brain Activity, Personality Traits and Affect: Electrocortical Activity in Reaction to Affective Film Stimuli

    Science.gov (United States)

    Makvand Hosseini, Sh.; Azad Fallah, P.; Rasoolzadeh Tabatabaei, S. K.; Ghannadyan Ladani, S. H.; Heise, C.

    We studied the patterns of activation over the cerebral cortex in reaction to affective film stimuli in four groups of extroverts, introverts, neurotics and emotionally stables. Measures of extraversion and neuroticism were collected and resting EEG was recorded from 40 right handed undergraduate female students (19-23) on one occasion for five 30s periods in baseline condition and in affective states. Mean log-transformed absolute alpha power was extracted from 12 electrode sites and analyzed. Patterns of activation were different in personality groups. Different patterns of asymmetries were observed in personality groups in reaction to affective stimuli. Results were partly consistent with approach and withdrawal model and provided supportive evidence for the role of right frontal asymmetry in negative affects in two groups (introverts and emotionally stables) as well as the role of right central asymmetry (increase on right and decrease on left) in active affective states (anxiety and happiness) in all personality groups. Results were also emphasized on the role of decrease activity relative to baseline in cortical regions (bilaterally in frontal and unilaterally in left parietal and temporal regions) in moderating of positive and negative emotion.

  8. The effects of 6-gingerol on proliferation, differentiation, and maturation of osteoblast-like MG-63 cells

    Energy Technology Data Exchange (ETDEWEB)

    Fan, J.Z.; Yang, X.; Bi, Z.G. [Department of Orthopedic Surgery, First Affiliated Hospital, Harbin Medicine University, Harbin (China)

    2015-04-28

    We investigated whether 6-gingerol affects the maturation and proliferation of osteoblast-like MG63 cells in vitro. Osteoblast-like MG63 cells were treated with 6-gingerol under control conditions, and experimental inflammation was induced by tumor necrosis factor-α (TNF-α). Expression of different osteogenic markers and cytokines was analyzed by real-time PCR, Western blotting, and enzyme-linked immunosorbent assay. In addition, alkaline phosphatase (ALP) enzyme activity and biomineralization as markers for differentiation were measured. Treatment with 6-gingerol resulted in insignificant effects on the proliferation rate. 6-Gingerol induced the differentiation of osteoblast-like cells with increased transcription levels of osteogenic markers, upregulated ALP enzyme activity, and enhanced mineralized nodule formation. Stimulation with TNF-α led to enhanced interleukin-6 and nuclear factor-κB expression and downregulated markers of osteoblastic differentiation. 6-Gingerol reduced the degree of inflammation in TNF-α-treated MG-63 cells. In conclusion, 6-gingerol stimulated osteoblast differentiation in normal physiological and inflammatory settings, and therefore, 6-gingerol represents a promising agent for treating osteoporosis or bone inflammation.

  9. Three-dimensional culture of rat calvarial osteoblasts in porous biodegradable polymers

    Science.gov (United States)

    Ishaug-Riley, S. L.; Crane-Kruger, G. M.; Yaszemski, M. J.; Mikos, A. G.

    1998-01-01

    Neonatal rat calvarial osteoblasts were cultured in 90% porous, 75:25 poly(DL-lactic-co-glycolic acid) (PLGA) foam scaffolds for up to 56 days to examine the effects of the cell seeding density, scaffold pore size, and foam thickness on the proliferation and function of the cells in this three-dimensional environment. Osteoblasts were seeded at either 11.1 x 10(5) or 22.1 x 10(5) cells per cm2 onto PLGA scaffolds having pore sizes in the range of 150-300 or 500-710 microm with a thickness of either 1.9 or 3.2 mm. After 1 day in culture, 75.6 and 68.6% of the seeded cells attached and proliferated on the 1.9 mm thick scaffolds of 150-300 microm pore size for the low and high seeding densities, respectively. The number of osteoblasts continued to increase throughout the study and eventually leveled off near 56 days, as indicated by a quantitative DNA assay. Osteoblast/foam constructs with a low cell seeding density achieved comparable DNA content and alkaline phosphatase (ALPase) activity after 14 days, and mineralization results after 56 days to those with a high cell seeding density. A maximum penetration depth of osseous tissue of 220+/-40 microm was reached after 56 days in the osteoblast/foam constructs of 150-300 microm pore size initially seeded with a high cell density. For constructs of 500-710 microm pore size, the penetration depth was 190+/-40 microm under the same conditions. Scaffold pore size and thickness did not significantly affect the proliferation or function of osteoblasts as demonstrated by DNA content, ALPase activity, and mineralized tissue formation. These data show that comparable bone-like tissues can be engineered in vitro over a 56 day period using different rat calvarial osteoblast seeding densities onto biodegradable polymer scaffolds with pore sizes in the range of 150-710 microm. When compared with the results of a previous study where similar polymer scaffolds were seeded and cultured with marrow stromal cells, this study

  10. Abdominal Fat and Sarcopenia in Women Significantly Alter Osteoblasts Homeostasis In Vitro by a WNT/β-Catenin Dependent Mechanism

    Directory of Open Access Journals (Sweden)

    Francesca Wannenes

    2014-01-01

    Full Text Available Obesity and sarcopenia have been associated with mineral metabolism derangement and low bone mineral density (BMD. We investigated whether imbalance of serum factors in obese or obese sarcopenic patients could affect bone cell activity in vitro. To evaluate and characterize potential cellular and molecular changes of human osteoblasts, cells were exposed to sera of four groups of patients: (1 affected by obesity with normal BMD (O, (2 affected by obesity with low BMD (OO, (3 affected by obesity and sarcopenia (OS, and (4 affected by obesity, sarcopenia, and low BMD (OOS as compared to subjects with normal body weight and normal BMD (CTL. Patients were previously investigated and characterized for body composition, biochemical and bone turnover markers. Then, sera of different groups of patients were used to incubate human osteoblasts and evaluate potential alterations in cell homeostasis. Exposure to OO, OS, and OOS sera significantly reduced alkaline phosphatase, osteopontin, and BMP4 expression compared to cells exposed to O and CTL, indicating a detrimental effect on osteoblast differentiation. Interestingly, sera of all groups of patients induced intracellular alteration in Wnt/β-catenin molecular pathway, as demonstrated by the significant alteration of specific target genes expression and by altered β-catenin cellular compartmentalization and GSK3β phosphorylation. In conclusion our results show for the first time that sera of obese subjects with low bone mineral density and sarcopenia significantly alter osteoblasts homeostasis in vitro, indicating potential detrimental effects of trunk fat on bone formation and skeletal homeostasis.

  11. Chronic alcohol abuse and endosseous implants: Linkage of in vitro osteoblast dysfunction to titanium osseointegration rate

    International Nuclear Information System (INIS)

    Chronic alcohol consumption is associated with pathological effects on bone, and it is correlated with the increasing risk of osteoporosis and fractures. The negative effects of alcohol intake also influence bone repair processes and the osseointegration of implants. The aim of the present in vitro study was to investigate the proliferation and synthetic activity of osteoblasts isolated from the trabecular bone of rats previously exposed to 7-week intermittent exposure to ethanol vapour (EE-OB), and sham-aged rats (SA-OB), when cultured on standard commercially pure Ti (cpTi). Osteoblast proliferation (WST-1), alkaline phosphatase (ALP), osteocalcin (OC), collagen type I (CICP), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and transforming growth factor-β1 (TGF-β1) were measured at 1, 7, and 14 days of culture. Our results showed a decrease in the cell viability and synthetic activity of osteoblasts exposed to ethanol when cultured on cpTi. Moreover, the release of local regulatory factors from osteoblasts was imbalanced: TGF-β1 production was reduced and TNF-α and IL-6 were up-regulated. These in vitro data suggest that alcohol abuse affects bone repair and decreases the ability to form bone around standard cpTi. Innovative surfaces and adjuvant therapies could be useful when implants are required in alcoholics

  12. Short time administration of antirheumatic drugs - Methotrexate as a strong inhibitor of osteoblast's proliferation in vitro

    Directory of Open Access Journals (Sweden)

    Annussek Tobias

    2012-09-01

    Full Text Available Abstract Introduction Due to increasing use of disease modifying antirheumatic drugs (DMARDs as first line therapy in rheumatic diseases, dental and maxillofacial practitioner should be aware of drug related adverse events. Especially effects on bone-metabolism and its cells are discussed controversially. Therefore we investigate the in vitro effect of short time administration of low dose methotrexate (MTX on osteoblasts as essential part of bone remodelling cells. Methods Primary bovine osteoblasts (OBs were incubated with various concentrations of MTX, related to tissue concentrations, over a period of fourteen days by using a previously established standard protocol. The effect on cell proliferation as well as mitochondrial activity was assessed by using 3-(4, 5-dimethylthiazol-2-yl 2, 5-diphenyltetrazolium bromide (MTT assay, imaging and counting of living cells. Additionally, immunostaining of extracellular matrix proteins was used to survey osteogenic differentiation. Results All methods indicate a strong inhibition of osteoblast`s proliferation by short time administration of low dose MTX within therapeutically relevant concentrations of 1 to 1000nM, without affecting cell differentiation of middle-stage differentiated OBs in general. More over a significant decrease of cell numbers and mitochondrial activity was found at these MTX concentrations. The most sensitive method seems to be the MTT-assay. MTX-concentration of 0,01nM and concentrations below had no inhibitory effects anymore. Conclusion Even low dose methotrexate acts as a potent inhibitor of osteoblast’s proliferation and mitochondrial metabolism in vitro, without affecting main differentiation of pre-differentiated osteoblasts. These results suggest possible negative effects of DMARDs concerning bone healing and for example osseointegration of dental implants. Especially the specifics of the jaw bone with its high vascularisation and physiological high tissue metabolism

  13. IL-1β-induced matrix metalloproteinase-13 is activated by a disintegrin and metalloprotease-28-regulated proliferation of human osteoblast-like cells

    Energy Technology Data Exchange (ETDEWEB)

    Ozeki, Nobuaki; Kawai, Rie; Yamaguchi, Hideyuki; Hiyama, Taiki; Kinoshita, Katsue; Hase, Naoko; Nakata, Kazuhiko [Department of Endodontics, School of Dentistry, Aichi Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya, Aichi 464-8651 (Japan); Kondo, Ayami [Department of Medicinal Biochemistry, School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto, Chikusa-ku, Nagoya, Aichi 464-8650 (Japan); Mogi, Makio, E-mail: makio@dpc.agu.ac.jp [Department of Medicinal Biochemistry, School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto, Chikusa-ku, Nagoya, Aichi 464-8650 (Japan); Nakamura, Hiroshi [Department of Endodontics, School of Dentistry, Aichi Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya, Aichi 464-8651 (Japan)

    2014-04-15

    We reported previously that matrix metalloproteinase (MMP)-13 accelerates bone remodeling in oral periradicular lesions, and indicated a potentially unique role for MMP-13 in wound healing and regeneration of alveolar bone. The ADAM (a disintegrin and metalloprotease) family is a set of multifunctional cell surface and secreted glycoproteins, of which ADAM-28 has been localized in bone and bone-like tissues. In this study, we show that interleukin (IL)-1β induces the expression of MMP-13 and ADAM-28 in homogeneous α7 integrin-positive human skeletal muscle stem cell (α7{sup +}hSMSC)-derived osteoblast-like (α7{sup +}hSMSC-OB) cells, and promotes proliferation while inhibiting apoptosis in these cells. At higher concentrations, however, IL-1β failed to induce the expression of these genes and caused an increase in apoptosis. We further employed ADAM-28 small interfering RNA (siRNA) to investigate whether IL-1β-induced MMP-13 expression is linked to this IL-1β-mediated changes in cell proliferation and apoptosis. Silencing ADAM-28 expression potently suppressed IL-1β-induced MMP-13 expression and activity, decreased cell proliferation and increased apoptosis in α7{sup +}hSMSC-OB cells. In contrast, MMP-13 siRNA had no effect on ADAM-28 expression, suggesting ADAM-28 regulates MMP-13. Exogenous MMP-13 induced α7{sup +}hSMSC-OB cell proliferation and could rescue ADAM-28 siRNA-induced apoptosis, and we found that proMMP-13 is partially cleaved into its active form by ADAM-28 in vitro. Overall, our results suggest that IL-1β-induced MMP-13 expression and changes in cell proliferation and apoptosis in α7{sup +}hSMSC-OB cells are regulated by ADAM-28. - Highlights: • IL-1β induces the MMP-13 and ADAM-28 expression in human osteoblast-like cells. • IL-1β-induced MMP-13 expression increases proliferation and decreased apoptosis. • MMP-13 expression induced by IL-1β is regulated by ADAM-28. • proMMP-13 appears to be cleaved into its active form via

  14. IL-1β-induced matrix metalloproteinase-13 is activated by a disintegrin and metalloprotease-28-regulated proliferation of human osteoblast-like cells

    International Nuclear Information System (INIS)

    We reported previously that matrix metalloproteinase (MMP)-13 accelerates bone remodeling in oral periradicular lesions, and indicated a potentially unique role for MMP-13 in wound healing and regeneration of alveolar bone. The ADAM (a disintegrin and metalloprotease) family is a set of multifunctional cell surface and secreted glycoproteins, of which ADAM-28 has been localized in bone and bone-like tissues. In this study, we show that interleukin (IL)-1β induces the expression of MMP-13 and ADAM-28 in homogeneous α7 integrin-positive human skeletal muscle stem cell (α7+hSMSC)-derived osteoblast-like (α7+hSMSC-OB) cells, and promotes proliferation while inhibiting apoptosis in these cells. At higher concentrations, however, IL-1β failed to induce the expression of these genes and caused an increase in apoptosis. We further employed ADAM-28 small interfering RNA (siRNA) to investigate whether IL-1β-induced MMP-13 expression is linked to this IL-1β-mediated changes in cell proliferation and apoptosis. Silencing ADAM-28 expression potently suppressed IL-1β-induced MMP-13 expression and activity, decreased cell proliferation and increased apoptosis in α7+hSMSC-OB cells. In contrast, MMP-13 siRNA had no effect on ADAM-28 expression, suggesting ADAM-28 regulates MMP-13. Exogenous MMP-13 induced α7+hSMSC-OB cell proliferation and could rescue ADAM-28 siRNA-induced apoptosis, and we found that proMMP-13 is partially cleaved into its active form by ADAM-28 in vitro. Overall, our results suggest that IL-1β-induced MMP-13 expression and changes in cell proliferation and apoptosis in α7+hSMSC-OB cells are regulated by ADAM-28. - Highlights: • IL-1β induces the MMP-13 and ADAM-28 expression in human osteoblast-like cells. • IL-1β-induced MMP-13 expression increases proliferation and decreased apoptosis. • MMP-13 expression induced by IL-1β is regulated by ADAM-28. • proMMP-13 appears to be cleaved into its active form via ADAM-28

  15. Quercus infectoria Gall Extract Enhanced the Proliferation and Activity of Human Fetal Osteoblast Cell Line (hFOB 1.19)

    OpenAIRE

    Hapidin, Hermizi; ROZELAN, Dalila; ABDULLAH, Hasmah; WAN HANAFFI, Wan Nurhidayah; Soelaiman, Ima Nirwana

    2015-01-01

    Background: The present study investigated the effects of Quercus infectoria (QI) gall extract on the proliferation, alkaline phosphatase (ALP), osteocalcin, and the morphology of a human fetal osteoblast cell line (hFOB 1.19).

  16. Microgravity induces pelvic bone loss through osteoclastic activity, osteocytic osteolysis, and osteoblastic cell cycle inhibition by CDKN1a/p21.

    Science.gov (United States)

    Blaber, Elizabeth A; Dvorochkin, Natalya; Lee, Chialing; Alwood, Joshua S; Yousuf, Rukhsana; Pianetta, Piero; Globus, Ruth K; Burns, Brendan P; Almeida, Eduardo A C

    2013-01-01

    Bone is a dynamically remodeled tissue that requires gravity-mediated mechanical stimulation for maintenance of mineral content and structure. Homeostasis in bone occurs through a balance in the activities and signaling of osteoclasts, osteoblasts, and osteocytes, as well as proliferation and differentiation of their stem cell progenitors. Microgravity and unloading are known to cause osteoclast-mediated bone resorption; however, we hypothesize that osteocytic osteolysis, and cell cycle arrest during osteogenesis may also contribute to bone loss in space. To test this possibility, we exposed 16-week-old female C57BL/6J mice (n = 8) to microgravity for 15-days on the STS-131 space shuttle mission. Analysis of the pelvis by µCT shows decreases in bone volume fraction (BV/TV) of 6.29%, and bone thickness of 11.91%. TRAP-positive osteoclast-covered trabecular bone surfaces also increased in microgravity by 170% (p = 0.004), indicating osteoclastic bone degeneration. High-resolution X-ray nanoCT studies revealed signs of lacunar osteolysis, including increases in cross-sectional area (+17%, p = 0.022), perimeter (+14%, p = 0.008), and canalicular diameter (+6%, p = 0.037). Expression of matrix metalloproteinases (MMP) 1, 3, and 10 in bone, as measured by RT-qPCR, was also up-regulated in microgravity (+12.94, +2.98 and +16.85 fold respectively, p<0.01), with MMP10 localized to osteocytes, and consistent with induction of osteocytic osteolysis. Furthermore, expression of CDKN1a/p21 in bone increased 3.31 fold (p<0.01), and was localized to osteoblasts, possibly inhibiting the cell cycle during tissue regeneration as well as conferring apoptosis resistance to these cells. Finally the apoptosis inducer Trp53 was down-regulated by -1.54 fold (p<0.01), possibly associated with the quiescent survival-promoting function of CDKN1a/p21. In conclusion, our findings identify the pelvic and femoral region of the mouse skeleton as an active site of rapid bone

  17. Alpha-adrenergic blocker mediated osteoblastic stem cell differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Yoon Jung [Craniomaxillofacial Reconstructive Sciences Major, College of Dentistry, Seoul National University, Seoul 110-749 (Korea, Republic of); Lee, Jue Yeon [Craniomaxillofacial Reconstructive Sciences Major, College of Dentistry, Seoul National University, Seoul 110-749 (Korea, Republic of); Research Center, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul (Korea, Republic of); Lee, Seung Jin [Department of Industrial Pharmacy, College of Pharmacy, Ewha Womans University, Seoul (Korea, Republic of); Research Center, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul (Korea, Republic of); Chung, Chong-Pyoung [Department of Periodontology, School of Dentistry, Seoul National University, Seoul (Korea, Republic of); Research Center, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul (Korea, Republic of); Park, Yoon Jeong, E-mail: parkyj@snu.ac.kr [Craniomaxillofacial Reconstructive Sciences Major, College of Dentistry, Seoul National University, Seoul 110-749 (Korea, Republic of); Research Center, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul (Korea, Republic of)

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer Doxazocin directly up-regulated bone metabolism at a low dose. Black-Right-Pointing-Pointer Doxazocin induced osteoblastic stem cell differentiation without affecting cell proliferation. Black-Right-Pointing-Pointer This osteogenic stem cell differentiation is mediated by ERK-signal dependent pathway. -- Abstract: Recent researches have indicated a role for antihypertensive drugs including alpha- or beta-blockers in the prevention of bone loss. Some epidemiological studies reported the protective effects of those agents on fracture risk. However, there is limited information on the association with those agents especially at the mechanism of action. In the present study, we investigated the effects of doxazosin, an alpha-blocker that is clinically used for the treatment of benign prostatic hyperplasia (BPH) along with antihypertensive medication, on the osteogenic stem cell differentiation. We found that doxazosin increased osteogenic differentiation of human mesenchymal stem cells, detected by Alizarin red S staining and calcein. Doxazosin not only induced expression of alkaline phosphatase, type I collagen, osteopontin, and osteocalcin, it also resulted in increased phosphorylation of extracellular signal-regulated kinase (ERK1/2), a MAP kinase involved in osteoblastic differentiation. Treatment with U0126, a MAP kinase inhibitor, significantly blocked doxazosin-induced osteoblastic differentiation. Unrelated to activation of osteogenic differentiation by doxazosin, we found that there were no significant changes in adipogenic differentiation or in the expression of adipose-specific genes, including peroxisome proliferator-activated receptor {gamma}, aP2, or LPL. In this report, we suggest that doxazosin has the ability to increase osteogenic cell differentiation via ERK1/2 activation in osteogenic differentiation of adult stem cells, which supports the protective effects of antihypertensive drug on fracture risk and

  18. Effect of nanocoating with rhamnogalacturonan-I on surface properties and osteoblasts response.

    Science.gov (United States)

    Gurzawska, Katarzyna; Svava, Rikke; Syberg, Susanne; Yihua, Yu; Haugshøj, Kenneth Brian; Damager, Iben; Ulvskov, Peter; Christensen, Leif Højslet; Gotfredsen, Klaus; Jørgensen, Niklas Rye

    2012-03-01

    Long-term stability of titanium implants are dependent on a variety of factors. Nanocoating with organic molecules is one of the methods used to improve osseointegration. Therefore, the aim of this study is to evaluate the in vitro effect of nanocoating with pectic rhamnogalacturonan-I (RG-I) on surface properties and osteoblasts response. Three different RG-Is from apple and lupin pectins were modified and coated on amino-functionalized tissue culture polystyrene plates (aminated TCPS). Surface properties were evaluated by scanning electron microscopy, contact angle measurement, atomic force microscopy, and X-ray photoelectron spectroscopy. The effects of nanocoating on proliferation, matrix formation and mineralization, and expression of genes (real-time PCR) related to osteoblast differentiation and activity were tested using human osteoblast-like SaOS-2 cells. It was shown that RG-I coatings affected the surface properties. All three RG-I induced bone matrix formation and mineralization, which was also supported by the finding that gene expression levels of alkaline phosphatase, osteocalcin, and collagen type-1 were increased in cells cultured on the RG-I coated surface, indicating a more differentiated osteoblastic phenotype. This makes RG-I coating a promising and novel candidate for nanocoatings of implants. PMID:22213456

  19. Titanium With Nanotopography Induces Osteoblast Differentiation by Regulating Endogenous Bone Morphogenetic Protein Expression and Signaling Pathway.

    Science.gov (United States)

    M S Castro-Raucci, Larissa; S Francischini, Marcelo; N Teixeira, Lucas; P Ferraz, Emanuela; B Lopes, Helena; T de Oliveira, Paulo; Hassan, Mohammad Q; Losa, Adalberto L; Beloti, Marcio M

    2016-07-01

    We aimed at evaluating the effect of titanium (Ti) with nanotopography (Nano) on the endogenous expression of BMP-2 and BMP-4 and the relevance of this process to the nanotopography-induced osteoblast differentiation. MC3T3-E1 cells were grown on Nano and machined (Machined) Ti surfaces and the endogenous BMP-2/4 expression and the effect of BMP receptor BMPR1A silencing in both osteoblast differentiation and expression of genes related to TGF-β/BMP signaling were evaluated. Nano supported higher BMP-2 gene and protein expression and upregulated the osteoblast differentiation compared with Machined Ti surface. The BMPR1A silencing inhibited the osteogenic potential induced by Nano Ti surface as indicated by reduced alkaline phosphatase (ALP), osteocalcin and RUNX2 gene expression, RUNX2 protein expression and ALP activity. In addition, the expression of genes related to TGF-β/BMP signaling was deeply affected by BMPR1A-silenced cells grown on Nano Ti surface. In conclusion, we have demonstrated for the first time that nanotopography induces osteoblast differentiation, at least in part, by upregulating the endogenous production of BMP-2 and modulating BMP signaling pathway. J. Cell. Biochem. 117: 1718-1726, 2016. © 2015 Wiley Periodicals, Inc. PMID:26681207

  20. Do Real Estate Loans Affect Economic Activity?

    OpenAIRE

    Z. Onder; S.Ozyildirim Gunalay; S. Ozyildirim; Y. Gunalay

    2007-01-01

    Real estate, especially housing, has an important share in the nationís wealth. For example, real estate constitutes 48.8% of total wealth in the world in 1991. Moreover, housing construction and its financing are two important factors that affect economic development in developed and developing countries. Although the mortgage system has been developing in the 1980s and 1990s in the developing economies, the impact of mortgage credits on the economy has been ignored in the literature. The on...

  1. Estradiol influences the mechanical properties of human fetal osteoblasts through cytoskeletal changes

    International Nuclear Information System (INIS)

    Highlights: ► Estradiol induced stiffness changes of osteoblasts were quantified using AFM. ► Estradiol causes significant decrease in the stiffness of osteoblasts. ► Decreased stiffness was caused by decreased density of f-actin network. ► Stiffness changes were not associated with mineralized matrix of osteoblasts. ► Estradiol increases inherent alkaline phosphatase activity of osteoblasts. -- Abstract: Estrogen is known to have a direct effect on bone forming osteoblasts and bone resorbing osteoclasts. The cellular and molecular effects of estrogen on osteoblasts and osteoblasts-like cells have been extensively studied. However, the effect of estrogen on the mechanical property of osteoblasts has not been studied yet. It is important since mechanical property of the mechanosensory osteoblasts could be pivotal to its functionality in bone remodeling. This is the first study aimed to assess the direct effect of estradiol on the apparent elastic modulus (E∗) and corresponding cytoskeletal changes of human fetal osteoblasts (hFOB 1.19). The cells were cultured in either medium alone or medium supplemented with β-estradiol and then subjected to Atomic Force Microscopy indentation (AFM) to determine E∗. The underlying changes in cytoskeleton were studied by staining the cells with TRITC-Phalloidin. Following estradiol treatment, the cells were also tested for proliferation, alkaline phosphatase activity and mineralization. With estradiol treatment, E∗ of osteoblasts significantly decreased by 43–46%. The confocal images showed that the changes in f-actin network observed in estradiol treated cells can give rise to the changes in the stiffness of the cells. Estradiol also increases the inherent alkaline phosphatase activity of the cells. Estradiol induced stiffness changes of osteoblasts were not associated with changes in the synthesized mineralized matrix of the cells. Thus, a decrease in osteoblast stiffness with estrogen treatment was

  2. Ectomycorrhizal activity as affected by soil liming

    Energy Technology Data Exchange (ETDEWEB)

    Andersson, Solbritt

    1996-05-01

    Acidification of the forest soils in southern Sweden due to atmospheric deposition has become evident during recent decades. To counteract further acidification, liming of forests in the most affected areas has been proposed. Most forest trees in the temperate and boreal forest ecosystems live in symbiosis with ectomycorrhizal fungi, and their uptake of mineral nutrients from the soil is greatly influenced by the symbiosis. In this thesis effects of liming on ectomycorrhiza have been studied in relation to effects on root colonization, fungal growth and nitrogen uptake. In field experiments the effects of liming on ectomycorrhizal colonization of root tips were variable, possibly due to different soil types and climatic variations. However, a changed mycorrhizal community structure could be detected. Laboratory studies also showed that the substrate may influence the outcome of lime applications; the nutrient status of the substrate had a marked effect on how mycelial growth was affected by liming. Under the experimental conditions used in the studies presented in this thesis, liming reduced the uptake of nitrogen and phosphorus by both mycorrhizal and non-mycorrhizal plants. The amount of extractable nitrogen and phosphorus in the peat was also reduced by liming. The latter could be due to either microbial or chemical immobilization. The lime induced decrease in nitrogen uptake was stronger in non-mycorrhizal plants than in mycorrhizal plants. Thus, the mycorrhizal plants had a higher ability to deal with the negative effects of liming on nitrogen availability. This was not the case for phosphorus. The lime induced decrease in phosphorus uptake was stronger for mycorrhizal plants, and in the highest lime treatment there was no significant difference between the mycorrhizal and the non-mycorrhizal spruce plants. 76 refs, 2 figs, 1 tab

  3. Irisin Enhances Osteoblast Differentiation In Vitro

    Directory of Open Access Journals (Sweden)

    Graziana Colaianni

    2014-01-01

    Full Text Available It has been recently demonstrated that exercise activity increases the expression of the myokine Irisin in skeletal muscle, which is able to drive the transition of white to brown adipocytes, likely following a phenomenon of transdifferentiation. This new evidence supports the idea that muscle can be considered an endocrine organ, given its ability to target adipose tissue by promoting energy expenditure. In accordance with these new findings, we hypothesized that Irisin is directly involved in bone metabolism, demonstrating its ability to increase the differentiation of bone marrow stromal cells into mature osteoblasts. Firstly, we confirmed that myoblasts from mice subjected to 3 weeks of free wheel running increased Irisin expression compared to nonexercised state. The conditioned media (CM collected from myoblasts of exercised mice induced osteoblast differentiation in vitro to a greater extent than those of mice housed in resting conditions. Furthermore, the differentiated osteoblasts increased alkaline phosphatase and collagen I expression by an Irisin-dependent mechanism. Our results show, for the first time, that Irisin directly targets osteoblasts, enhancing their differentiation. This finding advances notable perspectives in future studies which could satisfy the ongoing research of exercise-mimetic therapies with anabolic action on the skeleton.

  4. Ionizing radiation affects active ileal electrolyte transport

    International Nuclear Information System (INIS)

    Exposure to ionizing radiation has pronounced effects on gastrointestinal physiology eliciting the fluid and electrolyte loss of the gastrointestinal syndrome. This study reports on the effect of whole-body cobalt-60 exposure on active electrolyte transport by rabbit ileum in an effort to quantify these changes and to define the mechanism by which electrolyte transport is altered. The short-circuit current (lsc), a measure of active electrolyte transport, was determined for ileal segments isolated from rabbits radiated with 5 to 100 Gy and compared to those from sham irradiated control 1 to 96 hours after exposure. One hour after exposure there was no apparent effect of radiation. However by 24 hours, there was a significant increase in lsc of segments from animals exposed to doses of 7.5 Gy and greater. The lsc remained elevated during the 96 hours for 10 and 12 Gy whereas at 7.5 Gy it returned to control values by 72 hours. The response of the tissue to a secretagogue, theophylline, was reduced 72 hours post-irradiation. By 96 hours after exposure, the response to an actively transported amino acid, alanine, was also reduced. These results indicate that radiation-induced fluid and electrolyte loss is not simply a consequence of denudiation of the intestine but due in part to alterations in cellular transport processes

  5. Human osteoblasts from younger normal and osteoporotic donors show differences in proliferation and TGF beta-release in response to cyclic strain.

    Science.gov (United States)

    Neidlinger-Wilke, C; Stalla, I; Claes, L; Brand, R; Hoellen, I; Rübenacker, S; Arand, M; Kinzl, L

    1995-12-01

    Mechanical stimulation of bone tissue by physical activity stimulates bone formation in normal bone and may attenuate bone loss of osteoporotic patients. However, altered responsiveness of osteoblasts in osteoporotic bone to mechanical stimuli may contribute to osteoporotic bone involution. The purpose of the present study was to investigate whether osteoblasts from osteoporotic patients and normal donors show differences in proliferation and TGF beta production in responses to cyclic strain. Human osteoblasts isolated from collagenase-treated bone explants of 10 osteoporotic patients (average age 70 +/- 6 yr) and 8 normal donors (average age 54 +/- 10 yr) were plated into elastic rectangular silicone dishes. Subconfluent cultures were stimulated by cyclic strain (1%, 1 Hz) in electromechanical cell stretching apparatus at three consecutive days for each 30 min. The cultures were assayed for proliferation, alkaline phosphatase activity and TGF beta release in each three parallel cultures. In all experiments, osteoblasts grown in the same elastic dishes but without mechanical stimulation served as controls. Significant differences between stimulated cultures and unstimulated controls were determined by a paired two-tailed Wilcoxon test. In comparison to the unstimulated controls, osteoblasts from normal donors significantly increased proliferation (p = 0.025) and TGF beta secretion (p = 0.009) into the conditioned culture medium. In contrast, osteoblasts from osteoporotic donors failed to increase both proliferation (p > 0.05) and TGF beta release (p > 0.05) in response to cyclic strain. Alkaline phosphatase activity was not significantly affected (p > 0.05) in normal as well as osteoporotic bone derived osteoblasts. These findings suggest a different responsiveness to 1% cyclic strain of osteoblasts isolated from normal and osteoporotic bone that could be influenced by both the disease of osteoporosis and the higher average age of the osteoporotic patient group

  6. Effects of IL-23 and IL-27 on osteoblasts and osteoclasts: inhibitory effects on osteoclast differentiation.

    Science.gov (United States)

    Kamiya, Sadahiro; Nakamura, Chika; Fukawa, Takeshi; Ono, Katsuhiro; Ohwaki, Toshiyuki; Yoshimoto, Takayuki; Wada, Seiki

    2007-01-01

    Interleukin (IL)-23 and IL-27 are IL-6/IL-12 family members that play a role in the regulation of T helper 1 cell differentiation. Cytokines are known to be involved in the bone remodeling process, although the effects of IL-23 and IL-27 have not been clarified. In this study, we examined the possible roles of these cytokines on osteoblast phenotypes and osteoclastogenesis. We found that IL-27 induced signal transducers and activators of transcription 3 activation in osteoblasts. However, neither IL-23 nor IL-27 showed any significant effects on alkaline phosphatase activity, receptor activator of nuclear factor kappaB ligand (RANKL) expression, mRNA expression such as alkaline phosphatase type I procollagen, or the proliferation of osteoblasts. Osteoclastogenesis from bone marrow cells induced by soluble RANKL was partially inhibited by IL-23 and IL-27 with reduced multinucleated cell numbers, but these interleukins did not affect the proliferation of osteoclast progenitor cells. These results indicate that IL-23 and IL-27 could partly modify cell fusion or the survival of multinucleated osteoclasts. On the other hand, partially purified T cells, which are activated by 2 microg/ml anti-CD3 antibody, completely inhibited osteoclastogenesis by M-CSF/RANKL. On using T cells activated with 0.2 microg/ml anti-CD3 antibody, in which osteoclastogenesis was partially inhibited, the interleukins had additive effects for inhibiting osteoclastogenesis. Although the consequences of phosphorylated signals in osteoblasts have not been identified, IL-23 and IL-27, partly and indirectly through activated T cells, inhibited osteoclastogenesis, indicating that these interleukins may protect against bone destructive autoimmune disorders. PMID:17704992

  7. Adenosine Triphosphate stimulates differentiation and mineralization in human osteoblast-like Saos-2 cells.

    Science.gov (United States)

    Cutarelli, Alessandro; Marini, Mario; Tancredi, Virginia; D'Arcangelo, Giovanna; Murdocca, Michela; Frank, Claudio; Tarantino, Umberto

    2016-05-01

    In the last years adenosine triphosphate (ATP) and subsequent purinergic system activation through P2 receptors were investigated highlighting their pivotal role in bone tissue biology. In osteoblasts ATP can regulate several activities like cell proliferation, cell death, cell differentiation and matrix mineralization. Since controversial results exist, in this study we analyzed the ATP effects on differentiation and mineralization in human osteoblast-like Saos-2 cells. We showed for the first time the altered functional activity of ATP receptors. Despite that, we found that ATP can reduce cell proliferation and stimulate osteogenic differentiation mainly in the early stages of in vitro maturation as evidenced by the enhanced expression of alkaline phosphatase (ALP), Runt-related transcription factor 2 (Runx2) and Osteocalcin (OC) genes and by the increased ALP activity. Moreover, we found that ATP can affect mineralization in a biphasic manner, at low concentrations ATP always increases mineral deposition while at high concentrations it always reduces mineral deposition. In conclusion, we show the osteogenic effect of ATP on both early and late stage activities like differentiation and mineralization, for the first time in human osteoblastic cells. PMID:27189526

  8. Pyk2 and Megakaryocytes Regulate Osteoblast Differentiation and Migration Via Distinct and Overlapping Mechanisms.

    Science.gov (United States)

    Eleniste, Pierre P; Patel, Vruti; Posritong, Sumana; Zero, Odette; Largura, Heather; Cheng, Ying-Hua; Himes, Evan R; Hamilton, Matthew; Baughman, Jenna; Kacena, Melissa A; Bruzzaniti, Angela

    2016-06-01

    Osteoblast differentiation and migration are necessary for bone formation during bone remodeling. Mice lacking the proline-rich tyrosine kinase Pyk2 (Pyk2-KO) have increased bone mass, in part due to increased osteoblast proliferation. Megakaryocytes (MKs), the platelet-producing cells, also promote osteoblast proliferation in vitro and bone-formation in vivo via a pathway that involves Pyk2. In the current study, we examined the mechanism of action of Pyk2, and the role of MKs, on osteoblast differentiation and migration. We found that Pyk2-KO osteoblasts express elevated alkaline phosphatase (ALP), type I collagen and osteocalcin mRNA levels as well as increased ALP activity, and mineralization, confirming that Pyk2 negatively regulates osteoblast function. Since Pyk2 Y402 phosphorylation is important for its catalytic activity and for its protein-scaffolding functions, we expressed the phosphorylation-mutant (Pyk2(Y402F) ) and kinase-mutant (Pyk2(K457A) ) in Pyk2-KO osteoblasts. Both Pyk2(Y402F) and Pyk2(K457A) reduced ALP activity, whereas only kinase-inactive Pyk2(K457A) inhibited Pyk2-KO osteoblast migration. Consistent with a role for Pyk2 on ALP activity, co-culture of MKs with osteoblasts led to a decrease in the level of phosphorylated Pyk2 (pY402) as well as a decrease in ALP activity. Although, Pyk2-KO osteoblasts exhibited increased migration compared to wild-type osteoblasts, Pyk2 expression was not required necessary for the ability of MKs to stimulate osteoblast migration. Together, these data suggest that osteoblast differentiation and migration are inversely regulated by MKs via distinct Pyk2-dependent and independent signaling pathways. Novel drugs that distinguish between the kinase-dependent or protein-scaffolding functions of Pyk2 may provide therapeutic specificity for the control of bone-related diseases. J. Cell. Biochem. 117: 1396-1406, 2016. © 2015 Wiley Periodicals, Inc. PMID:26552846

  9. Ultrastructural and metabolic changes in osteoblasts exposed to uranyl nitrate

    International Nuclear Information System (INIS)

    Exposure to uranium is an occupational hazard to workers who continually handle uranium and an environmental risk to the population at large. Since the cellular and molecular pathways of uranium toxicity in osteoblast cells are still unknown, the aim of the present work was to evaluate the adverse effects of uranyl nitrate (UN) on osteoblasts both in vivo and in vitro. Herein we studied the osteoblastic ultrastructural changes induced by UN in vivo and analyzed cell proliferation, generation of reactive oxygen species (ROS), apoptosis, and alkaline phosphatase (APh) activity in osteoblasts exposed to various UN concentrations (0.1, 1, 10, and 100 μM) in vitro. Cell proliferation was quantified by means of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, ROS was determined using the nitro blue tetrazolium test, apoptosis was morphologically determined using Hoechst 3332 and APh activity was assayed spectrophotometrically. Electron microscopy revealed that the ultrastructure of active and inactive osteoblasts exposed to uranium presented cytoplasmic and nuclear alterations. In vitro, 1-100 μM UN failed to modify cell proliferation ratio and to induce apoptosis. ROS generation increased in a dose-dependent manner in all tested doses. APh activity was found to decrease in 1-100 μM UN-treated cells vs. controls. Our results show that UN modifies osteoblast cell metabolism by increasing ROS generation and reducing APh activity, suggesting that ROS may play a more complex role in cell physiology than simply causing oxidative damage. (orig.)

  10. Ultrastructural and metabolic changes in osteoblasts exposed to uranyl nitrate

    Energy Technology Data Exchange (ETDEWEB)

    Tasat, D.R. [Universidad Nacional de San Martin, Escuela de Ciencia y Tecnologia, Pcia de Bs.As. (Argentina); Universidad de Buenos Aires, Catedra de Histologia y Embriologia, Facultad de Odontologia, Buenos Aires (Argentina); Orona, N.S. [Universidad Nacional de San Martin, Escuela de Ciencia y Tecnologia, Pcia de Bs.As. (Argentina); Mandalunis, P.M. [Universidad de Buenos Aires, Catedra de Histologia y Embriologia, Facultad de Odontologia, Buenos Aires (Argentina); Cabrini, R.L. [Comision Nacional de Energia Atomica, Departamento de Radiobiologia, Buenos Aires (Argentina); Ubios, A.M. [Comision Nacional de Energia Atomica, Departamento de Radiobiologia, Buenos Aires (Argentina); Universidad de Buenos Aires, Catedra de Histologia y Embriologia, Facultad de Odontologia, Buenos Aires (Argentina)

    2007-05-15

    Exposure to uranium is an occupational hazard to workers who continually handle uranium and an environmental risk to the population at large. Since the cellular and molecular pathways of uranium toxicity in osteoblast cells are still unknown, the aim of the present work was to evaluate the adverse effects of uranyl nitrate (UN) on osteoblasts both in vivo and in vitro. Herein we studied the osteoblastic ultrastructural changes induced by UN in vivo and analyzed cell proliferation, generation of reactive oxygen species (ROS), apoptosis, and alkaline phosphatase (APh) activity in osteoblasts exposed to various UN concentrations (0.1, 1, 10, and 100 {mu}M) in vitro. Cell proliferation was quantified by means of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, ROS was determined using the nitro blue tetrazolium test, apoptosis was morphologically determined using Hoechst 3332 and APh activity was assayed spectrophotometrically. Electron microscopy revealed that the ultrastructure of active and inactive osteoblasts exposed to uranium presented cytoplasmic and nuclear alterations. In vitro, 1-100 {mu}M UN failed to modify cell proliferation ratio and to induce apoptosis. ROS generation increased in a dose-dependent manner in all tested doses. APh activity was found to decrease in 1-100 {mu}M UN-treated cells vs. controls. Our results show that UN modifies osteoblast cell metabolism by increasing ROS generation and reducing APh activity, suggesting that ROS may play a more complex role in cell physiology than simply causing oxidative damage. (orig.)

  11. Bezafibrate enhances proliferation and differentiation of osteoblastic MC3T3-E1 cells via AMPK and eNOS activation

    OpenAIRE

    Zhong, Xing; Xiu, Ling-ling; Wei, Guo-hong; Liu, Yuan-Yuan; Su, Lei; Cao, Xiao-pei; Li, Yan-Bing; Xiao, Hai-peng

    2011-01-01

    Aim: To investigate the effects of bezafibrate on the proliferation and differentiation of osteoblastic MC3T3-E1 cells, and to determine the signaling pathway underlying the effects. Methods: MC3T3-E1 cells, a mouse osteoblastic cell line, were used. Cell viability and proliferation were examined using MTT assay and colorimetric BrdU incorporation assay, respectively. NO production was evaluated using the Griess reagent. The mRNA expression of ALP, collagen I, osteocalcin, BMP-2, and Runx-2 w...

  12. Biologically active extracts with kidney affections applications

    Science.gov (United States)

    Pascu (Neagu), Mihaela; Pascu, Daniela-Elena; Cozea, Andreea; Bunaciu, Andrei A.; Miron, Alexandra Raluca; Nechifor, Cristina Aurelia

    2015-12-01

    This paper is aimed to select plant materials rich in bioflavonoid compounds, made from herbs known for their application performances in the prevention and therapy of renal diseases, namely kidney stones and urinary infections (renal lithiasis, nephritis, urethritis, cystitis, etc.). This paper presents a comparative study of the medicinal plant extracts composition belonging to Ericaceae-Cranberry (fruit and leaves) - Vaccinium vitis-idaea L. and Bilberry (fruit) - Vaccinium myrtillus L. Concentrated extracts obtained from medicinal plants used in this work were analyzed from structural, morphological and compositional points of view using different techniques: chromatographic methods (HPLC), scanning electronic microscopy, infrared, and UV spectrophotometry, also by using kinetic model. Liquid chromatography was able to identify the specific compounds of the Ericaceae family, present in all three extracts, arbutosid, as well as specific components of each species, mostly from the class of polyphenols. The identification and quantitative determination of the active ingredients from these extracts can give information related to their therapeutic effects.

  13. Affective Response to Physical Activity: Testing for Measurement Invariance of the Physical Activity Affect Scale across Active and Non-Active Individuals

    Science.gov (United States)

    Carpenter, Laura C.; Tompkins, Sara Anne; Schmiege, Sarah J.; Nilsson, Renea; Bryan, Angela

    2010-01-01

    Affective responses to physical activity are assumed to play a role in exercise initiation and maintenance. The Physical Activity Affect Scale measures four dimensions of an individual's affective response to exercise. Group differences in the interpretation of scale items can impact the interpretability of mean differences, underscoring the need…

  14. The effect of plasma-nitrided titanium surfaces on osteoblastic cell adhesion, proliferation, and differentiation.

    Science.gov (United States)

    Ferraz, Emanuela P; Sa, Juliana C; de Oliveira, Paulo T; Alves, Clodomiro; Beloti, Marcio M; Rosa, Adalberto L

    2014-04-01

    In this study, we evaluated the effect of new plasma-nitrided Ti surfaces on the progression of osteoblast cultures, including cell adhesion, proliferation and differentiation. Ti surfaces were treated using two plasma-nitriding protocols, hollow cathode for 3 h (HC 3 h) and 1 h (HC 1 h) and planar for 1 h. Untreated Ti surfaces were used as control. Cells derived from human alveolar and rat calvarial bones were cultured on Ti surfaces for periods of up to 14 days and the following parameters were evaluated: cell morphology, adhesion, spreading and proliferation, alkaline phosphatase (ALP) activity, extracellular matrix mineralization, and gene expression of key osteoblast markers. Plasma-nitriding treatments resulted in Ti surfaces with distinct physicochemical characteristics. The cell adhesion and ALP activity were higher on plasma-nitrided Ti surfaces compared with untreated one, whereas cell proliferation and extracellular matrix mineralization were not affected by the treatments. In addition, the plasma-nitrided Ti surfaces increased the ALP, reduced the osteocalcin and did not affect the Runx2 gene expression. We have shown that HC 3 h and planar Ti surfaces slightly favored the osteoblast differentiation process, and then these surfaces should be considered for further investigation using preclinical models. PMID:23625878

  15. Nemo-like kinase (NLK) expression in osteoblastic cells and suppression of osteoblastic differentiation

    International Nuclear Information System (INIS)

    Mitogen-activated protein kinases (MAPKs) regulate proliferation and differentiation in osteoblasts. The vertebral homologue of nemo, nemo-like kinase (NLK), is an atypical MAPK that targets several signaling components, including the T-cell factor/lymphoid enhancer factor (TCF/Lef1) transcription factor. Recent studies have shown that NLK forms a complex with the histone H3-K9 methyltransferase SETDB1 and suppresses peroxisome proliferator-activated receptor (PPAR)-gamma:: action in the mesenchymal cell line ST2. Here we investigated whether NLK regulates osteoblastic differentiation. We showed that NLK mRNA is expressed in vivo in osteoblasts at embryonic day 18.5 (E18.5) mouse calvariae. By using retrovirus vectors, we performed forced expression of NLK in primary calvarial osteoblasts (pOB cells) and the mesenchymal cell line ST2. Wild-type NLK (NLK-WT) suppressed alkaline phosphatase activity and expression of bone marker genes such as alkaline phosphatase, type I procollagen, runx2, osterix, steopontin and osteocalcin in these cells. NLK-WT also decreased type I collagen protein expression in pOB and ST2 cells. Furthermore, mineralized nodule formation was reduced in pOB cells overexpressing NLK-WT. In contrast, kinase-negative form of NLK (NLK-KN) did not suppress or partially suppress ALP activity and bone marker gene expression in pOB and ST2 cells. NLK-KN did not suppress nodule formation in pOB cells. In addition to forced expression, suppression of endogenous NLK expression by siRNA increased bone marker gene expression in pOB and ST2 cells. Finally, transcriptional activity analysis of gene promoters revealed that NLK-WT suppressed Wnt1 activation of TOP flash promoter and Runx2 activation of the osteocalcin promoter. Taken together, these results suggest that NLK negatively regulates osteoblastic differentiation.

  16. Nemo-like kinase (NLK) expression in osteoblastic cells and suppression of osteoblastic differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Nifuji, Akira, E-mail: nifuji-a@tsurumi-u.ac.jp [Transcriptome profiling group, National Institute of Radiological Sciences, Chiba (Japan); Department of Pharmacology, Tsurumi University School of Dental Medicine, Yokohama (Japan); Ideno, Hisashi [Transcriptome profiling group, National Institute of Radiological Sciences, Chiba (Japan); Ohyama, Yoshio [Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo (Japan); Takanabe, Rieko; Araki, Ryoko; Abe, Masumi [Transcriptome profiling group, National Institute of Radiological Sciences, Chiba (Japan); Noda, Masaki [Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo (Japan); Shibuya, Hiroshi [Department of Molecular Cell Biology, Medical Research Institute and School of Biomedical Science, Tokyo Medical and Dental University, Tokyo (Japan)

    2010-04-15

    Mitogen-activated protein kinases (MAPKs) regulate proliferation and differentiation in osteoblasts. The vertebral homologue of nemo, nemo-like kinase (NLK), is an atypical MAPK that targets several signaling components, including the T-cell factor/lymphoid enhancer factor (TCF/Lef1) transcription factor. Recent studies have shown that NLK forms a complex with the histone H3-K9 methyltransferase SETDB1 and suppresses peroxisome proliferator-activated receptor (PPAR)-gamma:: action in the mesenchymal cell line ST2. Here we investigated whether NLK regulates osteoblastic differentiation. We showed that NLK mRNA is expressed in vivo in osteoblasts at embryonic day 18.5 (E18.5) mouse calvariae. By using retrovirus vectors, we performed forced expression of NLK in primary calvarial osteoblasts (pOB cells) and the mesenchymal cell line ST2. Wild-type NLK (NLK-WT) suppressed alkaline phosphatase activity and expression of bone marker genes such as alkaline phosphatase, type I procollagen, runx2, osterix, steopontin and osteocalcin in these cells. NLK-WT also decreased type I collagen protein expression in pOB and ST2 cells. Furthermore, mineralized nodule formation was reduced in pOB cells overexpressing NLK-WT. In contrast, kinase-negative form of NLK (NLK-KN) did not suppress or partially suppress ALP activity and bone marker gene expression in pOB and ST2 cells. NLK-KN did not suppress nodule formation in pOB cells. In addition to forced expression, suppression of endogenous NLK expression by siRNA increased bone marker gene expression in pOB and ST2 cells. Finally, transcriptional activity analysis of gene promoters revealed that NLK-WT suppressed Wnt1 activation of TOP flash promoter and Runx2 activation of the osteocalcin promoter. Taken together, these results suggest that NLK negatively regulates osteoblastic differentiation.

  17. Bezafibrate enhances proliferation and differentiation of osteoblastic MC3T3-E1 cells via AMPK and eNOS activation

    Institute of Scientific and Technical Information of China (English)

    Xing ZHONG; Ling-ling XIU; Guo-hong WEI; Yuan-yuan LIU; Lei SU; Xiao-pei CAO; Yan-bing LI; Hai-peng XIAO

    2011-01-01

    Aim: To investigate the effects of bezafibrate on the proliferation and differentiation of osteoblastic MC3T3-E1 cells, and to determine the signaling pathway underlying the effects.Methods: MC3T3-E1 cells, a mouse osteoblastic cell line, were used. Cell viability and proliferation were examined using MTT assay and colorimetric BrdU incorporation assay, respectively. NO production was evaluated using the Griess reagent. The mRNA expression of ALP, collagen I, osteocalcin, BMP-2, and Runx-2 was measured using real-time PCR. Western blot analysis was used to detect the expression of AMPK and eNOS proteins.Results: Bezafibrate increased the viability and proliferation of MC3T3-E1 cells in a dose- and time-dependent manner. Bezafibrate (100 μmol/L) significantly enhanced osteoblastic mineralization and expression of the differentiation markers ALP, collagen I and osteocalcin. Bezaflbrate (100 μmol/L) increased phosphorylation of AMPK and eNOS, which led to an increase of NO production by 4.08-fold, and upregulating BMP-2 and Runx-2 mRNA expression. These effects could be blocked by AMPK inhibitor compound C (5 μmol/L), or the PPARβ inhibitor GSK0660 (0.5 μmol/L), but not by the PPARa inhibitor MK886 (10 μmol/L). Furthermore, GSK0660, compound C, or N-nitro-L-arginine methyl ester hydrochloride (L-NAME, 1 mmol/L) could reverse the stimulatory effects of bezafibrate (100 pmol/L) on osteoblast proliferation and differentiation, whereas MK886 only inhibited bezafibrate-induced osteoblast prolifera-tion.Conclusion: Bezafibrate stimulates proliferation and differentiation of MC3T3-E1 cells, mainly via a PPARβ-dependent mechanism. The drug might be beneficial for osteoporosis by promoting bone formation.

  18. Osteoblast cell response to surface-modified carbon nanotubes

    International Nuclear Information System (INIS)

    In order to investigate the interaction of cells with modified multi-walled carbon nanotubes (MWCNTs) for their potential biomedical applications, the MWCNTs were chemically modified with carboxylic acid groups (–COOH), polyvinyl alcohol (PVA) polymer and biomimetic apatite on their surfaces. Additionally, human osteoblast MG-63 cells were cultured in the presence of the surface-modified MWCNTs. The metabolic activities of osteoblastic cells, cell proliferation properties, as well as cell morphology were studied. The surface modification of MWCNTs with biomimetic apatite exhibited a significant increase in the cell viability of osteoblasts, up to 67.23%. In the proliferation phases, there were many more cells in the biomimetic apatite-modified MWCNT samples than in the MWCNTs–COOH. There were no obvious changes in cell morphology in osteoblastic MG-63 cells cultured in the presence of these chemically-modified MWCNTs. The surface modification of MWCNTs with apatite achieves an effective enhancement of their biocompatibility.

  19. Effects of pyrite bioleaching solution of Acidithiobacillus ferrooxidans on viability, differentiation and mineralization potentials of rat osteoblasts.

    Science.gov (United States)

    Zhou, Jian; Chen, Ke-Ming; Zhi, De-Juan; Xie, Qin-Jian; Xian, Cory J; Li, Hong-Yu

    2015-12-01

    Iron pyrite, an important component of traditional Chinese medicine, has a poor solubility, bioavailability, and patient compliance due to a high dose required and associated side effects, all of which have limited its clinical applications and experimental studies on its action mechanisms in improving fracture healing. This study investigated Acidithiobacillus ferrooxidans (A.f)-bioleaching of two kinds of pyrites and examined bioactivities of the derived solutions in viability and osteogenic differentiation in rat calvarial osteoblasts. A.f bioleaching improved element contents (Fe, Mn, Zn, Cu, and Se) in the derived solutions and the solutions concentration-dependently affected osteoblast viability and differentiation. While the solutions had no effects at low concentrations and inhibited the osteoblast alkaline phosphatase (ALP) activity at high concentrations, they improved ALP activity at their optimal concentrations. The improved osteoblast differentiation and osteogenic function at optimal concentrations were also revealed by levels of ALP cytochemical staining, calcium deposition, numbers and areas of mineralized nodules formed, mRNA and protein expression levels of osteogenesis-related genes (osteocalcin, Bmp-2, Runx-2, and IGF-1), and Runx-2 nuclear translocation. Data from this study will be useful in offering new strategies for improving pyrite bioavailability and providing a mechanistic explanation for the beneficial effects of pyrite in improving bone healing. PMID:26283321

  20. Akt1 in osteoblasts and osteoclasts controls bone remodeling.

    Directory of Open Access Journals (Sweden)

    Naohiro Kawamura

    Full Text Available Bone mass and turnover are maintained by the coordinated balance between bone formation by osteoblasts and bone resorption by osteoclasts, under regulation of many systemic and local factors. Phosphoinositide-dependent serine-threonine protein kinase Akt is one of the key players in the signaling of potent bone anabolic factors. This study initially showed that the disruption of Akt1, a major Akt in osteoblasts and osteoclasts, in mice led to low-turnover osteopenia through dysfunctions of both cells. Ex vivo cell culture analyses revealed that the osteoblast dysfunction was traced to the increased susceptibility to the mitochondria-dependent apoptosis and the decreased transcriptional activity of runt-related transcription factor 2 (Runx2, a master regulator of osteoblast differentiation. Notably, our findings revealed a novel role of Akt1/forkhead box class O (FoxO 3a/Bim axis in the apoptosis of osteoblasts: Akt1 phosphorylates the transcription factor FoxO3a to prevent its nuclear localization, leading to impaired transactivation of its target gene Bim which was also shown to be a potent proapoptotic molecule in osteoblasts. The osteoclast dysfunction was attributed to the cell autonomous defects of differentiation and survival in osteoclasts and the decreased expression of receptor activator of nuclear factor-kappaB ligand (RANKL, a major determinant of osteoclastogenesis, in osteoblasts. Akt1 was established as a crucial regulator of osteoblasts and osteoclasts by promoting their differentiation and survival to maintain bone mass and turnover. The molecular network found in this study will provide a basis for rational therapeutic targets for bone disorders.

  1. 16 CFR 801.3 - Activities in or affecting commerce.

    Science.gov (United States)

    2010-01-01

    ... 16 Commercial Practices 1 2010-01-01 2010-01-01 false Activities in or affecting commerce. 801.3... in or affecting commerce. Section 7A(a)(1) is satisfied if any entity included within the acquiring person, or any entity included within the acquired person, is engaged in commerce or in any...

  2. Increased Tartrate-Resistant Acid Phosphatase Expression in Osteoblasts and Osteocytes in Experimental Osteoporosis in Rats

    OpenAIRE

    Solberg, Lene B.; Brorson, Sverre-Henning; Stordalen, Gunhild A.; Bækkevold, Espen S; Andersson, Göran; Reinholt, Finn P.

    2014-01-01

    Tartrate-resistant acid phosphatase (TRAP) is known as an osteoclast marker, but osteoblasts and osteocytes in the vicinity of bone remodeling sites also express TRAP. Cell culture studies suggest that osteoblasts endocytose osteoclastic TRAP for inactivation. To evaluate whether changes in osteoclast activity could alter TRAP expression in osteoblasts and/or osteocytes in vivo, we studied the ovariectomized and vitamin D-deficient rat (Ovx-D) and rats healing from rickets. Bone sections were...

  3. The effects of bone pâté on human osteoblasts cell cultures.

    Science.gov (United States)

    Quaranta, Nicola; Buccoliero, Cinzia; De Luca, Concetta; Mori, Giorgio; Brunetti, Giacomina; Colucci, Silvia; Colaianni, Graziana; Grano, Maria

    2016-06-01

    The aim of the present study was to evaluate the effect of bone pate on human osteoblast differentiation by measuring cell viability, alkaline phosphatase activity and expression of the transcription factors and of the major components of the extracellular matrix. Although bone paté has been used in ear surgery for many years and when placed in contact with mastoid and external auditory canal bone become viable, the cellular mechanisms that lead to its osteointegration have never been described. Bone paté taken from four patients subjected to mastoidectomy and affected by middle ear and mastoid cholesteatoma was placed in contact with osteoblast-like cell cultures. Four experimental conditions were obtained: cell cultures treated with bone patè, with bone paté mixed with fibrin glue, with fibrin glue and untreated. After 24 h, the viability of the cells was evaluated; after 1 week, alkaline phosphatase activity and the expression of transcription factors and bone matrix proteins were assessed by quantitative polymerase chain reaction. After 24 h osteoblasts showed increased viability when treated with bone paté (19 % increase) and bone pate mixed with fibrin glue (34 % increase). After 1 week, the number of alkaline phosphatase positive cells increased by 97 and 94 % in cultures treated with bone paté alone and bone pate mixed with fibrin glue. Treatment with bone patè upregulated transcription factors and components of the extracellular matrix. The present data show that bone paté has a high osteoinductive potential on human osteoblasts, enhancing their activity. PMID:26133919

  4. The Preventive Effect of Biochanin A on Bone Loss in Ovariectomized Rats: Involvement in Regulation of Growth and Activity of Osteoblasts and Osteoclasts

    OpenAIRE

    Shu-Jem Su; Yao-Tsung Yeh; Huey-Wen Shyu

    2013-01-01

    Biochanin A (BCA) is a major isoflavone abundant in red clover (Trifolium pretense). The protective effect of BCA on bone loss in an ovariectomized (OVX) animal model has never been clarified. The objective of this study was to investigate the biological effects of BCA on bone loss in OVX rats in vivo and on the development of osteoblasts and osteoclasts in vitro. Ovariectomy resulted in a marked increase in body weight and a decrease in femoral bone mineral density and trabecular bone volume...

  5. How do oil prices affect oilrig activity? : an empirical investigation

    OpenAIRE

    2004-01-01

    Resume "How do oil prices affect oilrig activity? An empirical investigation" by Guro Børnes Ringlund. Supervisors: Knut Einar Rosendahl and Terje Skjerpen. In this thesis, I analyse the relationship between oilrig activity and oil price changes for several oil-producing regions in the world. Rig activity is a preparation for future production of oil, through exploration for new fields or development of existing fields, and is thus an indicator for the future level of oil production. ...

  6. Aging impairs osteoblast differentiation of mesenchymal stem cells grown on titanium by favoring adipogenesis

    Science.gov (United States)

    ABUNA, Rodrigo Paolo Flores; STRINGHETTA-GARCIA, Camila Tami; FIORI, Leonardo Pimentel; DORNELLES, Rita Cassia Menegati; ROSA, Adalberto Luiz; BELOTI, Marcio Mateus

    2016-01-01

    ABSTRACT Aging negatively affects bone/titanium implant interactions. Our hypothesis is that the unbalance between osteogenesis and adipogenesis induced by aging may be involved in this phenomenon. Objective We investigated the osteoblast and adipocyte differentiation of mesenchymal stem cells (MSCs) from young and aged rats cultured on Ti. Material and Methods Bone marrow MSCs derived from 1-month and 21-month rats were cultured on Ti discs under osteogenic conditions for periods of up to 21 days and osteoblast and adipocyte markers were evaluated. Results Cell proliferation, alkaline phosphatase (ALP) activity, extracellular matrix mineralization and gene expression of RUNX2, osterix, ALP, bone sialoprotein, osteopontin, and osteocalcin were reduced in cultures of 21-month rats compared with 1-month rats grown on Ti. Gene expression of PPAR-γ , adipocyte protein 2, and resistin and lipid accumulation were increased in cultures of 21-month rats compared with 1-month rats grown on the same conditions. Conclusions These results indicate that the lower osteogenic potential of MSCs derived from aged rats compared with young rats goes along with the higher adipogenic potential in cultures grown on Ti surface. This unbalance between osteoblast and adipocyte differentiation should be considered in dental implant therapy to the elderly population. PMID:27556209

  7. Effects of alpha-calcitonin gene-related peptide on osteoprotegerin and receptor activator of nuclear factor-κB ligand expression in MG-63 osteoblast-like cells exposed to polyethylene particles

    Directory of Open Access Journals (Sweden)

    Kauther Max D

    2010-11-01

    Full Text Available Abstract Background Recent studies demonstrated an impact of the nervous system on particle-induced osteolysis, the major cause of aseptic loosening of joint replacements. Methods In this study of MG-63 osteoblast-like cells we analyzed the influence of ultra-high molecular weight polyethylene (UHMWPE particles and the neurotransmitter alpha-calcitonin gene-related peptide (CGRP on the osteoprotegerin/receptor activator of nuclear factor-κB ligand/receptor activator of nuclear factorκB (OPG/RANKL/RANK system. MG-63 cells were stimulated by different UHMWPE particle concentrations (1:100, 1:500 and different doses of alpha-CGRP (10-7 M, 10-9 M, 10-11 M. RANKL and OPG mRNA expression and protein levels were measured by RT-PCR and Western blot. Results Increasing particle concentrations caused an up-regulation of RANKL after 72 hours. Alpha-CGRP showed a dose-independent depressive effect on particle-induced expression of RANKL mRNA in both cell-particle ratios. RANKL gene transcripts were significantly (P -7 M lead to an up-regulation of OPG protein. Conclusion In conclusion, a possible osteoprotective influence of the neurotransmitter alpha-CGRP on particle stimulated osteoblast-like cells could be shown. Alpha-CGRP might be important for bone metabolism under conditions of particle-induced osteolysis.

  8. Macrophage and osteoblast responses to biphasic calcium phosphate microparticles.

    Science.gov (United States)

    Fellah, Borhane Hakim; Delorme, Bruno; Sohier, Jérôme; Magne, David; Hardouin, Pierre; Layrolle, Pierre

    2010-06-15

    The aim of this work was to investigate in vitro the biological events leading to ectopic bone formation in contact with microporous biphasic calcium phosphate (BCP) ceramics. After implantation, microparticles may arise from their degradation and induce an inflammatory response involving macrophages. The secretion of pro-inflammatory cytokines may affect the differentiation of osteoblasts. Mouse macrophage-like (J774) and osteoblast-like (MC3T3-E1) cells were cultured in the presence of BCP microparticles of different sizes (response initiated by BCP microparticles may have both detrimental and beneficial effects on osteogenesis. PMID:20014296

  9. OSTEOBLAST ADHESION OF BREAST CANCER CELLS WITH SCANNING ACOUSTIC MICROSCOPY

    Energy Technology Data Exchange (ETDEWEB)

    Chiaki Miyasaka; Robyn R. Mercer; Andrea M. Mastro; Ken L. Telschow

    2005-03-01

    Breast cancer frequently metastasizes to the bone. Upon colonizing bone tissue, the cancer cells stimulate osteoclasts (cells that break bone down), resulting in large lesions in the bone. The breast cancer cells also affect osteoblasts (cells that build new bone). Conditioned medium was collected from a bone-metastatic breast cancer cell line, MDA-MB-231, and cultured with an immature osteoblast cell line, MC3T3-E1. Under these conditions the osteoblasts acquired a changed morphology and appeared to adherer in a different way to the substrate and to each other. To characterize cell adhesion, MC3T3-E1 osteoblasts were cultured with or without MDA-MB-231 conditioned medium for two days, and then assayed with a mechanical scanning acoustic reflection microscope (SAM). The SAM indicated that in normal medium the MC3T3-E1 osteoblasts were firmly attached to their plastic substrate. However, MC3T3-E1 cells cultured with MDA-MB-231 conditioned medium displayed both an abnormal shape and poor adhesion at the substrate interface. The cells were fixed and stained to visualize cytoskeletal components using optical microscopic techniques. We were not able to observe these differences until the cells were quite confluent after 7 days of culture. However, using the SAM, we were able to detect these changes within 2 days of culture with MDA-MB-231 conditioned medium

  10. Quercetin Partially Preserves Development of Osteoblast Phenotype in Fetal Rat Calvaria Cells in an Oxidative Stress Environment.

    Science.gov (United States)

    Messer, Jonathan G; La, Stephanie; Hopkins, Robin G; Kipp, Deborah E

    2016-12-01

    Studies are needed to improve understanding of the osteoblast antioxidant response, and the balance between oxidative homeostasis and osteoblast differentiation. The flavonol quercetin aglycone (QRC) up-regulates the osteoblast antioxidant response in vitro without suppressing osteoblast phenotype, suggesting that QRC may preserve osteoblast phenotypic development in cells subsequently exposed to oxidative stress, which suppresses osteoblast differentiation. The aims of this study were to assess the extent that QRC pretreatment preserved development of the osteoblast phenotype in cells subsequently cultured with hydrogen peroxide, an oxidative stressor, and to characterize alterations in the osteoblast antioxidant response and in key antioxidant signaling pathways. We hypothesized that pretreatment with QRC would preserve phenotypic development after hydrogen peroxide treatment, suppress the hydrogen peroxide-induced antioxidant response, and that the antioxidant response would involve alterations in Nrf2 and ERK1/2 signaling. Results showed that treating fetal rat calvarial osteoblasts for 4 days (D5-9) with 300 μM hydrogen peroxide resulted in fewer alkaline phosphatase-positive cells and mineralized nodules, altered cell morphology, and significantly lower osteoblast phenotypic gene expression (P stress response coincided with alterations in phosphorylated ERK1/2, but not Nrf2. These results suggest that QRC suppresses hydrogen peroxide-induced activation of the antioxidant response, and partially preserves osteoblast phenotypic development. J. Cell. Physiol. 231: 2779-2788, 2016. © 2016 Wiley Periodicals, Inc. PMID:27028516

  11. Monitoring Affect States during Effortful Problem Solving Activities

    Science.gov (United States)

    D'Mello, Sidney K.; Lehman, Blair; Person, Natalie

    2010-01-01

    We explored the affective states that students experienced during effortful problem solving activities. We conducted a study where 41 students solved difficult analytical reasoning problems from the Law School Admission Test. Students viewed videos of their faces and screen captures and judged their emotions from a set of 14 states (basic…

  12. Changes in the insulin-like growth factor-system may contribute to in vitro age-related impaired osteoblast functions

    DEFF Research Database (Denmark)

    Kveiborg, Marie; Flyvbjerg, Allan; Rattan, Suresh;

    2000-01-01

    Age-related bone loss is thought to be due to impaired osteoblast functions. Insulin-like growth factors (IGFs) have been shown to be important stimulators of bone formation and osteoblast activities in vitro and in vivo. We tested the hypothesis that in vitro osteoblast senescence is associated ...

  13. ent-Kaurane diterpenoids from Croton tonkinensis stimulate osteoblast differentiation.

    Science.gov (United States)

    Dao, Trong-Tuan; Lee, Kwang-Youl; Jeong, Hyung-Min; Nguyen, Phi-Hung; Tran, Tien Lam; Thuong, Phuong-Thien; Nguyen, Bich-Thu; Oh, Won-Keun

    2011-12-27

    Four new ent-kaurane diterpenoids (1-4) were isolated from the leaves of Croton tonkinensis by bioactivity-guided fractionation using an in vitro osteoblast differentiation assay. Their structures were identified as ent-11β-acetoxykaur-16-en-18-ol (1), ent-11α-hydroxy-18-acetoxykaur-16-ene (2), ent-14β-hydroxy-18-acetoxykaur-16-ene (3), and ent-7α-hydroxy-18-acetoxykaur-16-ene (4). Compounds 1-4 significantly increased alkaline phosphatase activity and osteoblastic gene promoter activity. Compounds 1-3 also increased the levels of ALP and collagen type I alpha mRNA in C2C12 cells in a dose-dependent manner. These results suggest that ent-kaurane diterpenoids from C. tonkinensis have a direct stimulatory effect on osteoblast differentiation and may be potential therapeutic molecules against bone diseases such as osteoporosis. PMID:22085418

  14. NFAT Signaling in Osteoblasts Regulates the Hematopoietic Niche in the Bone Microenvironment

    Directory of Open Access Journals (Sweden)

    Cheryl L. Sesler

    2013-01-01

    Full Text Available Osteoblasts support hematopoietic cell development, including B lymphopoiesis. We have previously shown that the nuclear factor of activated T cells (NFAT negatively regulates osteoblast differentiation and bone formation. Interestingly, in smooth muscle, NFAT has been shown to regulate the expression of vascular cellular adhesion molecule-1 (VCAM-1, a mediator of cell adhesion and signaling during leukocyte development. To examine whether NFAT signaling in osteoblasts regulates hematopoietic development in vivo, we generated a mouse model expressing dominant-negative NFAT driven by the 2.3 kb fragment of the collagen-αI promoter to disrupt NFAT activity in osteoblasts (dnNFATOB. Bone histomorphometry showed that dnNFATOB mice have significant increases in bone volume (44% and mineral apposition rate (131% and decreased trabecular thickness (18%. In the bone microenvironment, dnNFATOB mice displayed a significant increase (87% in Lineage−cKit+Sca-1+ (LSK cells and significant decreases in B220+CD19−IgM− pre-pro-B cells (41% and B220+CD19+IgM+ immature B cells (40%. Concurrent with these findings, LSK cell differentiation into B220+ cells was inhibited when cocultured on differentiated primary osteoblasts harvested from dnNFATOB mice. Gene expression and protein levels of VCAM-1 in osteoblasts decreased in dnNFATOB mice compared to controls. These data suggest that osteoblast-specific NFAT activity mediates early B lymphopoiesis, possibly by regulating VCAM-1 expression on osteoblasts.

  15. Collagen-derived dipeptide prolyl-hydroxyproline promotes differentiation of MC3T3-E1 osteoblastic cells

    International Nuclear Information System (INIS)

    Highlights: • Pro-Hyp did not affect MC3T3-E1 cell proliferation and matrix mineralization. • Pro-Hyp significantly increased alkaline phosphatase activity. • Pro-Hyp significantly upregulated gene expression of Runx2, Osterix, and Col1α1. - Abstract: Prolyl-hydroxyproline (Pro-Hyp) is one of the major constituents of collagen-derived dipeptides. The objective of this study was to investigate the effects of Pro-Hyp on the proliferation and differentiation of MC3T3-E1 osteoblastic cells. Addition of Pro-Hyp did not affect MC3T3-E1 cell proliferation and matrix mineralization but alkaline phosphatase activity was significantly increased. Furthermore, cells treated with Pro-Hyp significantly upregulated gene expression of Runx2, Osterix, and Col1α1. These results indicate that Pro-Hyp promotes osteoblast differentiation. This study demonstrates for the first time that Pro-Hyp has a positive effect on osteoblast differentiation with upregulation of Runx2, Osterix, and Collα1 gene expression

  16. Collagen-derived dipeptide prolyl-hydroxyproline promotes differentiation of MC3T3-E1 osteoblastic cells

    Energy Technology Data Exchange (ETDEWEB)

    Kimira, Yoshifumi, E-mail: kimira@josai.ac.jp [Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama 350-0295 (Japan); Ogura, Kana; Taniuchi, Yuri [Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama 350-0295 (Japan); Kataoka, Aya; Inoue, Naoki; Sugihara, Fumihito [Nitta Gelatin Inc., Peptide Division, 2-22 Futamata, Yao, Osaka 581-0024 (Japan); Nakatani, Sachie; Shimizu, Jun; Wada, Masahiro; Mano, Hiroshi [Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama 350-0295 (Japan)

    2014-10-24

    Highlights: • Pro-Hyp did not affect MC3T3-E1 cell proliferation and matrix mineralization. • Pro-Hyp significantly increased alkaline phosphatase activity. • Pro-Hyp significantly upregulated gene expression of Runx2, Osterix, and Col1α1. - Abstract: Prolyl-hydroxyproline (Pro-Hyp) is one of the major constituents of collagen-derived dipeptides. The objective of this study was to investigate the effects of Pro-Hyp on the proliferation and differentiation of MC3T3-E1 osteoblastic cells. Addition of Pro-Hyp did not affect MC3T3-E1 cell proliferation and matrix mineralization but alkaline phosphatase activity was significantly increased. Furthermore, cells treated with Pro-Hyp significantly upregulated gene expression of Runx2, Osterix, and Col1α1. These results indicate that Pro-Hyp promotes osteoblast differentiation. This study demonstrates for the first time that Pro-Hyp has a positive effect on osteoblast differentiation with upregulation of Runx2, Osterix, and Collα1 gene expression.

  17. Histone demethylase Jmjd3 regulates osteoblast apoptosis through targeting anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bim.

    Science.gov (United States)

    Yang, Di; Okamura, Hirohiko; Teramachi, Jumpei; Haneji, Tatsuji

    2016-04-01

    Posttranslational modifications including histone methylation regulate gene transcription through directly affecting the structure of chromatin. Trimethylation of histone H3K27 (H3K27me3) contributes to gene silencing and the histone demethylase Jumonji domain-containing 3 (Jmjd3) specifically removes the methylation of H3K27me3, followed by the activation of gene expression. In the present study, we explored the roles of Jmjd3 in regulating osteoblast apoptosis. Knockdown of Jmjd3 promoted osteoblast apoptosis induced by serum deprivation with decreased mitochondrial membrane potential and increased levels of caspase-3 activation, PARP cleavage, and DNA fragmentation. B cell lymphoma-2 (Bcl-2), an anti-apoptotic protein, was down-regulated by knockdown of Jmjd3 through retaining H3K27me3 on its promoter region. Knockdown of Jmjd3 increased the pro-apoptotic activity of Bim through inhibiting ERK-dependent phosphorylation of Bim. Protein kinase D1 (PKD1), which stimulates ERK phosphorylation, decreased in the Jmjd3-knockdown cells and introduction of PKD1 relieved osteoblast apoptosis in the Jmjd3-knockdown cells through increasing ERK-regulated Bim phosphorylation. These results suggest that Jmjd3 regulates osteoblast apoptosis through targeting Bcl-2 expression and Bim phosphorylation. PMID:26795455

  18. Surface microcracks signal osteoblasts to regulate alignment and bone formation.

    Science.gov (United States)

    Shu, Yutian; Baumann, Melissa J; Case, Eldon D; Irwin, Regina K; Meyer, Sarah E; Pearson, Craig S; McCabe, Laura R

    2014-11-01

    Microcracks are present in bone and can result from fatigue damage due to repeated, cyclically applied stresses. From a mechanical point, microcracks can dissipate strain energy at the advancing tip of a crack to improve overall bone toughness. Physiologically, microcracks are thought to trigger bone remodeling. Here, we examine the effect of microcracks specifically on osteoblasts, which are bone-forming cells, by comparing cell responses on microcracked versus non-microcracked hydroxyapatite (HA) specimens. Osteoblast attachment was found to be greater on microcracked HA specimens (pmicrocracks on HA. Cells also displayed a preferential attachment that was 75 to 90 μm away from the microcrack indent. After 21 days of culture, osteoblast maturation was notably enhanced on the HA with microcracks, as indicated by increased alkaline phosphatase activity and gene expression. Furthermore, examination of bone deposition by confocal laser scanning microscopy indicated preferential mineralization at microcrack indentation sites. Dissolution studies indicate that the microcracks increase calcium release, which could contribute to osteoblast responses. Our findings suggest that microcracks signal osteoblast attachment and bone formation/healing. PMID:25280696

  19. Bioenergetics and mitochondrial transmembrane potential during differentiation of cultured osteoblasts

    Science.gov (United States)

    Komarova, S. V.; Ataullakhanov, F. I.; Globus, R. K.

    2000-01-01

    To evaluate the relationship between osteoblast differentiation and bioenergetics, cultured primary osteoblasts from fetal rat calvaria were grown in medium supplemented with ascorbate to induce differentiation. Before ascorbate treatment, the rate of glucose consumption was 320 nmol. h(-1). 10(6) cells(-1), respiration was 40 nmol. h(-1). 10(6) cells(-1), and the ratio of lactate production to glucose consumption was approximately 2, indicating that glycolysis was the main energy source for immature osteoblasts. Ascorbate treatment for 14 days led to a fourfold increase in respiration, a threefold increase in ATP production, and a fivefold increase in ATP content compared with that shown in immature cells. Confocal imaging of mitochondria stained with a transmembrane potential-sensitive vital dye showed that mature cells possessed abundant amounts of high-transmembrane-potential mitochondria, which were concentrated near the culture medium-facing surface. Acute treatment of mature osteoblasts with metabolic inhibitors showed that the rate of glycolysis rose to maintain the cellular energy supply constant. Thus progressive differentiation coincided with changes in cellular metabolism and mitochondrial activity, which are likely to play key roles in osteoblast function.

  20. Combined Effects of Soy Isoflavones and β-Carotene on Osteoblast Differentiation

    Directory of Open Access Journals (Sweden)

    Yoriko Nishide

    2015-10-01

    Full Text Available Soy isoflavones, genistein, daidzein and its metabolite equol, as well as β-carotene have been reported to be effective for maintaining bone health. However, it remains to be elucidated whether combining soy isoflavones with β-carotene is beneficial to bone formation. This study investigated the combined effect of soy isoflavones and β-carotene on the differentiation of MC3T3-E1 preosteoblastic cells. Daidzein and genistein alone did not affect cell growth but increased alkaline phosphatase (ALP activity. Beta-carotene alone inhibited cell growth and markedly enhanced ALP activity. Soy isoflavones combined with β-carotene resulted in higher ALP activity than treatment with isoflavones or β-carotene alone. We observed significant main effects of β-carotene on the enhanced expression of Runx2, ALP, and ostepontin mRNA, whereas there was a significant main effect of soy isoflavones on the expression of osterix mRNA. To investigate how β-carotene affected osteoblast differentiation, MC3T3-E1 cells were treated with retinoic acid receptor (RAR pan-antagonist combined with β-carotene. Osteopontin and ALP mRNA expression levels, which were increased following treatment with β-carotene, were significantly suppressed by the RAR pan-antagonist. This suggests treatment with β-carotene enhanced early osteoblastic differentiation, at least in part via RAR signaling. These results indicate that a combination of isoflavones and β-carotene may be useful for maintaining a positive balance of bone turnover by inducing osteoblast differentiation.

  1. Purification of a peptide from seahorse, that inhibits TPA-induced MMP, iNOS and COX-2 expression through MAPK and NF-kappaB activation, and induces human osteoblastic and chondrocytic differentiation.

    Science.gov (United States)

    Ryu, BoMi; Qian, Zhong-Ji; Kim, Se-Kwon

    2010-03-30

    Ongoing efforts to search for naturally occurring, bioactive substances for the amelioration of arthritis have led to the discovery of natural products with substantial bioactive properties. The seahorse (Hippocampus kuda Bleeler), a telelost fish, is one source of known beneficial products, yet has not been utilized for arthritis research. In the present work, we have purified and characterized a bioactive peptide from seahorse hydrolysis. Among the hydrolysates tested, pronase E-derived hydrolysate exhibited the highest alkaline phosphatase (ALP) activity, a phenotype marker of osteoblast and chondrocyte differentiation. After its separation from the hydrolysate by several purification steps, the peptide responsible for the ALP activity was isolated and its sequence was identified as LEDPFDKDDWDNWK (1821Da). We have shown that the isolated peptide induces differentiation of osteoblastic MG-63 and chondrocytic SW-1353 cells by measuring ALP activity, mineralization and collagen synthesis. Our results indicate that the peptide acts during early to late stages of differentiation in MG-63 and SW-1353 cells. We also assessed the concentration dependence of the peptide's inhibition of MMP (-1, -3 and -13), iNOS and COX-2 expression after treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), a common form of phorbol ester. The peptide also inhibited NO production in MG-63 and SW-1353 cells. To elucidate the mechanisms by which the peptide acted, we examined its effects on TPA-induced MAPKs/NF-kappaB activation and determined that the peptide treatment significantly reduced p38 kinase/NF-kappaB in MG-63 cells and MAPKs/NF-kappaB in SW-1353 cells. PMID:20004183

  2. Toxicity of uranium and lead on osteoblastic bone cells

    Energy Technology Data Exchange (ETDEWEB)

    Milgram, S.; Thiebault, C.; Carriere, M.; Gouget, B. [CEA Saclay, CNRS, UMR9956, Lab Pierre Sue, F-91191 Gif Sur Yvette, (France); Malaval, L. [INSERM, 42023 Saint Etienne (France)

    2007-07-01

    Bone is one of the main retention organs affected by uranium (U) and lead (Pb). Intoxications have been documented to inhibit bone formation and impair bone modeling and remodeling. However, only few studies dealt with cellular and molecular mechanisms of their toxicity. The purpose of this study was to investigate the acute cytotoxicity of U and Pb and their phenotypic effects on ROS17/2.8 osteoblastic cells. The most likely forms of the toxics in contact with cells after blood contamination were selected for cell exposure. Results show that whatever their speciation, bone cells are always more sensitive to Pb than to U. Moreover, Pb is toxic when it is left free in the exposure medium or when it is complexed with bicarbonate, cysteine or citrate, but not with albumin or phosphate. U is more cytotoxic when it is complexed with transferrin than with bicarbonate. A direct correlation between toxicity and cellular accumulation could be observed. Beside, exposure of U or Pb to bone cells induces a speciation-dependant variation of RNA expression of two markers of bone formation and mineralization: osteocalcin (OCN) and bone sialoprotein (BSP). OCN and BSP-expression could be activated in sub-toxic condition, respectively, by Pb-albumin (1.6-fold) and U-bicarbonate (2.3-fold). In the meantime, U-transferrin and Pb-citrate lead to an inhibition of the two markers. This study shows a complex mechanism of toxicity of two heavy metals with a significant phenotypic impact on osteoblastic cells highly dependant on metal speciation which controls cell accumulation. (authors)

  3. How does the anthropogenic activity affect the spring discharge?

    Science.gov (United States)

    Hao, Yonghong; Zhang, Juan; Wang, Jiaojiao; Li, Ruifang; Hao, Pengmei; Zhan, Hongbin

    2016-09-01

    Karst hydrological process has largely been altered by climate change and human activity. In many places throughout the world, human activity (e.g. groundwater pumping and dewatering from mining) has intensified and surpassed climate change, where human activity becomes the primary factor that affects groundwater system. But it is still largely unclear how the human activity affects spring discharge in magnitude and periodicity. This study investigates the effects of anthropogenic activity on spring discharge, using the Xin'an Springs of China as an example. The Xin'an Spring discharge were divided into two time periods: the pre-development period from 1956 to 1971 and the post-development period from 1972 to 2013. We confirm the dividing time (i.e. 1971) of these two periods using the Wilcoxon rank-sum test. Then the wavelet transform and wavelet coherence were used to analyze the karst hydrological processes for the two periods respectively. We analyze the correlations of precipitation and the Xin'an spring discharge with the monsoons including the Indian Summer Monsoon (ISM) and the West North Pacific Monsoon (WNPM) and the climate teleconnections including El Niño Southern Oscillation (ENSO) and Pacific Decadal Oscillation (PDO), respectively. The results indicated that the spring discharge was attenuated about 19.63% under the influence of human activity in the Xin'an Springs basin. However, human activity did not alter the size of the resonance frequencies between the spring discharge and the monsoons. In contrast, it reinforced the periodicities of the monsoons-driven spring discharge. It suggested that human has adapted to the major climate periodicities, and human activity had the same rhyme with the primary climate periodicity. In return, human activity enhances the correlation between the monsoons and the spring discharge.

  4. Calcium signals and calcium channels in osteoblastic cells

    Science.gov (United States)

    Duncan, R. L.; Akanbi, K. A.; Farach-Carson, M. C.

    1998-01-01

    Calcium (Ca2+) channels are present in non-excitable as well as in excitable cells. In bone cells of the osteoblast lineage, Ca2+ channels play fundamental roles in cellular responses to external stimuli including both mechanical forces and hormonal signals. They are also proposed to modulate paracrine signaling between bone-forming osteoblasts and bone-resorbing osteoclasts at local sites of bone remodeling. Calcium signals are characterized by transient increases in intracellular Ca2+ levels that are associated with activation of intracellular signaling pathways that control cell behavior and phenotype, including patterns of gene expression. Development of Ca2+ signals is a tightly regulated cellular process that involves the concerted actions of plasma membrane and intracellular Ca2+ channels, along with Ca2+ pumps and exchangers. This review summarizes the current state of knowledge concerning the structure, function, and role of Ca2+ channels and Ca2+ signals in bone cells, focusing on the osteoblast.

  5. Chondrocytic Atf4 regulates osteoblast differentiation and function via Ihh

    OpenAIRE

    Wang, Weiguang; Lian, Na; Ma., Yun; LI, LINGZHEN; Gallant, Richard C.; Elefteriou, Florent; Yang, Xiangli

    2012-01-01

    Atf4 is a leucine zipper-containing transcription factor that activates osteocalcin (Ocn) in osteoblasts and indian hedgehog (Ihh) in chondrocytes. The relative contribution of Atf4 in chondrocytes and osteoblasts to the regulation of skeletal development and bone formation is poorly understood. Investigations of the Atf4–/–;Col2a1-Atf4 mouse model, in which Atf4 is selectively overexpressed in chondrocytes in an Atf4-null background, demonstrate that chondrocyte-derived Atf4 regulates osteog...

  6. Estradiol influences the mechanical properties of human fetal osteoblasts through cytoskeletal changes

    Energy Technology Data Exchange (ETDEWEB)

    Muthukumaran, Padmalosini [Department of Bioengineering, National University of Singapore (Singapore); Lim, Chwee Teck [Department of Bioengineering, National University of Singapore (Singapore); Department of Mechanical Engineering, National University of Singapore (Singapore); Mechanobiology Institute, National University of Singapore (Singapore); Singapore-MIT Alliance for Research and Technology (SMART), National University of Singapore (Singapore); Lee, Taeyong, E-mail: bielt@nus.edu.sg [Department of Bioengineering, National University of Singapore (Singapore)

    2012-07-06

    Highlights: Black-Right-Pointing-Pointer Estradiol induced stiffness changes of osteoblasts were quantified using AFM. Black-Right-Pointing-Pointer Estradiol causes significant decrease in the stiffness of osteoblasts. Black-Right-Pointing-Pointer Decreased stiffness was caused by decreased density of f-actin network. Black-Right-Pointing-Pointer Stiffness changes were not associated with mineralized matrix of osteoblasts. Black-Right-Pointing-Pointer Estradiol increases inherent alkaline phosphatase activity of osteoblasts. -- Abstract: Estrogen is known to have a direct effect on bone forming osteoblasts and bone resorbing osteoclasts. The cellular and molecular effects of estrogen on osteoblasts and osteoblasts-like cells have been extensively studied. However, the effect of estrogen on the mechanical property of osteoblasts has not been studied yet. It is important since mechanical property of the mechanosensory osteoblasts could be pivotal to its functionality in bone remodeling. This is the first study aimed to assess the direct effect of estradiol on the apparent elastic modulus (E{sup Asterisk-Operator }) and corresponding cytoskeletal changes of human fetal osteoblasts (hFOB 1.19). The cells were cultured in either medium alone or medium supplemented with {beta}-estradiol and then subjected to Atomic Force Microscopy indentation (AFM) to determine E{sup Asterisk-Operator }. The underlying changes in cytoskeleton were studied by staining the cells with TRITC-Phalloidin. Following estradiol treatment, the cells were also tested for proliferation, alkaline phosphatase activity and mineralization. With estradiol treatment, E{sup Asterisk-Operator} of osteoblasts significantly decreased by 43-46%. The confocal images showed that the changes in f-actin network observed in estradiol treated cells can give rise to the changes in the stiffness of the cells. Estradiol also increases the inherent alkaline phosphatase activity of the cells. Estradiol induced stiffness

  7. Myeloma cells suppress osteoblasts through sclerostin secretion

    International Nuclear Information System (INIS)

    Wingless-type (Wnt) signaling through the secretion of Wnt inhibitors Dickkopf1, soluble frizzled-related protein-2 and -3 has a key role in the decreased osteoblast (OB) activity associated with multiple myeloma (MM) bone disease. We provide evidence that another Wnt antagonist, sclerostin, an osteocyte-expressed negative regulator of bone formation, is expressed by myeloma cells, that is, human myeloma cell lines (HMCLs) and plasma cells (CD138+ cells) obtained from the bone marrow (BM) of a large number of MM patients with bone disease. We demonstrated that BM stromal cells (BMSCs), differentiated into OBs and co-cultured with HMCLs showed, compared with BMSCs alone, reduced expression of major osteoblastic-specific proteins, decreased mineralized nodule formation and attenuated the expression of members of the activator protein 1 transcription factor family (Fra-1, Fra-2 and Jun-D). Moreover, in the same co-culture system, the addition of neutralizing anti-sclerostin antibodies restored OB functions by inducing nuclear accumulation of β-catenin. We further demonstrated that the upregulation of receptor activator of nuclear factor κ-B ligand and the downregulation of osteoprotegerin in OBs were also sclerostin mediated. Our data indicated that sclerostin secretion by myeloma cells contribute to the suppression of bone formation in the osteolytic bone disease associated to MM

  8. Establishment of a new model for culturing rabbit osteoblasts in vitro

    International Nuclear Information System (INIS)

    To establish an experimental model for culturing rabbit osteoblasts in vitro, the osteoblasts were isolated from the calvarial bone of a 15-day old rabbit using a method of culturing the bone pieces in a medium after they had been digested by an enzyme for 15 min. The acquired cells were assayed by cell morphology, alkaline phosphatase activity and production of a mineralized matrix. The results showed that the cells had the morphologic characteristics and some biological behaviours of osteoblasts. Based on the primary isolation of osteoblasts from bone and combining digestion with explants, a novel model for culturing rabbit osteoblasts in vitro was established, which is easy, efficient and effective. This model can be used in many studies of osteogenesis mechanisms and bone replacement materials. (communication)

  9. Autocrine effects of neuromedin B stimulate the proliferation of rat primary osteoblasts.

    Science.gov (United States)

    Saito, Hiroki; Nakamachi, Tomoya; Inoue, Kazuhiko; Ikeda, Ryuji; Kitamura, Kazuo; Minamino, Naoto; Shioda, Seiji; Miyata, Atsuro

    2013-05-01

    Neuromedin B (NMB) is a mammalian bombesin-like peptide that regulates exocrine/endocrine secretion, smooth muscle contraction, body temperature, and the proliferation of some cell types. Here, we show that mRNA encoding Nmb and its receptor (Nmbr) are expressed in rat bone tissue. Immunohistochemical analysis demonstrated that NMB and NMBR colocalize in osteoblasts, epiphyseal chondrocytes, and proliferative chondrocytes of growth plates from mouse hind limbs. Then, we investigated the effect of NMB on the proliferation of rat primary cultured osteoblasts. Proliferation assays and 5-bromo-2'-deoxyuridine incorporation assays demonstrated that NMB augments the cell number and enhances DNA synthesis in osteoblasts. Pretreatment with the NMBR antagonist BIM23127 inhibited NMB-induced cell proliferation and DNA synthesis. Western blot analysis showed that NMB activates ERK1/2 MAPK signaling in osteoblasts. Pretreatment with the MAPK/ERK kinase inhibitor U0126 attenuated NMB-induced cell proliferation and DNA synthesis. We also investigated the effects of molecules that contribute to osteoblast proliferation and differentiation on Nmb expression in osteoblasts. Real-time PCR analysis demonstrated that 17β-estradiol (E2) and transforming growth factor β1 increase and decrease Nmb mRNA expression levels respectively. Finally, proliferation assays revealed that the NMBR antagonist BIM23127 suppresses E2-induced osteoblast proliferation. These results suggest that NMB/NMBR signaling plays an autocrine or paracrine role in osteoblast proliferation and contributes to the regulation of bone formation. PMID:23428580

  10. Active house: A contemporary housing model for flood affected population

    Directory of Open Access Journals (Sweden)

    Stratimirović Tatjana

    2015-01-01

    Full Text Available The effectiveness of architectural knowledge in the struggle for a better future can be seen in the attitude that a good design or a good architectural solution, does not belong solely to the privileged ones as an improvement of the basic requirements, rather quite the opposite, that it is created as a response to a need. The goal of physical and emotional wellbeing, combined with a long term strategy for reducing the negative impact of the built environment by converting it into a positive influence upon the natural ecosystem, brings together and advances bioclimatic principles, architectural design and sustainable construction in the contemporary housing model dubbed the Active House. The Active House Workshop was held, as part of a wider student initiative New Housing Models for Flood Affected Population, at the University of Belgrade - Faculty of Architecture. The purpose of the campaign was to provide help to flood affected communities and assistance in efforts for repairing buildings in Serbia, hit by the severe floods of May 2014. Students came up with nine design solutions for small family homes, which incorporate the principles of Active House into existing construction techniques. In an architectural context, when concerning repair work after flooding, the need to consider problems related to contemporary living conditions through the ‘active’ category is seen in a new understanding of nature which allows the replacement of a passive restoration model, with an active models for designing in interaction with the environment.

  11. Osteoblastogenesis and Role of Osteoblasts in Calcıum Homeostasis and Remodeling of Bone

    Directory of Open Access Journals (Sweden)

    Neslihan Başcıl Tütüncü

    2008-05-01

    Full Text Available Bone remodeling is very important for repair of microfractures and fatigue damage and prevention of excessive aging and its consequences. Bone remodeling lasts for about 6-9 months. During this period osteoclasts resorb damaged bone and osteoblasts synthesize new bone. The lifespan of mature osteoclasts is about 15 days and for osteoblasts 3 months. Therefore, the time required for the remodeling of a given segement of bone is much longer than the lifespan of its cells which perform remodeling. A supply of new osteoblasts and osteoclasts are therefore needed for succesful remodeling by the basic multicellular unit. The major event that triggers osteogenesis is the transition of mesenchymal stem cells into bone differentiating osteoblast cells. Osteoblast commitment and differentation are controlled by complex activities. Many factors are involved in the regulation of osteoblastogenesis. Bone morphogenetic proteins and the Wnt glycoproteins play crucial roles in signaling osteoblast commitment and differentiation, and are the only known factors capable of initiating osteoblastogenesis from uncommitted progenitors. They can initiate commitment of mesenchymal cells to osteoblastic lineage. The initial cell division is asymmetric, giving rise to another stem cell and a committed osteoprogenitor. After commitment to the osteoblastic lineage, a osteoprogenitor cell gives rise to the transit-amplifying compartment. At this stage osteoprogenitor cells proliferate intensively. After this stage, the cells are more differentiated and give rise to preosteoblasts which express both STRO1, alkaline phosphatase, pyrophosphate, and type 1 collagen. Preosteoblasts are committed to the osteoblast lineage with extensive replicative capacity, but have no self-renewal capacity. Preosteoblasts form the intermediate stage of osteoblastogenesis. The mature osteoblasts express osteopontin, alkaline phosphatase, bone sialoprotein, and osteocalcin. This stage is

  12. Lack of effect of adenosine on the function of rodent osteoblasts and osteoclasts in vitro.

    Science.gov (United States)

    Hajjawi, Mark O R; Patel, Jessal J; Corcelli, Michelangelo; Arnett, Timothy R; Orriss, Isabel R

    2016-06-01

    Extracellular ATP, signalling through P2 receptors, exerts well-documented effects on bone cells, inhibiting mineral deposition by osteoblasts and stimulating the formation and resorptive activity of osteoclasts. The aims of this study were to determine the potential osteotropic effects of adenosine, the hydrolysis product of ATP, on primary bone cells in vitro. We determined the effect of exogenous adenosine on (1) the growth, alkaline phosphatase (TNAP) activity and bone-forming ability of osteoblasts derived from the calvariae of neonatal rats and mice and the marrow of juvenile rats and (2) the formation and resorptive activity of osteoclasts from juvenile mouse marrow. Reverse transcription polymerase chain reaction (RT-PCR) analysis showed marked differences in the expression of P1 receptors in osteoblasts from different sources. Whilst mRNA for the A1 and A2B receptors was expressed by all primary osteoblasts, A2A receptor expression was limited to rat bone marrow and mouse calvarial osteoblasts and the A3 receptor to rat bone marrow osteoblasts. We found that adenosine had no detectable effects on cell growth, TNAP activity or bone formation by rodent osteoblasts in vitro. The analogue 2-chloroadenosine, which is hydrolysed more slowly than adenosine, had no effects on rat or mouse calvarial osteoblasts but increased TNAP activity and bone formation by rat bone marrow osteoblasts by 30-50 % at a concentration of 1 μM. Osteoclasts were found to express the A2A, A2B and A3 receptors; however, neither adenosine (≤100 μM) nor 2-chloroadenosine (≤10 μM) had any effect on the formation or resorptive activity of mouse osteoclasts in vitro. These results suggest that adenosine, unlike ATP, is not a major signalling molecule in the bone. PMID:26861849

  13. Vitamin a is a negative regulator of osteoblast mineralization.

    Directory of Open Access Journals (Sweden)

    Thomas Lind

    Full Text Available An excessive intake of vitamin A has been associated with an increased risk of fractures in humans. In animals, a high vitamin A intake leads to a reduction of long bone diameter and spontaneous fractures. Studies in rodents indicate that the bone thinning is due to increased periosteal bone resorption and reduced radial growth. Whether the latter is a consequence of direct effects on bone or indirect effects on appetite and general growth is unknown. In this study we therefore used pair-feeding and dynamic histomorphometry to investigate the direct effect of a high intake of vitamin A on bone formation in rats. Although there were no differences in body weight or femur length compared to controls, there was an approximately halved bone formation and mineral apposition rate at the femur diaphysis of rats fed vitamin A. To try to clarify the mechanism(s behind this reduction, we treated primary human osteoblasts and a murine preosteoblastic cell line (MC3T3-E1 with the active metabolite of vitamin A; retinoic acid (RA, a retinoic acid receptor (RAR antagonist (AGN194310, and a Cyp26 inhibitor (R115866 which blocks endogenous RA catabolism. We found that RA, via RARs, suppressed in vitro mineralization. This was independent of a negative effect on osteoblast proliferation. Alkaline phosphatase and bone gamma carboxyglutamate protein (Bglap, Osteocalcin were drastically reduced in RA treated cells and RA also reduced the protein levels of Runx2 and Osterix, key transcription factors for progression to a mature osteoblast. Normal osteoblast differentiation involved up regulation of Cyp26b1, the major enzyme responsible for RA degradation, suggesting that a drop in RA signaling is required for osteogenesis analogous to what has been found for chondrogenesis. In addition, RA decreased Phex, an osteoblast/osteocyte protein necessary for mineralization. Taken together, our data indicate that vitamin A is a negative regulator of osteoblast mineralization.

  14. Rac1 and Cdc42 GTPases regulate shear stress-driven β-catenin signaling in osteoblasts

    International Nuclear Information System (INIS)

    Highlights: •Shear stress increased TCF/LEF activity and stimulated β-catenin nuclear localization. •Rac1, Cdc42, and RhoA displayed distinct dynamic activity patterns under flow. •Rac1 and Cdc42, but not RhoA, regulate shear stress-driven TCF/LEF activation. •Cytoskeleton did not significantly affect shear stress-induced TCF/LEF activation. -- Abstract: Beta-catenin-dependent TCF/LEF (T-cell factor/lymphocyte enhancing factor) is known to be mechanosensitive and an important regulator for promoting bone formation. However, the functional connection between TCF/LEF activity and Rho family GTPases is not well understood in osteoblasts. Herein we investigated the molecular mechanisms underlying oscillatory shear stress-induced TCF/LEF activity in MC3T3-E1 osteoblast cells using live cell imaging. We employed fluorescence resonance energy transfer (FRET)-based and green fluorescent protein (GFP)-based biosensors, which allowed us to monitor signal transduction in living cells in real time. Oscillatory (1 Hz) shear stress (10 dynes/cm2) increased TCF/LEF activity and stimulated translocation of β-catenin to the nucleus with the distinct activity patterns of Rac1 and Cdc42. The shear stress-induced TCF/LEF activity was blocked by the inhibition of Rac1 and Cdc42 with their dominant negative mutants or selective drugs, but not by a dominant negative mutant of RhoA. In contrast, constitutively active Rac1 and Cdc42 mutants caused a significant enhancement of TCF/LEF activity. Moreover, activation of Rac1 and Cdc42 increased the basal level of TCF/LEF activity, while their inhibition decreased the basal level. Interestingly, disruption of cytoskeletal structures or inhibition of myosin activity did not significantly affect shear stress-induced TCF/LEF activity. Although Rac1 is reported to be involved in β-catenin in cancer cells, the involvement of Cdc42 in β-catenin signaling in osteoblasts has not been identified. Our findings in this study demonstrate that

  15. Rac1 and Cdc42 GTPases regulate shear stress-driven β-catenin signaling in osteoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Wan, Qiaoqiao; Cho, Eunhye [Department of Biomedical Engineering, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202 (United States); Yokota, Hiroki [Department of Biomedical Engineering, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202 (United States); Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN 46202 (United States); Na, Sungsoo, E-mail: sungna@iupui.edu [Department of Biomedical Engineering, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202 (United States)

    2013-04-19

    Highlights: •Shear stress increased TCF/LEF activity and stimulated β-catenin nuclear localization. •Rac1, Cdc42, and RhoA displayed distinct dynamic activity patterns under flow. •Rac1 and Cdc42, but not RhoA, regulate shear stress-driven TCF/LEF activation. •Cytoskeleton did not significantly affect shear stress-induced TCF/LEF activation. -- Abstract: Beta-catenin-dependent TCF/LEF (T-cell factor/lymphocyte enhancing factor) is known to be mechanosensitive and an important regulator for promoting bone formation. However, the functional connection between TCF/LEF activity and Rho family GTPases is not well understood in osteoblasts. Herein we investigated the molecular mechanisms underlying oscillatory shear stress-induced TCF/LEF activity in MC3T3-E1 osteoblast cells using live cell imaging. We employed fluorescence resonance energy transfer (FRET)-based and green fluorescent protein (GFP)-based biosensors, which allowed us to monitor signal transduction in living cells in real time. Oscillatory (1 Hz) shear stress (10 dynes/cm{sup 2}) increased TCF/LEF activity and stimulated translocation of β-catenin to the nucleus with the distinct activity patterns of Rac1 and Cdc42. The shear stress-induced TCF/LEF activity was blocked by the inhibition of Rac1 and Cdc42 with their dominant negative mutants or selective drugs, but not by a dominant negative mutant of RhoA. In contrast, constitutively active Rac1 and Cdc42 mutants caused a significant enhancement of TCF/LEF activity. Moreover, activation of Rac1 and Cdc42 increased the basal level of TCF/LEF activity, while their inhibition decreased the basal level. Interestingly, disruption of cytoskeletal structures or inhibition of myosin activity did not significantly affect shear stress-induced TCF/LEF activity. Although Rac1 is reported to be involved in β-catenin in cancer cells, the involvement of Cdc42 in β-catenin signaling in osteoblasts has not been identified. Our findings in this study demonstrate

  16. Effects of phytoestrogens and environmental estrogens on osteoblastic differentiation in MC3T3-E1 cells

    International Nuclear Information System (INIS)

    Phytoestrogens and environmental estrogens, which have in part some structural similarity to 17β-estradiol, are reported to act as agonists/antagonists of estrogen in animals and humans. Estrogen is known to play an important role in maintaining bone mass, since the concentration of serum estrogen decreases after menopause and the estrogen deficiency results in bone loss. In this study, we report the effects of phytoestrogens (genistein, daidzein, and coumestrol) and environmental estrogens (bisphenol A (BPA), p-n-nonylphenol (NP) and bis(2-ethylhexyl)phthalate (DEHP)) on osteoblast differentiation using MC3T3-E1 cells, a mouse calvaria osteoblast-like cell line. Coumestrol (10-10 to 10-6 M) slightly enhanced cell proliferation, while neither the other phytoestrogens (daidzein, genistein) nor environmental estrogens increased cell proliferation. Alkaline phosphatase (ALP) activity and cellular calcium (Ca) and phosphorus (P) contents were increased by phytoestrogens and BPA; however, neither NP nor DEHP affected those osteoblastic indicators. The effects of estrogenic potency, using the cell proliferation of MCF-7 cells, an estrogen receptor (ER)-positive human breast cancer cell line, indicate that coumestrol has the highest estrogenic potency among those phytoestrogens and environmental estrogens. The estrogenic potency of NP and DEHP were lower than the others. In conclusion, phytoestrogens, such as coumestrol, genistein and daidzein, and BPA increased ALP activity and enhanced bone mineralization in MC3T3-E1 cells, suggesting that not only phytoestrogen but also BPA, an environmental estrogen, is implicated in bone metabolism

  17. Environmental layout complexity affects neural activity during navigation in humans.

    Science.gov (United States)

    Slone, Edward; Burles, Ford; Iaria, Giuseppe

    2016-05-01

    Navigating large-scale surroundings is a fundamental ability. In humans, it is commonly assumed that navigational performance is affected by individual differences, such as age, sex, and cognitive strategies adopted for orientation. We recently showed that the layout of the environment itself also influences how well people are able to find their way within it, yet it remains unclear whether differences in environmental complexity are associated with changes in brain activity during navigation. We used functional magnetic resonance imaging to investigate how the brain responds to a change in environmental complexity by asking participants to perform a navigation task in two large-scale virtual environments that differed solely in interconnection density, a measure of complexity defined as the average number of directional choices at decision points. The results showed that navigation in the simpler, less interconnected environment was faster and more accurate relative to the complex environment, and such performance was associated with increased activity in a number of brain areas (i.e. precuneus, retrosplenial cortex, and hippocampus) known to be involved in mental imagery, navigation, and memory. These findings provide novel evidence that environmental complexity not only affects navigational behaviour, but also modulates activity in brain regions that are important for successful orientation and navigation. PMID:26990572

  18. Chinese herbal compound affects osteoblast proliferation and bone mineral density%中药复方对成骨细胞增殖及骨密度的影响*

    Institute of Scientific and Technical Information of China (English)

    颜军礼; 李蒙; 李瑞玉; 吴立萍; 孙艳浮; 李会龙

    2013-01-01

      背景:复方中药是由多种矿物植物动物等物质组成的复方药物,在治疗骨代谢疾病方面,能通过多途径,多靶点发挥综合治疗作用。  目的:研究复方中药对成骨细胞增殖及骨密度的影响,探讨复方中药对骨质疏松症的药理作用。  方法:回顾性分析以往经实验研究证实的某些复方中药对骨质疏松症动物模型成骨细胞增殖分化及骨密度的影响,分析影响复方中药促进骨形成的因素,通过对成骨细胞体外培养方法进行筛选,找出促进细胞增殖分化、提高骨密度的药物适宜剂量,与化学药物结果进行对照研究。  结果与结论:复方中药能够促进成骨细胞增殖分化、提高骨密度,在治疗骨质疏松症方面具有标本兼治、毒副作用小等优点,但复方中药在改善骨密度水平方面不及化学药物,并且应通过长期、大样本的临床研究,降低骨质疏松性骨折风险的干预作用。%BACKGROUND:Compound Chinese medicine is a kind of compound drugs with the combination of minerals, plants and animals, which play the multi-target integrated treatment effects in the treatment of bone metabolic disease through various methods. OBJECTIVE:To research the effect of compound traditional Chinese medicine on the proliferation and bone mineral density of osteoblasts, and to explore the pharmacological effect of compound traditional Chinese medicine in the treatment of osteoporosis. METHODS: A retrospective analysis was performed to analyze the effect of some compound traditional Chinese medicines on the proliferation and bone mineral density of osteoblasts that identified in the previous studies, in order to analyze the factors of compound traditional Chinese medicines that can promote the bone formation. The appropriate dose of the drugs that can promote cel proliferation and differentiation and improve the bone mineral density was screened out

  19. Integrin αv in the mechanical response of osteoblast lineage cells

    Energy Technology Data Exchange (ETDEWEB)

    Kaneko, Keiko [Department of Bone and Joint Disease, National Center for Geriatrics and Gerontology, Obu, Aichi 474-8511 (Japan); Ito, Masako [Medical Work-Life-Balance Center, Nagasaki University Hospital, Nagasaki 852-8501 (Japan); Naoe, Yoshinori [Department of Mechanism of Aging, National Center for Geriatrics and Gerontology, Obu, Aichi 474-8511 (Japan); Lacy-Hulbert, Adam [Department of Pediatrics, Massachusetts General Hospital, Boston, MA 02114 (United States); Ikeda, Kyoji, E-mail: kikeda@ncgg.go.jp [Department of Bone and Joint Disease, National Center for Geriatrics and Gerontology, Obu, Aichi 474-8511 (Japan)

    2014-05-02

    Highlights: • Deletion of integrin αv in osteoblast lineage results in an impaired SOST response to loading in vivo. • c-Src–p130Cas–JNK–YAP/TAZ is activated via integrin αv on osteoblasts in response to FSS. • Deletion of integrin αv in osteoblasts results in impaired responses to mechanical stimulation. • Integrin αv is a key component of the mechanosensing machinery in bone. - Abstract: Although osteoblast lineage cells, especially osteocytes, are thought to be a primary mechanosensory cell in bone, the identity of the mechano-receptor and downstream mechano-signaling pathways remain largely unknown. Here we show using osteoblastic cell model of mechanical stimulation with fluid shear stress that in the absence of integrin αv, phosphorylation of the Src substrate p130Cas and JNK was impaired, culminating in an inhibition of nuclear translocation of YAP/TAZ and subsequent transcriptional activation of target genes. Targeted deletion of the integrin αv in osteoblast lineage cells results in an attenuated response to mechanical loading in terms of Sost gene expression, indicative of a role for integrin αv in mechanoreception in vivo. Thus, integrin αv may be integral to a mechanosensing machinery in osteoblastic cells and involved in activation of a Src–JNK–YAP/TAZ pathway in response to mechanical stimulation.

  20. Osthole-mediated cell differentiation through bone morphogenetic protein-2/p38 and extracellular signal-regulated kinase 1/2 pathway in human osteoblast cells.

    Science.gov (United States)

    Kuo, Po-Lin; Hsu, Ya-Ling; Chang, Cheng-Hsiung; Chang, Jiunn-Kae

    2005-09-01

    The survival of osteoblast cells is one of the determinants of the development of osteoporosis in patients. Osthole (7-methoxy-8-isopentenoxycoumarin) is a coumarin derivative present in many medicinal plants. By means of alkaline phosphatase (ALP) activity, osteocalcin, osteopontin, and type I collagen, enzyme-linked immunosorbent assay, we have shown that osthole exhibits a significant induction of differentiation in two human osteoblast-like cell lines, MG-63 and hFOB. Induction of differentiation by osthole was associated with increased bone morphogenetic protein (BMP)-2 production and the activations of SMAD1/5/8 and p38 and extracellular signal-regulated kinase (ERK) 1/2 kinases. Addition of purified BMP-2 protein did not increase the up-regulation of ALP activity and osteocalcin by osthole, whereas the BMP-2 antagonist noggin blocked both osthole and BMP-2-mediated ALP activity enhancement, indicating that BMP-2 production is required in osthole-mediated osteoblast maturation. Pretreatment of osteoblast cells with noggin abrogated p38 activation but only partially decreased ERK1/2 activation, suggesting that BMP-2 signaling is required in p38 activation and is partially involved in ERK1/2 activation in osthole-treated osteoblast cells. Cotreatment of p38 inhibitor SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole] or p38 small interfering RNA (siRNA) expression inhibited osthole-mediated activation of ALP but only slightly affected osteocalcin production. In contrast, the production of osteocalcin induced by osthole was inhibited by the mitogen-activated protein kinase kinase inhibitor PD98059 (2'-amino-3'-methoxyflavone) or by expression of an ERK2 siRNA. These data suggest that BMP-2/p38 pathway links to the early phase, whereas ERK1/2 pathway is associated with the later phase in osthole-mediated differentiation of osteoblast cells. In this study, we demonstrate that osthole is a promising agent for treating osteoporosis

  1. Effects of quercetin, a natural phenolic compound, in the differentiation of human mesenchymal stem cells (MSC) into adipocytes and osteoblasts.

    Science.gov (United States)

    Casado-Díaz, Antonio; Anter, Jaouad; Dorado, Gabriel; Quesada-Gómez, José Manuel

    2016-06-01

    Natural phenols may have beneficial properties against oxidative stress, which is associated with aging and major chronic aging-related diseases, such as loss of bone mineral mass (osteoporosis) and diabetes. The main aim of this study was to analyze the effect of quercetin, a major nutraceutical compound present in the "Mediterranean diet", on mesenchymal stem-cell (MSC) differentiation. Such cells were induced to differentiate into osteoblasts or adipocytes in the presence of two quercetin concentrations (0.1 and 10μM). Several physiological parameters and the expression of osteoblastogenesis and adipogenesis marker genes were monitored. Quercetin (10μM) inhibited cell proliferation, alkaline phosphatase (ALPL) activity and mineralization, down-regulating the expression of ALPL, collagen type I alpha 1 (COL1A1) and osteocalcin [bone gamma-carboxyglutamate protein (BGLAP)] osteoblastogenesis-related genes in MSC differentiating into osteoblasts. Moreover, in these cultures, CCAAT/enhancer-binding protein alpha (CEBPA) and peroxisome proliferator-activated receptor gamma 2 (PPARG2) adipogenic genes were induced, and cells differentiated into adipocytes were observed. Quercetin did not affect proliferation, but increased adipogenesis, mainly at 10-μM concentration in MSC induced to differentiate to adipocytes. β- and γ-catenin (plakoglobin) nuclear levels were reduced and increased, respectively, in quercetin-treated cultures. This suggests that the effect of high concentration of quercetin on MSC osteoblastic and adipogenic differentiation is mediated via Wnt/β-catenin inhibition. In conclusion, quercetin supplementation inhibited osteoblastic differentiation and promoted adipogenesis at the highest tested concentration. Such possible adverse effects of high quercetin concentrations should be taken into account in nutraceutical or pharmaceutical strategies using such flavonol. PMID:27142748

  2. Osteoblastic Osteosarcoma in a Rabbit

    OpenAIRE

    Ishikawa, Megumi; Kondo, Hirotaka; Onuma, Mamoru; Shibuya, Hisashi; Sato, Tsuneo

    2012-01-01

    An osteosarcoma developed in the tarsal joint region involving the distal tibia of a domestic rabbit (Oryctolagus cuniculus). Micrometastases were present in the lungs. Histologically the tumor was composed of ovoid to short-spindle cells with abundant giant cells, producing irregular islands of osteoids. The tumor cells were immunopositive with antiosteocalcin monoclonal antibody, consistent with their derivation from osteoblasts. According to review of 10 published cases, productive osteobl...

  3. Bone marrow osteoblast vulnerability to chemotherapy

    OpenAIRE

    Gencheva, Marieta; Hare, Ian; Kurian, Susan; Fortney, Jim; Piktel, Debbie; Wysolmerski, Robert; Gibson, Laura F.

    2013-01-01

    Osteoblasts are a major component of the bone marrow microenvironment which provide support for hematopoietic cell development. Functional disruption of any element of the bone marrow niche, including osteoblasts, can potentially impair hematopoiesis. We have studied the effect of two widely used drugs with different mechanisms of action, etoposide (VP16) and melphalan, on murine osteoblasts at distinct stages of maturation. VP16 and melphalan delayed maturation of preosteoblasts and altered ...

  4. Differential sensitivity of osteoblasts and bacterial pathogens to 405-nm light highlighting potential for decontamination applications in orthopedic surgery

    Science.gov (United States)

    Ramakrishnan, Praveen; Maclean, Michelle; MacGregor, Scott J.; Anderson, John G.; Grant, M. Helen

    2014-10-01

    Healthcare associated infections pose a major threat to patients admitted to hospitals and infection rates following orthopedic arthroplasty surgery are as high as 4%. A 405-nm high-intensity narrow spectrum light has been proven to reduce environmental contamination in hospital isolation rooms, and there is potential to develop this technology for application in arthroplasty surgery. Cultured rat osteoblasts were exposed to varying light intensities and it was found that exposures of up to a dose of 36 J/cm2 had no significant effect on cell viability [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay], function (alkaline phosphatase activity), and proliferation rate (BrdU cell proliferation assay). High irradiance exposures (54 J/cm2) significantly affected the cell viability indicating that the effects of 405-nm light on osteoblasts are dose dependent. Additionally, exposure of a variety of clinically related bacteria to a dose of 36 J/cm2 resulted in up to 100% kill. These results demonstrating the differential sensitivity of osteoblasts and bacteria to 405-nm light are an essential step toward developing the technique for decontamination in orthopedic surgery.

  5. Osteoblast CFTR inactivation reduces differentiation and osteoprotegerin expression in a mouse model of cystic fibrosis-related bone disease.

    Directory of Open Access Journals (Sweden)

    Michael S Stalvey

    Full Text Available Low bone mass and increased fracture risk are recognized complications of cystic fibrosis (CF. CF-related bone disease (CFBD is characterized by uncoupled bone turnover--impaired osteoblastic bone formation and enhanced osteoclastic bone resorption. Intestinal malabsorption, vitamin D deficiency and inflammatory cytokines contribute to CFBD. However, epidemiological investigations and animal models also support a direct causal link between inactivation of skeletal cystic fibrosis transmembrane regulator (CFTR, the gene that when mutated causes CF, and CFBD. The objective of this study was to examine the direct actions of CFTR on bone. Expression analyses revealed that CFTR mRNA and protein were expressed in murine osteoblasts, but not in osteoclasts. Functional studies were then performed to investigate the direct actions of CFTR on osteoblasts using a CFTR knockout (Cftr-/- mouse model. In the murine calvarial organ culture assay, Cftr-/- calvariae displayed significantly less bone formation and osteoblast numbers than calvariae harvested from wildtype (Cftr+/+ littermates. CFTR inactivation also reduced alkaline phosphatase expression in cultured murine calvarial osteoblasts. Although CFTR was not expressed in murine osteoclasts, significantly more osteoclasts formed in Cftr-/- compared to Cftr+/+ bone marrow cultures. Indirect regulation of osteoclastogenesis by the osteoblast through RANK/RANKL/OPG signaling was next examined. Although no difference in receptor activator of NF-κB ligand (Rankl mRNA was detected, significantly less osteoprotegerin (Opg was expressed in Cftr-/- compared to Cftr+/+ osteoblasts. Together, the Rankl:Opg ratio was significantly higher in Cftr-/- murine calvarial osteoblasts contributing to a higher osteoclastogenesis potential. The combined findings of reduced osteoblast differentiation and lower Opg expression suggested a possible defect in canonical Wnt signaling. In fact, Wnt3a and PTH-stimulated canonical Wnt

  6. FACTORS AFFECTED DECARBOXYLATION ACTIVITY OF ENTEROCOCCUS FAECIUM ISOLATED FROM RABBIT

    Directory of Open Access Journals (Sweden)

    František Buňka

    2012-04-01

    Full Text Available Normal 0 21 false false false SK JA X-NONE Biogenic amines (BA are basic nitrogenous compounds formed mainly by decarboxylation of amino acids. There are generated in course of microbial, vegetable and animal metabolisms. The aim of the study was to monitor factors affected production of biogenic amines by Enterococcus faecium, which is found in rabbit meat. Biogenic amines were analyzed by means of UPLC (ultrahigh performance liquid chromatography equipped with a UV/VIS DAD detector. Decarboxylation activity of E. faecium was mainly influenced by the cultivation temperature and the amount of NaCl in this study. E. faecium produced most of the monitored biogenic amines levels: tyramine ˂2500 mg.l-1; putrescine ˂30 mg.l-1; spermidine ˂10 mg.l-1 and cadaverine ˂5 mg.l-1.doi:10.5219/182

  7. The Poor Osteoblastic Functions Can Correct by Teriparatide.

    Science.gov (United States)

    2015-09-01

    A study of stimulation of poor osteoblastic function by teriparatide and monitored by bone markers. Eighty-one cases were enrolled with poor osteoblastic functions, which were classified with the values of both markers: NMID Osteocalcin was less than 10.81 ng/ml and the PINP was less than 15.88 ng/ml. Most CTx were below 0.1 ng/ml or at any level. Every case took oral bisphosphanates for at least 2 years. Average age was 68 years old; 79 cases are female the others male. The base line of bone markers: NMID osteocalcin, PINP and CTx are 9.3, 14.3 and 0.08 ng/ml, respectively. The duration of Teriparatide injections was 6 months, dose 20 microgram subcutaneous per day. The blood calcium, uric acid and renal profile were monitored too. The results of98 percent of cases showed that dramatic increase of osteoblastic products and at the second month; all markers increased above the normal values that are indicators of osteoblastic resumes and their activities. The hypercalcemia can occur during treatment (22.2%) and hyperuricemia 12.34%; therefore, blood chemistry and bone markers should be monitored every month as a precaution. PMID:26529823

  8. Expression of POEM, a positive regulator of osteoblast differentiation, is suppressed by TNF-{alpha}

    Energy Technology Data Exchange (ETDEWEB)

    Tsukasaki, Masayuki [Department of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555 (Japan); Yamada, Atsushi, E-mail: yamadaa@dent.showa-u.ac.jp [Department of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555 (Japan); Suzuki, Dai [Department of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555 (Japan); Aizawa, Ryo [Department of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555 (Japan); Department of Periodontology, School of Dentistry, Showa University, 2-1-1 Kitasenzoku, Ohta, Tokyo 145-8515 (Japan); Miyazono, Agasa [Department of Periodontology, School of Dentistry, Showa University, 2-1-1 Kitasenzoku, Ohta, Tokyo 145-8515 (Japan); Miyamoto, Yoichi; Suzawa, Tetsuo; Takami, Masamichi; Yoshimura, Kentaro [Department of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555 (Japan); Morimura, Naoko [Laboratory for Comparative Neurogenesis, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198 (Japan); Yamamoto, Matsuo [Department of Periodontology, School of Dentistry, Showa University, 2-1-1 Kitasenzoku, Ohta, Tokyo 145-8515 (Japan); Kamijo, Ryutaro [Department of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555 (Japan)

    2011-07-15

    Highlights: {yields} TNF-{alpha} inhibits POEM gene expression. {yields} Inhibition of POEM gene expression is caused by NF-{kappa}B activation by TNF-{alpha}. {yields} Over-expression of POEM recovers inhibition of osteoblast differentiation by TNF-{alpha}. -- Abstract: POEM, also known as nephronectin, is an extracellular matrix protein considered to be a positive regulator of osteoblast differentiation. In the present study, we found that tumor necrosis factor-{alpha} (TNF-{alpha}), a key regulator of bone matrix properties and composition that also inhibits terminal osteoblast differentiation, strongly inhibited POEM expression in the mouse osteoblastic cell line MC3T3-E1. TNF-{alpha}-induced down-regulation of POEM gene expression occurred in both time- and dose-dependent manners through the nuclear factor kappa B (NF-{kappa}B) pathway. In addition, expressions of marker genes in differentiated osteoblasts were down-regulated by TNF-{alpha} in a manner consistent with our findings for POEM, while over-expression of POEM recovered TNF-{alpha}-induced inhibition of osteoblast differentiation. These results suggest that TNF-{alpha} inhibits POEM expression through the NF-{kappa}B signaling pathway and down-regulation of POEM influences the inhibition of osteoblast differentiation by TNF-{alpha}.

  9. Expression of POEM, a positive regulator of osteoblast differentiation, is suppressed by TNF-α

    International Nuclear Information System (INIS)

    Highlights: → TNF-α inhibits POEM gene expression. → Inhibition of POEM gene expression is caused by NF-κB activation by TNF-α. → Over-expression of POEM recovers inhibition of osteoblast differentiation by TNF-α. -- Abstract: POEM, also known as nephronectin, is an extracellular matrix protein considered to be a positive regulator of osteoblast differentiation. In the present study, we found that tumor necrosis factor-α (TNF-α), a key regulator of bone matrix properties and composition that also inhibits terminal osteoblast differentiation, strongly inhibited POEM expression in the mouse osteoblastic cell line MC3T3-E1. TNF-α-induced down-regulation of POEM gene expression occurred in both time- and dose-dependent manners through the nuclear factor kappa B (NF-κB) pathway. In addition, expressions of marker genes in differentiated osteoblasts were down-regulated by TNF-α in a manner consistent with our findings for POEM, while over-expression of POEM recovered TNF-α-induced inhibition of osteoblast differentiation. These results suggest that TNF-α inhibits POEM expression through the NF-κB signaling pathway and down-regulation of POEM influences the inhibition of osteoblast differentiation by TNF-α.

  10. The In-Vitro Effects of Sea Cucumber (Stichopus sp1) Extract on Human Osteoblast Cell Line

    OpenAIRE

    Shahrulazua, A; Samsudin, AR; Iskandar, MA; Amran, AS

    2013-01-01

    Abstract Despite its claimed therapeutic effects, the action of sea cucumber (known as gamat in the Malay language) on human osteoblast cells is still unknown. We performed in vitro studies utilising extract of Stichopus sp1 (gamat) to elucidate its effects on cell viability and functional activity. We found an inverse relationship between gamat concentration and its effect on osteoblast cell viability (p

  11. Tartrate-resistant acid phosphatase (TRAP) co-localizes with receptor activator of NF-KB ligand (RANKL) and osteoprotegerin (OPG) in lysosomal-associated membrane protein 1 (LAMP1)-positive vesicles in rat osteoblasts and osteocytes

    OpenAIRE

    Solberg, L. B.; Stang, E.; Brorson, S.-H.; Andersson, G; Reinholt, F.P.

    2014-01-01

    Tartrate-resistant acid phosphatase (TRAP) is well known as an osteoclast marker; however, a recent study from our group demonstrated enhanced number of TRAP + osteocytes as well as enhanced levels of TRAP located to intracellular vesicles in osteoblasts and osteocytes in experimental osteoporosis in rats. Such vesicles were especially abundant in osteoblasts and osteocytes in cancellous bone as well as close to bone surface and intracortical remodeling sites. To further investigate TRAP in o...

  12. Gold nanoparticles stimulate differentiation and mineralization of primary osteoblasts through the ERK/MAPK signaling pathway

    International Nuclear Information System (INIS)

    Gold nanoparticles (AuNPs) have shown great promise for a variety of applications, including chemistry, biology, and medicine. Recently, AuNPs have found promising applications in cartilage and bone repair. However, to realize the above promised applications, more work needs to be carried out to clarify the interactions between biological systems and AuNPs. In the present study, primary osteoblasts were used to evaluate the biocompatibility of 20-nm and 40-nm AuNPs, including morphology, proliferation, differentiation, gene and protein expression, and the underlying mechanisms. The results demonstrated that AuNPs were taken up by osteoblasts and aggregated in perinuclear compartment and vescular structures, but no morphological changes were observed. AuNPs could significantly promote the proliferation of osteoblasts, enhance the ALP activities, and increase the number of bone nodules and calcium content in vitro. In addition, the expression of BMP-2, Runx-2, OCN and Col-1 was remarkably up-regulated in the presence of AuNPs. It is noteworthy that 20-nm AuNPs are more potent than 40-nm AuNPs in regulating osteoblast activities. Besides, AuNPs increased the level of ERK phosphorylation/total ERK, suggesting the activation of ERK/MAPK pathway is involved in above activities. In conclusion, AuNPs exhibited great biocompatibility with osteoblasts, and have tremendous potential to be used as drug and/or gene delivery carrier for bone and tissue engineering in the future. - Highlights: • AuNPs aggregated in perinuclear compartment and vescular structures of osteoblasts. • AuNPs up-regulated the expression of Runx-2, BMP-2, OCN and Col I of osteoblasts. • AuNPs enhanced osteoblast differentiation by activating the ERK/MAPK pathway. • The size of nanoparticles may be important to exhibit their biological effects. • AuNPs have tremendous potential in bone and tissue engineering in future

  13. Gold nanoparticles stimulate differentiation and mineralization of primary osteoblasts through the ERK/MAPK signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Dawei [Department of Pharmacology, Guangdong Medical College, Dongguan 523808 (China); Department of Biomedical Sciences, City University of Hong Kong, Kowloon Tong (Hong Kong); Liu, Dandan [College of Chemistry and Environmental Science, Chemical Biology Key Laboratory of Hebei Province, Hebei University, Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of the Ministry of Education, Baoding 071002 (China); Department of Biomedical Sciences, City University of Hong Kong, Kowloon Tong (Hong Kong); Zhang, Jinchao, E-mail: jczhang6970@163.com [College of Chemistry and Environmental Science, Chemical Biology Key Laboratory of Hebei Province, Hebei University, Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of the Ministry of Education, Baoding 071002 (China); Fong, Chichun [Department of Biomedical Sciences, City University of Hong Kong, Kowloon Tong (Hong Kong); Yang, Mengsu, E-mail: bhmyang@cityu.edu.hk [Department of Biomedical Sciences, City University of Hong Kong, Kowloon Tong (Hong Kong)

    2014-09-01

    Gold nanoparticles (AuNPs) have shown great promise for a variety of applications, including chemistry, biology, and medicine. Recently, AuNPs have found promising applications in cartilage and bone repair. However, to realize the above promised applications, more work needs to be carried out to clarify the interactions between biological systems and AuNPs. In the present study, primary osteoblasts were used to evaluate the biocompatibility of 20-nm and 40-nm AuNPs, including morphology, proliferation, differentiation, gene and protein expression, and the underlying mechanisms. The results demonstrated that AuNPs were taken up by osteoblasts and aggregated in perinuclear compartment and vescular structures, but no morphological changes were observed. AuNPs could significantly promote the proliferation of osteoblasts, enhance the ALP activities, and increase the number of bone nodules and calcium content in vitro. In addition, the expression of BMP-2, Runx-2, OCN and Col-1 was remarkably up-regulated in the presence of AuNPs. It is noteworthy that 20-nm AuNPs are more potent than 40-nm AuNPs in regulating osteoblast activities. Besides, AuNPs increased the level of ERK phosphorylation/total ERK, suggesting the activation of ERK/MAPK pathway is involved in above activities. In conclusion, AuNPs exhibited great biocompatibility with osteoblasts, and have tremendous potential to be used as drug and/or gene delivery carrier for bone and tissue engineering in the future. - Highlights: • AuNPs aggregated in perinuclear compartment and vescular structures of osteoblasts. • AuNPs up-regulated the expression of Runx-2, BMP-2, OCN and Col I of osteoblasts. • AuNPs enhanced osteoblast differentiation by activating the ERK/MAPK pathway. • The size of nanoparticles may be important to exhibit their biological effects. • AuNPs have tremendous potential in bone and tissue engineering in future.

  14. MicroRNA-194 promotes osteoblast differentiation via downregulating STAT1

    Energy Technology Data Exchange (ETDEWEB)

    Li, Jun [Department of Emergency, Shannxi Province People' s Hospital, Third Affiliated Hospital of Xi' an Jiaotong University, Xi' an 710052 (China); He, Xijing [Department of Orthopaedics, Second Affiliated Hospital of Xi' an Jiaotong University, Xi' an 710004 (China); Wei, Wenzhi [Department of Emergency, Shannxi Province People' s Hospital, Third Affiliated Hospital of Xi' an Jiaotong University, Xi' an 710052 (China); Zhou, Xiaobo, E-mail: xiaobozhouxa@163.com [Department of Immunology and Pathogenic Biology, Medical School, Xi' an Jiaotong University, Xi' an 710061 (China)

    2015-05-01

    Osteoblast differentiation is a vital process in maintaining bone homeostasis in which various transcriptional factors, signaling molecules, and microRNAs (miRNAs) are involved. Recently, signal transducer and activator of transcription 1 (STAT1) has been found to play an important role in regulating osteoblast differentiation. Here, we identified that STAT1 expression was regulated by miR-194. Using mouse bone mesenchymal stem cells (BMSCs), we found that miR-194 expression was significantly increased following osteoblast differentiation induction. Overexpression of miR-194 by lentivirus-mediated gene transfer markedly increased osteoblast differentiation, whereas inhibition of miR-194 significantly suppressed osteoblast differentiation of BMSCs. Using a dual-luciferase reporter assay, a direct interaction between miR-194 and the 3′-untranslated region (UTR) of STAT1 was confirmed. Additionally, miR-194 regulated mRNA and protein expression of STAT1 in BMSCs. Further analysis showed that miR-194 overexpression promoted the nuclear translocation of runt-related transcription factor 2 (Runx2), which is critical for osteoblast differentiation. In contrast, inhibition of miR-194 blocked the nuclear translocation of Runx2. Moreover, overexpression of STAT1 significantly blocked Runx2 nuclear translocation and osteoblast differentiation mediated by miR-194 overexpression. Taken together, our data suggest that miR-194 regulates osteoblast differentiation through modulating STAT1-mediated Runx2 nuclear translocation. - Highlights: • Overexpression of miR-194 significantly increased osteoblast differentiation. • miR-194 directly targeted the 3′- UTR of STAT1. • miR-194 regulated the expression of STAT1. • Overexpression of miR-194 promoted the nuclear translocation of Runx2.

  15. Relaxation training affects success and activation on a teaching test.

    Science.gov (United States)

    Helin, P; Hänninen, O

    1987-12-01

    We studied the effects of an audiocassette-relaxation training period (ART) and its timing on success at a teaching test (lecture type), on observed tension and on a number of physiological responses. The electrical activity of the upper trapezius muscle (EMG), heart rate (HR) and blood pressure (BP), of female and male instructor candidates, were examined before, during and after the teaching test as well as during its critique. The relaxation period (18 min) was presented either on the preceding night (ARTnt) or immediately before the teaching test (ARTimm). The influence of personality (types A-B and extrovert-introvert) was also studied. ART improved success at the teaching test in both sexes. In males (but not in females), ARTimm decreased EMG level during the test, but ARTnt increased EMG at the test period as compared to the control group. In females, both ARTnt and ARTimm lowered HR more than in the control group. ARTimm lowered systolic BP in both sexes. Personality types affected the ART responses; ART was more beneficial for type A than B subjects. PMID:3325481

  16. Mechanism involved in enhancement of osteoblast differentiation by hyaluronic acid

    Energy Technology Data Exchange (ETDEWEB)

    Kawano, Michinao [Division of Maxillofacial Diagnostic and Surgical Science, Department of Oral and Maxillofacial Surgery, Kyushu Dental College, Kitakyushu 803-8580 (Japan); Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental College, Kitakyushu 803-8580 (Japan); Ariyoshi, Wataru [Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental College, Kitakyushu 803-8580 (Japan); Iwanaga, Kenjiro [Division of Maxillofacial Diagnostic and Surgical Science, Department of Oral and Maxillofacial Surgery, Kyushu Dental College, Kitakyushu 803-8580 (Japan); Okinaga, Toshinori [Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental College, Kitakyushu 803-8580 (Japan); Habu, Manabu [Division of Maxillofacial Diagnostic and Surgical Science, Department of Oral and Maxillofacial Surgery, Kyushu Dental College, Kitakyushu 803-8580 (Japan); Yoshioka, Izumi [Division of Oral and Maxillofacial Surgery, Department of Medicine of Sensory and Motor Organs, University of Miyazaki, Kiyotake, Miyazaki 889-1692 (Japan); Tominaga, Kazuhiro [Division of Maxillofacial Diagnostic and Surgical Science, Department of Oral and Maxillofacial Surgery, Kyushu Dental College, Kitakyushu 803-8580 (Japan); Oral Bioresearch Center, Kyushu Dental College, Kitakyushu 803-8580 (Japan); Nishihara, Tatsuji, E-mail: tatsujin@kyu-dent.ac.jp [Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental College, Kitakyushu 803-8580 (Japan); Oral Bioresearch Center, Kyushu Dental College, Kitakyushu 803-8580 (Japan)

    2011-02-25

    Research highlights: {yields} In this study was to investigate the effects of HA on osteoblast differentiation induced by BMP-2. {yields} MG63 cells were incubated with BMP-2 and HA for various time periods. {yields} Phosphorylation of Smad 1/5/8, p38, and ERK proteins was determined by western blot analysis. To elucidate the nuclear translocation of phosphorylated Smad 1/5/8, stimulated cells were subjected to immunofluorescence microscopy. {yields} HA enhanced BMP-2 induces osteoblastic differentiation in MG63 cells via down-regulation of BMP-2 antagonists and ERK phosphorylation. -- Abstract: Objectives: Bone morphogenetic protein-2 (BMP-2) is expected to be utilized to fill bone defects and promote healing of fractures. However, it is unable to generate an adequate clinical response for use in bone regeneration. Recently, it was reported that glycosaminoglycans, including heparin, heparan sulfate, keratan sulfate, dermatan sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, and hyaluronic acid (HA), regulate BMP-2 activity, though the mechanism by which HA regulates osteogenic activities has not been fully elucidated. The aim of this study was to investigate the effects of HA on osteoblast differentiation induced by BMP-2. Materials and methods: Monolayer cultures of osteoblastic lineage MG63 cells were incubated with BMP-2 and HA for various time periods. To determine osteoblastic differentiation, alkaline phosphatase (ALP) activity in the cell lysates was quantified. Phosphorylation of Smad 1/5/8, p38, and ERK proteins was determined by Western blot analysis. To elucidate the nuclear translocation of phosphorylated Smad 1/5/8, stimulated cells were subjected to immunofluorescence microscopy. To further elucidate the role of HA in enhancement of BMP-2-induced Smad signaling, mRNA expressions of the BMP-2 receptor antagonists noggin and follistatin were detected using real-time RT-PCR. Results: BMP-2-induced ALP activation, Smad 1/5/8 phosphorylation, and

  17. Mechanism involved in enhancement of osteoblast differentiation by hyaluronic acid

    International Nuclear Information System (INIS)

    Research highlights: → In this study was to investigate the effects of HA on osteoblast differentiation induced by BMP-2. → MG63 cells were incubated with BMP-2 and HA for various time periods. → Phosphorylation of Smad 1/5/8, p38, and ERK proteins was determined by western blot analysis. To elucidate the nuclear translocation of phosphorylated Smad 1/5/8, stimulated cells were subjected to immunofluorescence microscopy. → HA enhanced BMP-2 induces osteoblastic differentiation in MG63 cells via down-regulation of BMP-2 antagonists and ERK phosphorylation. -- Abstract: Objectives: Bone morphogenetic protein-2 (BMP-2) is expected to be utilized to fill bone defects and promote healing of fractures. However, it is unable to generate an adequate clinical response for use in bone regeneration. Recently, it was reported that glycosaminoglycans, including heparin, heparan sulfate, keratan sulfate, dermatan sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, and hyaluronic acid (HA), regulate BMP-2 activity, though the mechanism by which HA regulates osteogenic activities has not been fully elucidated. The aim of this study was to investigate the effects of HA on osteoblast differentiation induced by BMP-2. Materials and methods: Monolayer cultures of osteoblastic lineage MG63 cells were incubated with BMP-2 and HA for various time periods. To determine osteoblastic differentiation, alkaline phosphatase (ALP) activity in the cell lysates was quantified. Phosphorylation of Smad 1/5/8, p38, and ERK proteins was determined by Western blot analysis. To elucidate the nuclear translocation of phosphorylated Smad 1/5/8, stimulated cells were subjected to immunofluorescence microscopy. To further elucidate the role of HA in enhancement of BMP-2-induced Smad signaling, mRNA expressions of the BMP-2 receptor antagonists noggin and follistatin were detected using real-time RT-PCR. Results: BMP-2-induced ALP activation, Smad 1/5/8 phosphorylation, and nuclear translocation

  18. Alkaline phosphatase in osteoblasts is down-regulated by pulsatile fluid flow

    Science.gov (United States)

    Hillsley, M. V.; Frangos, J. A.

    1997-01-01

    It is our hypothesis that interstitial fluid flow plays a role in the bone remodeling response to mechanical loading. The fluid flow-induced expression of three proteins (collagen, osteopontin, and alkaline phosphatase) involved in bone remodeling was investigated. Rat calvarial osteoblasts subjected to pulsatile fluid flow at an average shear stress of 5 dyne/cm2 showed decreased alkaline phosphatase (AP) mRNA expression after only 1 hour of flow. After 3 hours of flow, AP mRNA levels had decreased to 30% of stationary control levels and remained at this level for an additional 5 hours of flow. Steady flow (4 dyne/cm2 fluid shear stress), in contrast, resulted in a delayed and less dramatic decrease in AP mRNA expression to 63% of control levels after 8 hours of flow. The reduced AP mRNA expression under pulsatile flow conditions was followed by reduced AP enzyme activity after 24 hours. No changes in collagen or osteopontin mRNA expression were detected over 8 hours of pulsatile flow. This is the first time fluid flow has been shown to affect gene expression in osteoblasts.

  19. Centrifugation of Cultured Osteoblasts And Macrophages as a Model To Study How Gravity Regulates The Function of Skeletal Cells

    Science.gov (United States)

    Globus, Ruth K.; Searby, Nancy D.; Almeida, Eduardo A. C.; Sutijono, Darrell; Yu, Joon-Ho; Malouvier, Alexander; Doty, Steven B.; Morey-Holton, Emily; Weinstein, Steven L.; Dalton, Bonnie P. (Technical Monitor)

    2000-01-01

    Mechanical loading helps define the architecture of weight-bearing bone via the tightly regulated process of skeletal turnover. Turnover occurs by the concerted activity of osteoblasts, responsible for bone formation. and osteoclasts, responsible for bone resorption. Osteoclasts are specialized megakaryon macrophages, which differentiate from monocytes in response to resorption stimuli, such as reduced weight-bearing. Habitation in space dramatically alters musculoskeletal loading, which modulates both cell function and bone structure. Our long-term objective is to define the molecular and cellular mechanisms that mediate skeletal adaptations to altered gravity environments. Our experimental approach is to apply hypergravity loads by centrifugation to rodents and cultured cells. As a first step, we examined the influence of centrifugation on the structure of cancellous bone in rats to test the ability of hypergravity to change skeletal architecture. Since cancellous bone undergoes rapid turnover we expected the most dramatic structural changes to occur in the shape of trabeculae of weight-bearing, cancellous bone. To define the cellular responses to hypergravity loads, we exposed cultured osteoblasts and macrophages to centrifugation. The intraosseous and intramedullary pressures within long bones in vivo reportedly range from 12-40 mm Hg, which would correspond to 18-59 gravity (g) in our cultures. We assumed that hydrostatic pressure from the medium above the cell layer is at least one major component of the mechanical load generated by centrifuging cultured cells. and therefore we exposed the cells to 10-50g. In osteoblasts, we examined the structure of their actin and microtubule networks, production of prostaglandin E2 (PGE2), and cell survival. Analysis of the shape of the cytoskeletal networks provides evidence for the ability of centrifugation to affect cell structure, while the production of PGE2 serves as a convenient marker for mechanical stimulation. We

  20. Glucocorticoid Suppresses Connexin 43 Expression by Inhibiting the Akt/mTOR Signaling Pathway in Osteoblasts.

    Science.gov (United States)

    Shen, Chen; Kim, Mi Ran; Noh, Jeong Mi; Kim, Su Jin; Ka, Sun-O; Kim, Ji Hye; Park, Byung-Hyun; Park, Ji Hyun

    2016-07-01

    The inhibition of proliferation or functional alteration of osteoblasts by glucocorticoids (GCs) has been recognized as an important etiology of GC-induced osteoporosis (GIO). Connexin 43 (Cx43) is the most abundant connexin isoform in bone cells and plays important roles in bone remodeling. Despite the important role of Cx43 in bone homeostasis and the prevalence of GIO, the direct action of GCs on Cx43 expression in osteoblasts has been poorly described. The aim of the present study was to evaluate how GCs affect Cx43 expression in osteoblasts. Dexamethasone (Dex) treatment decreased expression of Cx43 RNA and protein in MC3T3-E1 mouse osteoblastic cells. Reduction of Cx43 expression by Dex was dependent on the glucocorticoid receptor (GR), as it was abolished by pretreatment with a GR blocker. Treatment with PTH (1-34), a medication used for GIO management, counteracted the suppression of Cx43 by Dex. Akt or mTOR signaling modulators revealed the involvement of the Akt/mTOR signaling pathway in Dex-induced reduction of Cx43 expression. Moreover, overexpression of Cx43 significantly attenuated Dex-inhibited cell viability and proliferation, as evidenced by MTT and bromodeoxyuridine (BrdU) incorporation assay of MC3T3-E1 cells. To account for possible species or cell type differences, human primary osteoblasts were treated with Dex and similar downregulation of Cx43 by Dex was observed. In addition, immunofluorescent staining for Cx43 further demonstrated an apparent decrease in Dex-treated human osteoblasts, while analysis of lucifer yellow propagation revealed reduced gap junction intercellular communication by Dex. Collectively, these findings indicate that GCs suppress Cx43 expression in osteoblasts via GR and the Akt/mTOR signaling pathway and overexpression of Cx43 may, at least in part, rescue osteoblasts from GC-induced reductions in proliferation. PMID:26914606

  1. Characterization of Vitamin k2 FUNTIONS IN Saos-2 osteoblasts

    OpenAIRE

    Strand, Siv Kristine Sola

    2012-01-01

    MK-7 as a potential treatment against osteoporosis, favoring bone formation, was explored in this thesis. Today most treatments for bone loss target suppression of osteoclastogenesis.Biological activity and cytotoxicity of vitamin K2, MK-7, in osteoblasts were investigated to give a better understanding of MK-7’s effect on bone formation. In order to determine these characteristics, rezasurin assays, qPCR assays and arachidonic acid release assays of Saos-2 cells were used throughout this stu...

  2. Prevention of osteocyte and osteoblast apoptosis by bisphosphonates and calcitonin

    OpenAIRE

    Plotkin, Lilian I.; Weinstein, Robert S; Parfitt, A. Michael; Roberson, Paula K.; Manolagas, Stavros C.; Bellido, Teresita

    1999-01-01

    Glucocorticoid-induced osteoporosis may be due, in part, to increased apoptosis of osteocytes and osteoblasts, and bisphosphonates (BPs) are effective in the management of this condition. We have tested the hypothesis that BPs suppress apoptosis in these cell types. Etidronate, alendronate, pamidronate, olpadronate, or amino-olpadronate (IG9402, a bisphosphonate that lacks antiresorptive activity) at 10–9 to 10–6 M prevented apoptosis of murine osteocytic MLO-Y4 cells, whether it was induced ...

  3. The transient receptor potential ion channel TRPV6 is expressed at low levels in osteoblasts and has little role in osteoblast calcium uptake.

    Directory of Open Access Journals (Sweden)

    Robert Little

    Full Text Available BACKGROUND: TRPV6 ion channels are key mediators of regulated transepithelial absorption of Ca2+ within the small intestine. Trpv6-/- mice were reported to have lower bone density than wild-type littermates and significant disturbances in calcium homeostasis that suggested a role for TRPV6 in osteoblasts during bone formation and mineralization. TRPV6 and molecules related to transepithelial Ca2+ transport have been reported to be expressed at high levels in human and mouse osteoblasts. RESULTS: Transmembrane ion currents in whole cell patch clamped SaOS-2 osteoblasts did not show sensitivity to ruthenium red, an inhibitor of TRPV5/6 ion channels, and 45Ca uptake was not significantly affected by ruthenium red in either SaOS-2 (P=0.77 or TE-85 (P=0.69 osteoblastic cells. In contrast, ion currents and 45Ca uptake were both significantly affected in a human bronchial epithelial cell line known to express TRPV6. TRPV6 was expressed at lower levels in osteoblastic cells than has been reported in some literature. In SaOS-2 TRPV6 mRNA was below the assay detection limit; in TE-85 TRPV6 mRNA was detected at 6.90±1.9×10(-5 relative to B2M. In contrast, TRPV6 was detected at 7.7±3.0×10(-2 and 2.38±0.28×10(-4 the level of B2M in human carcinoma-derived cell lines LNCaP and CaCO-2 respectively. In murine primary calvarial osteoblasts TRPV6 was detected at 3.80±0.24×10(-5 relative to GAPDH, in contrast with 4.3±1.5×10(-2 relative to GAPDH in murine duodenum. By immunohistochemistry, TRPV6 was expressed mainly in myleocytic cells of the murine bone marrow and was observed only at low levels in murine osteoblasts, osteocytes or growth plate cartilage. CONCLUSIONS: TRPV6 is expressed only at low levels in osteoblasts and plays little functional role in osteoblastic calcium uptake.

  4. Osteoblast-derived WNT16 represses osteoclastogenesis and prevents cortical bone fragility fractures.

    Science.gov (United States)

    Movérare-Skrtic, Sofia; Henning, Petra; Liu, Xianwen; Nagano, Kenichi; Saito, Hiroaki; Börjesson, Anna E; Sjögren, Klara; Windahl, Sara H; Farman, Helen; Kindlund, Bert; Engdahl, Cecilia; Koskela, Antti; Zhang, Fu-Ping; Eriksson, Emma E; Zaman, Farasat; Hammarstedt, Ann; Isaksson, Hanna; Bally, Marta; Kassem, Ali; Lindholm, Catharina; Sandberg, Olof; Aspenberg, Per; Sävendahl, Lars; Feng, Jian Q; Tuckermann, Jan; Tuukkanen, Juha; Poutanen, Matti; Baron, Roland; Lerner, Ulf H; Gori, Francesca; Ohlsson, Claes

    2014-11-01

    The WNT16 locus is a major determinant of cortical bone thickness and nonvertebral fracture risk in humans. The disability, mortality and costs caused by osteoporosis-induced nonvertebral fractures are enormous. We demonstrate here that Wnt16-deficient mice develop spontaneous fractures as a result of low cortical thickness and high cortical porosity. In contrast, trabecular bone volume is not altered in these mice. Mechanistic studies revealed that WNT16 is osteoblast derived and inhibits human and mouse osteoclastogenesis both directly by acting on osteoclast progenitors and indirectly by increasing expression of osteoprotegerin (Opg) in osteoblasts. The signaling pathway activated by WNT16 in osteoclast progenitors is noncanonical, whereas the pathway activated in osteoblasts is both canonical and noncanonical. Conditional Wnt16 inactivation revealed that osteoblast-lineage cells are the principal source of WNT16, and its targeted deletion in osteoblasts increases fracture susceptibility. Thus, osteoblast-derived WNT16 is a previously unreported key regulator of osteoclastogenesis and fracture susceptibility. These findings open new avenues for the specific prevention or treatment of nonvertebral fractures, a substantial unmet medical need. PMID:25306233

  5. Activity Clinic and Affects in Workplace Conflicts: Transformation through transferential activity

    Directory of Open Access Journals (Sweden)

    Livia Scheller

    2014-04-01

    Full Text Available This paper presents some reflections about an approach in work psychology: the Activity Clinic.After a brief introduction to the conceptual background of the “Activity Clinic”, it covers threedeeply interconnected themes. The first concerns the meaning attributed to the development of theaffects present in the work situation under analysis; the second discusses the reasons for theconflicts that are ultimately due to these affects; the third considers how a method of co-analysisof the activity can lead towards transformation of those conflicts.Our reflections refer to the process engendered by this methodological approach as one of“transferential activity”. The paper explains this process by empirically describing the“transport” of affects involved in the conflicts. The personal interpretation of the cause ofproblems gives way to the understanding that they are due to organizational dysfunction ratherthan to individual personalities. Measures can then be taken to break the deadlocks experiencedboth at the personal and collective level.

  6. Serine dipeptide lipids of Porphyromonas gingivalis inhibit osteoblast differentiation: Relationship to Toll-like receptor 2.

    Science.gov (United States)

    Wang, Yu-Hsiung; Nemati, Reza; Anstadt, Emily; Liu, Yaling; Son, Young; Zhu, Qiang; Yao, Xudong; Clark, Robert B; Rowe, David W; Nichols, Frank C

    2015-12-01

    Porphyromonas gingivalis is a periodontal pathogen strongly associated with loss of attachment and supporting bone for teeth. We have previously shown that the total lipid extract of P. gingivalis inhibits osteoblast differentiation through engagement of Toll-like receptor 2 (TLR2) and that serine dipeptide lipids of P. gingivalis engage both mouse and human TLR2. The purpose of the present investigation was to determine whether these serine lipids inhibit osteoblast differentiation in vitro and in vivo and whether TLR2 engagement is involved. Osteoblasts were obtained from calvaria of wild type or TLR2 knockout mouse pups that also express the Col2.3GFP transgene. Two classes of serine dipeptide lipids, termed Lipid 654 and Lipid 430, were tested. Osteoblast differentiation was monitored by cell GFP fluorescence and osteoblast gene expression and osteoblast function was monitored as von Kossa stained mineral deposits. Osteoblast differentiation and function were evaluated in calvarial cell cultures maintained for 21 days. Lipid 654 significantly inhibited GFP expression, osteoblast gene expression and mineral nodule formation and this inhibition was dependent on TLR2 engagement. Lipid 430 also significantly inhibited GFP expression, osteoblast gene expression and mineral nodule formation but these effects were only partially attributed to engagement of TLR2. More importantly, Lipid 430 stimulated TNF-α and RANKL gene expression in wild type cells but not in TLR2 knockout cells. Finally, osteoblast cultures were observed to hydrolyze Lipid 654 to Lipid 430 and this likely occurs through elevated PLA2 activity in the cultured cells. In conclusion, our results show that serine dipeptide lipids of P. gingivalis inhibit osteoblast differentiation and function at least in part through engagement of TLR2. The Lipid 430 serine class also increased the expression of genes that could increase osteoclast activity. We conclude that Lipid 654 and Lipid 430 have the potential

  7. Osteoblast growth behavior on porous-structure titanium surface

    Energy Technology Data Exchange (ETDEWEB)

    Tian Yuan; Ding Siyang; Peng Hui; Lu Shanming; Wang Guoping [Research Institute of Stomatology, Nanjing Medical University, Nanjing 210029 (China); Xia Lu, E-mail: shelueia@yahoo.com.cn [Research Institute of Stomatology, Nanjing Medical University, Nanjing 210029 (China); Wang Peizhi, E-mail: wangpzi@sina.com [Research Institute of Stomatology, Nanjing Medical University, Nanjing 210029 (China)

    2012-11-15

    Highlights: Black-Right-Pointing-Pointer Micro-arc oxidation technology formed a porous feature on titanium surface. Black-Right-Pointing-Pointer This porous surface accelerated adhesion, proliferation and differentiation compared with smooth surface. Black-Right-Pointing-Pointer Osteogenesis-related proteins and genes were up regulated by this porous surface. Black-Right-Pointing-Pointer It is anticipated that micro-arc oxidation surface could enhance osteoblastic activity and bone regeneration. - Abstract: A bioavailable surface generated by nano-technology could accelerate implant osteointegration, reduce healing time and enable implants to bear early loading. In this study, a nano-porous surface of titanium wafers was modified using micro-arc oxidation technique; surface of smooth titanium was used as control group. Surface characteristic was evaluated by investigating morphology, roughness and hydrophilicity of titanium wafers. In vitro studies, osteoblastic adhesion, proliferation and ALP activity, as well as gene and protein expressions relative to mineralization were assayed. Our results showed that a crater-liked nano-porous surface with greater roughness and better hydrophilicity were fabricated by micro-arc oxidation. It was further indicated that nano-porous surface could enhance adhesion, proliferation and ALP activity of osteoblasts compared with smooth surfaces. In addition, gene and protein expression of collagen-I, osteocalcin and osteopontin were also obviously increased. In summary, micro-arc oxidized techniques could form an irregular nano-porous morphology on implant surface which is favorable to improve osteoblastic function and prospected to be a potent modification of dental implant.

  8. Isoquercitrin and polyphosphate co-enhance mineralization of human osteoblast-like SaOS-2 cells via separate activation of two RUNX2 cofactors AFT6 and Ets1.

    Science.gov (United States)

    Wang, Xiaohong; Schröder, Heinz C; Feng, Qingling; Diehl-Seifert, Bärbel; Grebenjuk, Vladislav A; Müller, Werner E G

    2014-06-01

    Isoquercitrin, a dietary phytoestrogen, is a potential stimulator of bone mineralization used for prophylaxis of osteoporotic disorders. Here we studied the combined effects of isoquercitrin, a cell membrane permeable 3-O-glucoside of quercetin, and polyphosphate [polyP], a naturally occurring inorganic polymer inducing bone formation, on mineralization of human osteoblast-like SaOS-2 cells. Both compounds isoquercitrin and polyP induce at non-toxic concentrations the mineralization process of SaOS-2 cells. Co-incubation experiments revealed that isoquercitrin (at 0.1 and 0.3μM), if given simultaneously with polyP (as Ca(2+) salt; at 3, 10, 30 and 100μM) amplifies the mineralization-enhancing effect of the inorganic polymer. The biomineralization process induced by isoquercitrin and polyP is based on two different modes of action. After incubation of the cells with isoquercitrin or polyP the expression of the Runt-related transcription factor 2 [RUNX2] is significantly upregulated. In addition, isoquercitrin causes a strong increase of the steady-state-levels of the two co-activators of RUNX2, the activating transcription factor 6 [ATF6] and the Ets oncogene homolog 1 [Ets1]. The activating effect of isoquercitrin occurs via a signal transduction pathway involving ATF6, and by that, is independent from the induction cascade initiated by polyP. This conclusion is supported by the finding that isoquercitrin upregulates the expression of the gene encoding for osteocalcin, while polyP strongly increases the expression of the Ets1 gene and of the alkaline phosphatase. We show that the two compounds, polyP and isoquercitrin, have a co-enhancing effect on bone mineral formation and in turn might be of potential therapeutic value for prevention/treatment of osteoporosis. PMID:24726443

  9. Positive affect modulates activity in the visual cortex to images of high calorie foods.

    Science.gov (United States)

    Killgore, William D S; Yurgelun-Todd, Deborah A

    2007-05-01

    Activity within the visual cortex can be influenced by the emotional salience of a stimulus, but it is not clear whether such cortical activity is modulated by the affective status of the individual. This study used functional magnetic resonance imaging (fMRI) to examine the relationship between affect ratings on the Positive and Negative Affect Schedule and activity within the occipital cortex of 13 normal-weight women while viewing images of high calorie and low calorie foods. Regression analyses revealed that when participants viewed high calorie foods, Positive Affect correlated significantly with activity within the lingual gyrus and calcarine cortex, whereas Negative Affect was unrelated to visual cortex activity. In contrast, during presentations of low calorie foods, affect ratings, regardless of valence, were unrelated to occipital cortex activity. These findings suggest a mechanism whereby positive affective state may affect the early stages of sensory processing, possibly influencing subsequent perceptual experience of a stimulus. PMID:17464782

  10. Affect and Subsequent Physical Activity: An Ambulatory Assessment Study Examining the Affect-Activity Association in a Real-Life Context

    Science.gov (United States)

    Niermann, Christina Y. N.; Herrmann, Christian; von Haaren, Birte; van Kann, Dave; Woll, Alexander

    2016-01-01

    Traditionally, cognitive, motivational, and volitional determinants have been used to explain and predict health behaviors such as physical activity. Recently, the role of affect in influencing and regulating health behaviors received more attention. Affects as internal cues may automatically activate unconscious processes of behavior regulation. The aim of our study was to examine the association between affect and physical activity in daily life. In addition, we studied the influence of the habit of being physically active on this relationship. An ambulatory assessment study in 89 persons (33.7% male, 25 to 65 years, M = 45.2, SD = 8.1) was conducted. Affect was assessed in the afternoon on 5 weekdays using smartphones. Physical activity was measured continuously objectively using accelerometers and subjectively using smartphones in the evening. Habit strength was assessed at the beginning of the diary period. The outcomes were objectively and subjectively measured moderate-to-vigorous physical activity (MVPA) performed after work. Multilevel regression models were used to analyze the association between affect and after work MVPA. In addition, the cross-level interaction of habit strength and affect on after work MVPA was tested. Positive affect was positively related to objectively measured and self-reported after work MVPA: the greater the positive affect the more time persons subsequently spent on MVPA. An inverse relationship was found for negative affect: the greater the negative affect the less time persons spent on MVPA. The cross-level interaction effect was significant only for objectively measured MVPA. A strong habit seems to strengthen both the positive influence of positive affect and the negative influence of negative affect. The results of this study confirm previous results and indicate that affect plays an important role for the regulation of physical activity behavior in daily life. The results for positive affect were consistent. However, in

  11. Affect and subsequent physical activity: An ambulatory assessment study examining the affect-activity association in a real-life context

    Directory of Open Access Journals (Sweden)

    Christina eNiermann

    2016-05-01

    Full Text Available Traditionally, cognitive, motivational and volitional determinants have been used to explain and predict health behaviors such as physical activity. Recently, the role of affect in influencing and regulating health behaviors received more attention. Affects as internal cues may automatically activate unconscious processes of behavior regulation. The aim of our study was to examine the association between affect and physical activity in daily life. In addition, we studied the influence of the habit of being physically active on this relationship.An ambulatory assessment study in 89 persons (33.7% male, 25 to 65 years, M=45.2, SD=8.1 was conducted. Affect was assessed in the afternoon on 5 weekdays using smartphones. Physical activity was measured continuously objectively using accelerometers and subjectively using smartphones in the evening. Habit strength was assessed at the beginning of the diary period. The outcomes were objectively and subjectively measured moderate-to-vigorous physical activity (MVPA performed after work. Multilevel regression models were used to analyze the association between affect and after work MVPA. In addition, the cross-level interaction of habit strength and affect on after work MVPA was tested.Positive affect was positively related to objectively measured and self-reported after work MVPA: the greater the positive affect the more time persons subsequently spent on MVPA. An inverse relationship was found for negative affect: the greater the negative affect the less time persons spent on MVPA. The cross-level interaction effect was significant only for objectively measured MVPA. A strong habit seems to strengthen both the positive influence of positive affect and the negative influence of negative affect.The results of this study confirm previous results and indicate that affect plays an important role for the regulation of physical activity behavior in daily life. The results for positive affect were consistent

  12. Affect and Subsequent Physical Activity: An Ambulatory Assessment Study Examining the Affect-Activity Association in a Real-Life Context.

    Science.gov (United States)

    Niermann, Christina Y N; Herrmann, Christian; von Haaren, Birte; van Kann, Dave; Woll, Alexander

    2016-01-01

    Traditionally, cognitive, motivational, and volitional determinants have been used to explain and predict health behaviors such as physical activity. Recently, the role of affect in influencing and regulating health behaviors received more attention. Affects as internal cues may automatically activate unconscious processes of behavior regulation. The aim of our study was to examine the association between affect and physical activity in daily life. In addition, we studied the influence of the habit of being physically active on this relationship. An ambulatory assessment study in 89 persons (33.7% male, 25 to 65 years, M = 45.2, SD = 8.1) was conducted. Affect was assessed in the afternoon on 5 weekdays using smartphones. Physical activity was measured continuously objectively using accelerometers and subjectively using smartphones in the evening. Habit strength was assessed at the beginning of the diary period. The outcomes were objectively and subjectively measured moderate-to-vigorous physical activity (MVPA) performed after work. Multilevel regression models were used to analyze the association between affect and after work MVPA. In addition, the cross-level interaction of habit strength and affect on after work MVPA was tested. Positive affect was positively related to objectively measured and self-reported after work MVPA: the greater the positive affect the more time persons subsequently spent on MVPA. An inverse relationship was found for negative affect: the greater the negative affect the less time persons spent on MVPA. The cross-level interaction effect was significant only for objectively measured MVPA. A strong habit seems to strengthen both the positive influence of positive affect and the negative influence of negative affect. The results of this study confirm previous results and indicate that affect plays an important role for the regulation of physical activity behavior in daily life. The results for positive affect were consistent. However, in

  13. The bone resorption inhibitors odanacatib and alendronate affect post-osteoclastic events differently in ovariectomized rabbits.

    Science.gov (United States)

    Jensen, Pia Rosgaard; Andersen, Thomas Levin; Pennypacker, Brenda L; Duong, Le T; Delaissé, Jean-Marie

    2014-02-01

    Odanacatib (ODN) is a bone resorption inhibitor which differs from standard antiresorptives by its ability to reduce bone resorption without decreasing bone formation. What is the reason for this difference? In contrast with other antiresorptives, such as alendronate (ALN), ODN targets only the very last step of the resorption process. We hypothesize that ODN may therefore modify the remodeling events immediately following osteoclastic resorption. These events belong to the reversal phase and include recruitment of osteoblasts, which is critical for connecting bone resorption to formation. We performed a histomorphometric study of trabecular remodeling in vertebrae of estrogen-deficient rabbits treated or not with ODN or ALN, a model where ODN, but not ALN, was previously shown to preserve bone formation. In line with our hypothesis, we found that ODN treatment compared to ALN results in a shorter reversal phase, faster initiation of osteoid deposition on the eroded surfaces, and higher osteoblast recruitment. The latter is reflected by higher densities of mature bone forming osteoblasts and an increased subpopulation of cuboidal osteoblasts. Furthermore, we found an increase in the interface between osteoclasts and surrounding osteoblast-lineage cells. This increase is expected to favor the osteoclast-osteoblast interactions required for bone formation. Regarding bone resorption itself, we show that ODN, but not ALN, treatment results in shallower resorption lacunae, a geometry favoring bone stiffness. We conclude that, compared to standard antiresorptives, ODN shows distinctive effects on resorption geometry and on reversal phase activities which positively affect osteoblast recruitment and may therefore favor bone formation. PMID:24085265

  14. Oxidative Nanopatterning of Titanium Surface Influences mRNA and MicroRNA Expression in Human Alveolar Bone Osteoblastic Cells

    Science.gov (United States)

    Wimmers Ferreira, Maidy Rehder; Rodrigo Fernandes, Roger; Freire Assis, Amanda; Dernowsek, Janaína A.; Passos, Geraldo A.; Variola, Fabio; Fittipaldi Bombonato-Prado, Karina

    2016-01-01

    Titanium implants have been extensively used in orthopedic and dental applications. It is well known that micro- and nanoscale surface features of biomaterials affect cellular events that control implant-host tissue interactions. To improve our understanding of how multiscale surface features affect cell behavior, we used microarrays to evaluate the transcriptional profile of osteoblastic cells from human alveolar bone cultured on engineered titanium surfaces, exhibiting the following topographies: nanotexture (N), nano+submicrotexture (NS), and rough microtexture (MR), obtained by modulating experimental parameters (temperature and solution composition) of a simple yet efficient chemical treatment with a H2SO4/H2O2 solution. Biochemical assays showed that cell culture proliferation augmented after 10 days, and cell viability increased gradually over 14 days. Among the treated surfaces, we observed an increase of alkaline phosphatase activity as a function of the surface texture, with higher activity shown by cells adhering onto nanotextured surfaces. Nevertheless, the rough microtexture group showed higher amounts of calcium than nanotextured group. Microarray data showed differential expression of 716 mRNAs and 32 microRNAs with functions associated with osteogenesis. Results suggest that oxidative nanopatterning of titanium surfaces induces changes in the metabolism of osteoblastic cells and contribute to the explanation of the mechanisms that control cell responses to micro- and nanoengineered surfaces. PMID:27200092

  15. Overexpression of RANKL in osteoblasts: a possible mechanism of susceptibility to bone disease in cystic fibrosis.

    Science.gov (United States)

    Delion, Martial; Braux, Julien; Jourdain, Marie-Laure; Guillaume, Christine; Bour, Camille; Gangloff, Sophie; Pimpec-Barthes, Françoise Le; Sermet-Gaudelus, Isabelle; Jacquot, Jacky; Velard, Frédéric

    2016-09-01

    Bone fragility and loss are a significant cause of morbidity in patients with cystic fibrosis (CF), and the lack of effective therapeutic options means that treatment is more often palliative rather than curative. A deeper understanding of the pathogenesis of CF-related bone disease (CFBD) is necessary to develop new therapies. Defective CF transmembrane conductance regulator (CFTR) protein and chronic inflammation in bone are important components of the CFBD development. The receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG) drive the regulation of bone turnover. To investigate their roles in CFBD, we evaluated the involvement of defective CFTR in their production level in CF primary human osteoblasts with and without inflammatory stimulation, in the presence or not of pharmacological correctors of the CFTR. No major difference in cell ultrastructure was noted between cultured CF and non-CF osteoblasts, but a delayed bone matrix mineralization was observed in CF osteoblasts. Strikingly, resting CF osteoblasts exhibited strong production of RANKL protein, which was highly localized at the cell membrane and was enhanced in TNF (TNF-α) or IL-17-stimulated conditions. Under TNF stimulation, a defective response in OPG production was observed in CF osteoblasts in contrast to the elevated OPG production of non-CF osteoblasts, leading to an elevated RANKL-to-OPG protein ratio in CF osteoblasts. Pharmacological inhibition of CFTR chloride channel conductance in non-CF osteoblasts replicated both the decreased OPG production and the enhanced RANKL-to-OPG ratio. Interestingly, using CFTR correctors such as C18, we significantly reduced the production of RANKL by CF osteoblasts, in both resting and TNF-stimulated conditions. In conclusion, the overexpression of RANKL and high membranous RANKL localization in osteoblasts are related to defective CFTR, and may worsen bone resorption, leading to bone loss in patients with CF. Targeting

  16. Environmental polycyclic aromatic hydrocarbons affect androgen receptor activation in vitro

    DEFF Research Database (Denmark)

    Vinggaard, Anne Marie; Hnida, Christina; Larsen, John Christian

    2000-01-01

    of certain PAHs to activate the Ah receptor was assessed in H4IIE liver cancer cells, stably transfected with a luciferase reporter gene system. The positive control 2, 3,7, 8-tetrachlorodibenzodioxin (TCDD) caused a 13-14-fold induction of luciferase activity reaching maximum activity at 0.1 nM. DB...

  17. Environmental polycyclic aromatic hydrocarbons affect androgen receptor activation in vitro

    DEFF Research Database (Denmark)

    Vinggaard, Anne Marie; Hnida, Christina; Larsen, John Christian

    of certain PAHs to activate the Ah receptor was assessed in H4IIE liver cancer cells, stably transfected with a luciferase reporter gene system. The positive control 2, 3,7, 8-tetrachlorodibenzodioxin (TCDD) caused a 13-14-fold induction of luciferase activity reaching maximum activity at 0.1 nM. DB...

  18. The topographical properties of silica nanoparticle film preserve the osteoblast-like cell characteristics in vitro

    Science.gov (United States)

    Shim, Wooyoung; Lee, Seung Yun; Kim, Hyo-Sop; Kim, Jae-Ho

    2016-07-01

    The Transplantation of osteoblasts, along with an artificial implant, is experimentally considered as a therapeutics for degenerative bone diseases. However, osteoblasts have several limitations for application of transplantation in therapeutics, including a low-efficiency for bone mineralization and easy loss of characteristics in in vitro culture condition. In this study, we fabricated silica nano-particle (SNP) films using particles of different sizes to culture osteoblast-like cells for analysis the effect of topography on cellular behavior and characteristics. The physical parameters of films, such as intervals, height and roughness, were proportionally increased depending on the SNP diameter. When osteoblast-like cells were cultured on the various SNP films, the cell attachment rate on SNP-300 and SNP-700 was significantly decreased when it compared to tissue culture polystyrene (TCPS) group. In addition, the genes responsible for cell adhesion showed differential expression profiles in SNP films. The expression and activity of alkaline phosphatase were elevated in SNP-300 and SNP-700, and the extra-cellular matrix and osteoblast marker showed increased gene expression in these SNP films when compared to TCPS group. In the present study, we demonstrate that the topographical property of a nano-scale structure preserves the characteristics of osteoblast-like cells, and regulates the cellular behavior.

  19. Morphology and Differentiation of MG63 Osteoblast Cells on Saliva Contaminated Implant Surfaces

    Directory of Open Access Journals (Sweden)

    Neda Shams

    2015-11-01

    Full Text Available Objectives: Osteoblasts are the most important cells in the osseointegration process. Despite years of study on dental Implants, limited studies have discussed the effect of saliva on the adhesion process of osteoblasts to implant surfaces. The aim of this in vitro study was to evaluate the effect of saliva on morphology and differentiation of osteoblasts attached to implant surfaces.Materials and Methods: Twelve Axiom dental implants were divided into two groups. Implants of the case group were placed in containers, containing saliva, for 40 minutes. Then, all the implants were separately stored in a medium containing MG63 human osteoblasts for a week. Cell morphology and differentiation were assessed using a scanning electron microscope and their alkaline phosphatase (ALP activity was determined. The t-test was used to compare the two groups.Results: Scanning electron microscopic observation of osteoblasts revealed round or square cells with fewer and shorter cellular processes in saliva contaminated samples, whereas elongated, fusiform and well-defined cell processes were seen in the control group. ALP level was significantly lower in case compared to control group (P<0.05.Conclusion: Saliva contamination alters osteoblast morphology and differentiation and may subsequently interfere with successful osseointegration. Thus, saliva contamination of bone and implant must be prevented or minimized.

  20. Analysis of Osteoblast Differentiation on Polymer Thin Films Embedded with Carbon Nanotubes.

    Directory of Open Access Journals (Sweden)

    Jin Woo Lee

    Full Text Available Osteoblast differentiation can be modulated by variations in order of nanoscale topography. Biopolymers embedded with carbon nanotubes can cause various orders of roughness at the nanoscale and can be used to investigate the dynamics of extracellular matrix interaction with cells. In this study, clear relationship between the response of osteoblasts to integrin receptor activation, their phenotype, and transcription of certain genes on polymer composites embedded with carbon nanotubes was demonstrated. We generated an ultrathin nanocomposite film embedded with carbon nanotubes and observed improved adhesion of pre-osteoblasts, with a subsequent increase in their proliferation. The expression of genes encoding integrin subunits α5, αv, β1, and β3 was significantly upregulated at the early of time-point when cells initially attached to the carbon nanotube/polymer composite. The advantage of ultrathin nanocomposite film for pre-osteoblasts was demonstrated by staining for the cytoskeletal protein vinculin and cell nuclei. The expression of essential transcription factors for osteoblastogenesis, such as Runx2 and Sp7 transcription factor 7 (known as osterix, was upregulated after 7 days. Consequently, the expression of genes that determine osteoblast phenotype, such as alkaline phosphatase, type I collagen, and osteocalcin, was accelerated on carbon nanotube embedded polymer matrix after 14 days. In conclusion, the ultrathin nanocomposite film generated various orders of nanoscale topography that triggered processes related to osteoblast bone formation.

  1. Effects of β-Tricalcium Phosphate on the Growth of Rat Osteoblasts

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Calcium-phosphate materials, such as β- TCP could be degraded in vivo , and in the cultured cells in vitro. However, it is still unknown how the materials changed the activity of the cells. In order to understand the mechanism of β- TCP treatment on osteoblasts , cell proliferation rate measurement and microscopic observation were performed during co-culture of rat osteoblasts and β-TCP biomaterials. It was observed that low concentrations of β-TCP-treated cell proliferation rate was a little less than control by MTT assay, while higher concentrations of β- TCP-treated cell proliferation rate was more than control. The proliferation rate of osteoblast trented by β-TCP with bioglass had a similar effect with β-TCP-treated cell. Neither calcium chloride nor sodium monophosphate treatment could stimulate the cell proliferation at the concentration from 0.25 mM to 5 mM. During the treatment of β-TCP , particles could be found in the cells. It suggested that β-TCP could stimulate cell proliferation rate of rat osteoblast with or without bioglass, while Ca2 + and PO43- ( provided by calcium chloride and sodium monophosphate , separately), the degraded products of β- TCP had no significant effect on osteoblast cell growth in a wide range. The stimulating effect of β- TCP on osteoblast cell proliferation might relate to the encapsulation of the material particles into the cells.

  2. 78 FR 46418 - Proposed Information Collection (Obligation To Report Factors Affecting Entitlement) Activity...

    Science.gov (United States)

    2013-07-31

    ... AFFAIRS Proposed Information Collection (Obligation To Report Factors Affecting Entitlement) Activity... techniques or the use of other forms of information technology. Title: Obligation to Report Factors Affecting... dependents, may affect the amount of benefit that he or she receives or affect the right to receive...

  3. MEG brain activities reflecting affection for visual food stimuli.

    Science.gov (United States)

    Kuriki, Shinya; Miyamura, Takahiro; Uchikawa, Yoshinori

    2010-01-01

    This study aimed to explore the modulation of alpha rhythm in response to food pictures with distinct affection values. We examined the method to discriminate subject's state, i.e., whether he/she liked the article of food or not, from MEG signals detected over the head. Pictures of familiar foods were used as affective stimuli, while those pictures with complementary color phase were used as non-affective stimuli. Alpha band signals in a narrow frequency window around the spectral peak of individual subjects were wavelet analyzed and phase-locked component to the stimulus onset was obtained as a complex number. The amplitude of the phase-locked component was averaged during 0-1 s after stimulus onset for 30 epochs in a measurement session and across 76 channels of MEG sensor. In statistical test of individual subjects, significant difference was found in the real part of the averaged phase-locked amplitude between the normal-color and reverse-color pictures. These results suggest that affective information processing of food pictures is reflected in the synchronized component of narrow band alpha rhythm. PMID:21096510

  4. How Do Sociodemographics and Activity Participations Affect Activity-Travel? Comparative Study between Women and Men

    Directory of Open Access Journals (Sweden)

    Min Yang

    2014-01-01

    Full Text Available Activity-travel behaviors of women and men are different because they have different social and household responsibilities. However, studies concerning gender differences are mainly limited in developed countries. This paper concentrates on gender role-based differences in activity-travel behavior in a typical developing country, namely, China. Using data from 3656 cases collected through surveys conducted in Shangyu, data processing, method choice, and descriptive analysis were conducted. Binary and ordered logistic regression models segmented by gender were developed to evaluate the mechanism through which individual sociodemographics, household characteristics, and activity participations affect the number of trip chain types and activities for women and men. The results show that women aged 30 to 50 perform less subsistence activities. However, the difference between the different age groups of men is not as significant. In addition, men with bicycles and electric bicycles have more subsistence and maintenance activities, whereas women do not have these attributes. Moreover, women with children under schooling age make more maintenance trip chains but less leisure trip chains and activities, whereas men are free from this influence. Furthermore, both women and men perform more subsistence activities if the duration increases, and men have less influences than women do.

  5. Acerogenin A, a natural compound isolated from Acer nikoense Maxim, stimulates osteoblast differentiation through bone morphogenetic protein action

    International Nuclear Information System (INIS)

    Research highlights: → Acerogenin A stimulated osteoblast differentiation in osteogenic cells. → Acerogenin A-induced osteoblast differentiation was inhibited by noggin. → Acerogenin A increased Bmp-2, Bmp-4 and Bmp-7 mRNA expression in MC3T3-E1 cells. → Acerogenin A is a candidate agent for stimulating bone formation. -- Abstract: We investigated the effects of acerogenin A, a natural compound isolated from Acer nikoense Maxim, on osteoblast differentiation by using osteoblastic cells. Acerogenin A stimulated the cell proliferation of MC3T3-E1 osteoblastic cells and RD-C6 osteoblastic cells (Runx2-deficient cell line). It also increased alkaline phosphatase activity in MC3T3-E1 and RD-C6 cells and calvarial osteoblastic cells isolated from the calvariae of newborn mice. Acerogenin A also increased the expression of mRNAs related to osteoblast differentiation, including Osteocalcin, Osterix and Runx2 in MC3T3-E1 cells and primary osteoblasts: it also stimulated Osteocalcin and Osterix mRNA expression in RD-C6 cells. The acerogenin A treatment for 3 days increased Bmp-2, Bmp-4, and Bmp-7 mRNA expression levels in MC3T3-E1 cells. Adding noggin, a BMP specific-antagonist, inhibited the acerogenin A-induced increase in the Osteocalcin, Osterix and Runx2 mRNA expression levels. These results indicated that acerogenin A stimulates osteoblast differentiation through BMP action, which is mediated by Runx2-dependent and Runx2-independent pathways.

  6. Acerogenin A, a natural compound isolated from Acer nikoense Maxim, stimulates osteoblast differentiation through bone morphogenetic protein action

    Energy Technology Data Exchange (ETDEWEB)

    Kihara, Tasuku [Section of Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Section of Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Global Center of Excellence (GCOE) Program, International Research Center for Molecular Science in Tooth and Bone Diseases, Tokyo Medical and Dental University, Tokyo (Japan); Ichikawa, Saki [Section of Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Yonezawa, Takayuki; Lee, Ji-Won [Research Institute for Biological Functions, Chubu University, Kasugai, Aichi (Japan); Akihisa, Toshihiro [College of Science and Technology, Nihon University, Tokyo (Japan); Woo, Je Tae [Research Institute for Biological Functions, Chubu University, Kasugai, Aichi (Japan); Michi, Yasuyuki; Amagasa, Teruo [Section of Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Yamaguchi, Akira, E-mail: akira.mpa@tmd.ac.jp [Section of Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Global Center of Excellence (GCOE) Program, International Research Center for Molecular Science in Tooth and Bone Diseases, Tokyo Medical and Dental University, Tokyo (Japan)

    2011-03-11

    Research highlights: {yields} Acerogenin A stimulated osteoblast differentiation in osteogenic cells. {yields} Acerogenin A-induced osteoblast differentiation was inhibited by noggin. {yields} Acerogenin A increased Bmp-2, Bmp-4 and Bmp-7 mRNA expression in MC3T3-E1 cells. {yields} Acerogenin A is a candidate agent for stimulating bone formation. -- Abstract: We investigated the effects of acerogenin A, a natural compound isolated from Acer nikoense Maxim, on osteoblast differentiation by using osteoblastic cells. Acerogenin A stimulated the cell proliferation of MC3T3-E1 osteoblastic cells and RD-C6 osteoblastic cells (Runx2-deficient cell line). It also increased alkaline phosphatase activity in MC3T3-E1 and RD-C6 cells and calvarial osteoblastic cells isolated from the calvariae of newborn mice. Acerogenin A also increased the expression of mRNAs related to osteoblast differentiation, including Osteocalcin, Osterix and Runx2 in MC3T3-E1 cells and primary osteoblasts: it also stimulated Osteocalcin and Osterix mRNA expression in RD-C6 cells. The acerogenin A treatment for 3 days increased Bmp-2, Bmp-4, and Bmp-7 mRNA expression levels in MC3T3-E1 cells. Adding noggin, a BMP specific-antagonist, inhibited the acerogenin A-induced increase in the Osteocalcin, Osterix and Runx2 mRNA expression levels. These results indicated that acerogenin A stimulates osteoblast differentiation through BMP action, which is mediated by Runx2-dependent and Runx2-independent pathways.

  7. Mutation of the Zinc-Binding Metalloprotease Motif Affects Bacteroides fragilis Toxin Activity but Does Not Affect Propeptide Processing

    OpenAIRE

    Franco, Augusto A.; Buckwold, Simy L.; Shin, Jai W.; Ascon, Miguel; Sears, Cynthia L.

    2005-01-01

    To evaluate the role of the zinc-binding metalloprotease in Bacteroides fragilis toxin (BFT) processing and activity, the zinc-binding consensus sequences (H348, E349, H352, G355, H358, and M366) were mutated by site-directed-mutagenesis. Our results indicated that single point mutations in the zinc-binding metalloprotease motif do not affect BFT processing but do reduce or eliminate BFT biologic activity in vitro.

  8. Mutation of the Zinc-Binding Metalloprotease Motif Affects Bacteroides fragilis Toxin Activity but Does Not Affect Propeptide Processing

    Science.gov (United States)

    Franco, Augusto A.; Buckwold, Simy L.; Shin, Jai W.; Ascon, Miguel; Sears, Cynthia L.

    2005-01-01

    To evaluate the role of the zinc-binding metalloprotease in Bacteroides fragilis toxin (BFT) processing and activity, the zinc-binding consensus sequences (H348, E349, H352, G355, H358, and M366) were mutated by site-directed-mutagenesis. Our results indicated that single point mutations in the zinc-binding metalloprotease motif do not affect BFT processing but do reduce or eliminate BFT biologic activity in vitro. PMID:16041055

  9. Stress affects salivary alpha-Amylase activity in bonobos.

    Science.gov (United States)

    Behringer, Verena; Deschner, Tobias; Möstl, Erich; Selzer, Dieter; Hohmann, Gottfried

    2012-01-18

    Salivary alpha-Amylase (sAA) is a starch digesting enzyme. In addition to its function in the context of nutrition, sAA has also turned out to be useful for monitoring sympathetic nervous system activity. Recent studies on humans have found a relationship between intra-individual changes in sAA activity and physical and psychological stress. In studies on primates and other vertebrates, non-invasive monitoring of short-term stress responses is usually based on measurements of cortisol levels, which are indicative of hypothalamic-pituitary-adrenal activity. The few studies that have used both cortisol levels and sAA activity indicate that these two markers may respond differently and independently to different types of stress such that variation in the degree of the activation of different stress response systems might reflect alternative coping mechanisms or individual traits. Here, we present the first data on intra- and inter-individual variation of sAA activity in captive bonobos and compare the results with information from other ape species and humans. Our results indicate that sAA activity in the bonobo samples was significantly lower than in the human samples but within the range of other great ape species. In addition, sAA activity was significantly higher in samples collected at times when subjects had been exposed to stressors (judged by changes in behavioral patterns and cortisol levels) than in samples collected at other times. Our results indicate that bonobos possess functioning sAA and, as in other species, sAA activity is influenced by autonomic nervous system activity. Monitoring sAA activity could therefore be a useful tool for evaluating stress in bonobos. PMID:21945369

  10. The Macrophage Polarization Regulates MSC Osteoblast Differentiation in vitro.

    Science.gov (United States)

    Gong, Lei; Zhao, Yan; Zhang, Yi; Ruan, Zhi

    2016-01-01

    Bone repair is a complex yet highly organized process involving interactions between various cell types and the extracellular environment. Macrophages are not only activated in inflammation during early phases of repair processes, but they are also present in bone throughout the whole bone repair process. Bone marrow derived mesenchymal stem cells (MSCs) represent an attractive therapeutic for bone fracture with their expansion potential, osteogenic capability, and potential for injury. However, less is known about the interaction between macrophage and MSC during bone repair and regeneration. This study was aimed to investigate whether macrophages in different statuses can regulate MSC osteoblast differentiation in vitro. Using in vitro cell coculture of macrophage and MSC, it was shown that macrophage polarization can regulate MSC osteoblast differentiation. This was evidenced by increased alkaline phosphatase (ALP), osteogenic markers, and bone mineralization in M2 macrophage cocultured MSC but decreased in M1 counterpart. These results might be mediated by pro-regenerative cytokines, such as TGF-β, VEGF, and IFG-1, produced by M2 macrophages and detrimental inflammation cytokines, such as IL-6, IL-12, and TNF-α, produced by M1 macrophages. Taken together, this shows that macrophage polarization could be crucial for maintaining bone homeostasis and promoting bone repair by regulating the MSC osteoblast differentiation. PMID:26927345

  11. Lung cancer: district active treatment rates affect survival

    OpenAIRE

    CARTMAN, M.; Hatfield, A; Muers, M; Peake, M; Haward, R; Forman, D

    2002-01-01

    Design: A retrospective study of population based data held by the Northern & Yorkshire Cancer Registry and Information Service (NYCRIS), comparing active treatment rates for lung cancer with survival by districts.

  12. Oxidative Activity of Heated Coal Affected by Antypirogens

    Science.gov (United States)

    Torosyan, V. F.; Torosyan, E. S.; Borovikov, I. F.; Yakutova, V. A.

    2016-04-01

    The effect of antypirogens on chemical activity of heated coal is studied. It is proved that ammonium sulfate, calcium phosphate, calcium chloride, calcium nitrate and acid fluoride are the most effective antypirogens.

  13. Technology trends, energy prices affect worldwide rig activity

    International Nuclear Information System (INIS)

    The major worldwide offshore rig markets have improved slightly this year, while the onshore markets generally lagged slightly. Offshore rig utilization rates have remained strong worldwide, with some areas reaching nearly 100%. Total worldwide offshore rig (jack ups, semisubmersible, drillships, submersibles, and barges) utilization was about 86%. Offshore drilling activity is driven primarily by oil and natural gas price expectations. Natural gas prices tend to drive North American offshore drilling activity, including the shallow waters in the Gulf of Mexico. International offshore drilling activity and deepwater projects in the Gulf of Mexico are more closely tied to oil prices. The paper discusses US rig count, directional drilling activity, jack up rig demand, semisubmersibles demand, rig replacement costs, and new construction

  14. Disturbances of electrodynamic activity affect abortion in human

    OpenAIRE

    Jandová, A; Nedbalová, M.; Kobilková, J.; Čoček, A.; Dohnalová, A.; M. Cifra; Pokorný, J.

    2011-01-01

    Biochemical research of biological systems is highly developed, and it has disclosed a spectrum of chemical reactions, genetic processes, and the pathological development of various diseases. The fundamental hypothesis of physical processes in biological systems, in particular of coherent electrically polar vibrations and electromagnetic activity, was formulated by H. Fröhlich; he assumed connection of cancer process with degradation of coherent electromagnetic activity. But the questions of ...

  15. Carbon monoxide affects electrical and contractile activity of rat myocardium

    OpenAIRE

    Porokhnya Maria V; Haertdinov Nail N; Abramochkin Denis V; Zefirov Andrew L; Sitdikova Gusel F

    2011-01-01

    Abstract Background Carbon monoxide (CO) is a toxic gas, which also acts in the organism as a neurotransmitter. It is generated as a by-product of heme breakdown catalyzed by heme oxygenase. We have investigated changes in electrical and contractile activity of isolated rat atrial and ventricular myocardium preparations under the influence of CO. Methods Standard microelectrode technique was used for intracellular registration of electrical activity in isolated preparations of atrial and vent...

  16. Carbon monoxide affects electrical and contractile activity of rat myocardium

    Directory of Open Access Journals (Sweden)

    Porokhnya Maria V

    2011-06-01

    Full Text Available Abstract Background Carbon monoxide (CO is a toxic gas, which also acts in the organism as a neurotransmitter. It is generated as a by-product of heme breakdown catalyzed by heme oxygenase. We have investigated changes in electrical and contractile activity of isolated rat atrial and ventricular myocardium preparations under the influence of CO. Methods Standard microelectrode technique was used for intracellular registration of electrical activity in isolated preparations of atrial and ventricular myocardium. Contractions of atrial myocardial stripes were registered via force transducer. Results CO (10-4 - 10-3 M caused prominent decrease of action potential duration (APD in working atrial myocardium as well as significant acceleration of sinus rhythm. In addition CO reduced force of contractions and other parameters of contractile activity. Inhibitor of heme oxygenase zinc protoporphyrin IX exerts opposite effects: prolongation of action potential, reduction of sinus rhythm rate and enhancement of contractile function. Therefore, endogenous CO, which may be generated in the heart due to the presence of active heme oxygenase, is likely to exert the same effects as exogenous CO applied to the perfusing medium. In ventricular myocardium preparations exogenous CO also induced shortening of action potential, while zinc protoporphyrin IX produced the opposite effect. Conclusions Thus, endogenous or exogenous carbon monoxide may act as an important regulator of electrical and contractile cardiac activity.

  17. Lysyl Hydroxylase 3 Glucosylates Galactosylhydroxylysine Residues in Type I Collagen in Osteoblast Culture*

    Science.gov (United States)

    Sricholpech, Marnisa; Perdivara, Irina; Nagaoka, Hideaki; Yokoyama, Megumi; Tomer, Kenneth B.; Yamauchi, Mitsuo

    2011-01-01

    Lysyl hydroxylase 3 (LH3), encoded by Plod3, is the multifunctional collagen-modifying enzyme possessing LH, hydroxylysine galactosyltransferase (GT), and galactosylhydroxylysine-glucosyltransferase (GGT) activities. Although an alteration in type I collagen glycosylation has been implicated in several osteogenic disorders, the role of LH3 in bone physiology has never been investigated. To elucidate the function of LH3 in bone type I collagen modifications, we used a short hairpin RNA technology in a mouse osteoblastic cell line, MC3T3-E1; generated single cell-derived clones stably suppressing LH3 (short hairpin (Sh) clones); and characterized the phenotype. Plod3 expression and the LH3 protein levels in the Sh clones were significantly suppressed when compared with the controls, MC3T3-E1, and the clone transfected with an empty vector. In comparison with controls, type I collagen synthesized by Sh clones (Sh collagen) showed a significant decrease in the extent of glucosylgalactosylhydroxylysine with a concomitant increase of galactosylhydroxylysine, whereas the total number of hydroxylysine residues was essentially unchanged. In an in vitro fibrillogenesis assay, Sh collagen showed accelerated fibrillogenesis compared with the controls. In addition, when recombinant LH3-V5/His protein was generated in 293 cells and subjected to GGT/GT activity assay, it showed GGT but not GT activity against denatured type I collagen. The results from this study clearly indicate that the major function of LH3 in osteoblasts is to glucosylate galactosylhydroxylysine residues in type I collagen and that an impairment of this LH3 function significantly affects type I collagen fibrillogenesis. PMID:21220425

  18. Lysyl hydroxylase 3 glucosylates galactosylhydroxylysine residues in type I collagen in osteoblast culture.

    Science.gov (United States)

    Sricholpech, Marnisa; Perdivara, Irina; Nagaoka, Hideaki; Yokoyama, Megumi; Tomer, Kenneth B; Yamauchi, Mitsuo

    2011-03-18

    Lysyl hydroxylase 3 (LH3), encoded by Plod3, is the multifunctional collagen-modifying enzyme possessing LH, hydroxylysine galactosyltransferase (GT), and galactosylhydroxylysine-glucosyltransferase (GGT) activities. Although an alteration in type I collagen glycosylation has been implicated in several osteogenic disorders, the role of LH3 in bone physiology has never been investigated. To elucidate the function of LH3 in bone type I collagen modifications, we used a short hairpin RNA technology in a mouse osteoblastic cell line, MC3T3-E1; generated single cell-derived clones stably suppressing LH3 (short hairpin (Sh) clones); and characterized the phenotype. Plod3 expression and the LH3 protein levels in the Sh clones were significantly suppressed when compared with the controls, MC3T3-E1, and the clone transfected with an empty vector. In comparison with controls, type I collagen synthesized by Sh clones (Sh collagen) showed a significant decrease in the extent of glucosylgalactosylhydroxylysine with a concomitant increase of galactosylhydroxylysine, whereas the total number of hydroxylysine residues was essentially unchanged. In an in vitro fibrillogenesis assay, Sh collagen showed accelerated fibrillogenesis compared with the controls. In addition, when recombinant LH3-V5/His protein was generated in 293 cells and subjected to GGT/GT activity assay, it showed GGT but not GT activity against denatured type I collagen. The results from this study clearly indicate that the major function of LH3 in osteoblasts is to glucosylate galactosylhydroxylysine residues in type I collagen and that an impairment of this LH3 function significantly affects type I collagen fibrillogenesis. PMID:21220425

  19. Glutamate Receptor Agonists and Glutamate Transporter Antagonists Regulate Differentiation of Osteoblast Lineage Cells.

    Science.gov (United States)

    Xie, Wenjie; Dolder, Silvia; Siegrist, Mark; Wetterwald, Antoinette; Hofstetter, Willy

    2016-08-01

    Development and function of osteoblast lineage cells are regulated by a complex microenvironment consisting of the bone extracellular matrix, cells, systemic hormones and cytokines, autocrine and paracrine factors, and mechanical load. Apart from receptors that transduce extracellular signals into the cell, molecular transporters play a crucial role in the cellular response to the microenvironment. Transporter molecules are responsible for cellular uptake of nutritional components, elimination of metabolites, ion transport, and cell-cell communication. In this report, the expression of molecular transporters in osteoblast lineage cells was investigated to assess their roles in cell development and activity. Low-density arrays, covering membrane and vesicular transport molecules, were used to assess gene expression in osteoblasts representing early and late differentiation states. Receptors and transporters for the amino acid glutamate were found to be differentially expressed during osteoblast development. Glutamate is a neurotransmitter in the central nervous system, and the mechanisms of its release, signal transduction, and cellular reabsorption in the synaptic cleft are well understood. Less clear, however, is the control of equivalent processes in peripheral tissues. In primary osteoblasts, inhibition of glutamate transporters with nonselective inhibitors leads to an increase in the concentration of extracellular glutamate. This change was accompanied by a decrease in osteoblast proliferation, stimulation of alkaline phosphatase, and the expression of transcripts encoding osteocalcin. Enzymatic removal of extracellular glutamate abolished these pro-differentiation effects, as did the inhibition of PKC- and Erk1/2-signaling pathways. These findings demonstrate that glutamate signaling promotes differentiation and activation of osteoblast lineage cells. Consequently, the glutamate system may represent a putative therapeutic target to induce an anabolic response

  20. Icariine stimulates proliferation and differentiation of human osteoblasts by increasing production of bone morphogenetic protein 2

    Institute of Scientific and Technical Information of China (English)

    YIN Xiao-xue; CHEN Zhong-qiang; LIU Zhong-jun; MA Qing-jun; DANG Geng-ting

    2007-01-01

    Background lcariine is a flavonoid isolated from a traditional Chinese medicine Epimedium pubescens and is the main active compound of it. Recently, Epimedium pubescens was found to have a therapeutic effect on osteoporosis. But the mechanism is unclear. The aim of the study was to research the effect of lcariine on the proliferation and differentiation of human osteoblasts.Methods Human osteoblasts were obtained byinducing human marrow mesenchymal stem cells (hMSCs) directionally and were cultured in the presence of various concentrations of lcariine. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test was used to observe the effect of lcariine on cell proliferation. The activity of alkaline phosphatase (ALP) and the amount of calcified nodules were assayed to observe the effect on cell differentiation.The expression of bone morphogenetic protein 2 (BMP-2) mRNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR).Results Icariine (20 μg/ml) increased significantly the proliferation of human osteoblasts. And, lcariine (10 μg/ml and 20μg/ml) increased the activity of ALP and the amount of calcified nodules of human osteoblasts significantly (P<0.05).BMP-2 mRNA synthesis was elevated significantly in response to lcariine (20 μg/ml).Conclusions lcariine has a direct stimulatory effect on the proliferation and differentiation of cultured human osteoblastcells in vitro, which may be mediated by increasing production of BMP-2 in osteoblasts.

  1. Early life stress affects limited regional brain activity in depression.

    Science.gov (United States)

    Du, Lian; Wang, Jingjie; Meng, Ben; Yong, Na; Yang, Xiangying; Huang, Qingling; Zhang, Yan; Yang, Lingling; Qu, Yuan; Chen, Zhu; Li, Yongmei; Lv, Fajin; Hu, Hua

    2016-01-01

    Early life stress (ELS) can alter brain function and increases the risk of major depressive disorder (MDD) in later life. This study investigated whether ELS contributes to differences in regional brain activity between MDD patients and healthy controls (HC), as measured by amplitude of low-frequency fluctuation (ALFF)/fractional (f)ALFF. Eighteen first-episode, treatment-naïve MDD patients and HC were assessed with the Childhood Trauma Questionnaire and resting-state functional magnetic resonance imaging. We compared ALFF/fALFF between MDD patients and HC, with or without controlling for ELS, and determined whether ELS level was correlated with regional brain activity in each group. After regressing out ELS, we found that ALFF increased in bilateral amygdala and left orbital/cerebellum, while fALFF decreased in left inferior temporal and right middle frontal gyri in MDD patients relative to controls. ELS positively correlated with regional activity in the left cerebellum in MDD and in the right post-central/inferior temporal/superior frontal cingulate, inferior frontal gyrus and bilateral cerebellum in HC. Our findings indicate that there is only very limited region showing correlation between ELS and brain activity in MDD, while diverse areas in HC, suggesting ELS has few impacts on MDD patients. PMID:27138376

  2. Early life stress affects limited regional brain activity in depression

    Science.gov (United States)

    Du, Lian; Wang, Jingjie; Meng, Ben; Yong, Na; Yang, Xiangying; Huang, Qingling; Zhang, Yan; Yang, Lingling; Qu, Yuan; Chen, Zhu; Li, Yongmei; Lv, Fajin; Hu, Hua

    2016-01-01

    Early life stress (ELS) can alter brain function and increases the risk of major depressive disorder (MDD) in later life. This study investigated whether ELS contributes to differences in regional brain activity between MDD patients and healthy controls (HC), as measured by amplitude of low-frequency fluctuation (ALFF)/fractional (f)ALFF. Eighteen first-episode, treatment-naïve MDD patients and HC were assessed with the Childhood Trauma Questionnaire and resting-state functional magnetic resonance imaging. We compared ALFF/fALFF between MDD patients and HC, with or without controlling for ELS, and determined whether ELS level was correlated with regional brain activity in each group. After regressing out ELS, we found that ALFF increased in bilateral amygdala and left orbital/cerebellum, while fALFF decreased in left inferior temporal and right middle frontal gyri in MDD patients relative to controls. ELS positively correlated with regional activity in the left cerebellum in MDD and in the right post-central/inferior temporal/superior frontal cingulate, inferior frontal gyrus and bilateral cerebellum in HC. Our findings indicate that there is only very limited region showing correlation between ELS and brain activity in MDD, while diverse areas in HC, suggesting ELS has few impacts on MDD patients. PMID:27138376

  3. Inhibition of Nek2 by Small Molecules Affects Proteasome Activity

    Directory of Open Access Journals (Sweden)

    Lingyao Meng

    2014-01-01

    Full Text Available Background. Nek2 is a serine/threonine kinase localized to the centrosome. It promotes cell cycle progression from G2 to M by inducing centrosome separation. Recent studies have shown that high Nek2 expression is correlated with drug resistance in multiple myeloma patients. Materials and Methods. To investigate the role of Nek2 in bortezomib resistance, we ectopically overexpressed Nek2 in several cancer cell lines, including multiple myeloma lines. Small-molecule inhibitors of Nek2 were discovered using an in-house library of compounds. We tested the inhibitors on proteasome and cell cycle activity in several cell lines. Results. Proteasome activity was elevated in Nek2-overexpressing cell lines. The Nek2 inhibitors inhibited proteasome activity in these cancer cell lines. Treatment with these inhibitors resulted in inhibition of proteasome-mediated degradation of several cell cycle regulators in HeLa cells, leaving them arrested in G2/M. Combining these Nek2 inhibitors with bortezomib increased the efficacy of bortezomib in decreasing proteasome activity in vitro. Treatment with these novel Nek2 inhibitors successfully mitigated drug resistance in bortezomib-resistant multiple myeloma. Conclusion. Nek2 plays a central role in proteasome-mediated cell cycle regulation and in conferring resistance to bortezomib in cancer cells. Taken together, our results introduce Nek2 as a therapeutic target in bortezomib-resistant multiple myeloma.

  4. Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 is Expressed inOsteoblasts and Regulated by PTH

    Energy Technology Data Exchange (ETDEWEB)

    Sharma, Sonali; Mahalingam, Chandrika D.; Das, Varsha [Department of Internal Medicine/Endocrinology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Jamal, Shazia [Department of Oncology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Levi, Edi [Department of Oncology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Department of Pathology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Rishi, Arun K. [Department of Oncology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI 48201 (United States); VA Medical Center, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Datta, Nabanita S., E-mail: ndatta@med.wayne.edu [Department of Internal Medicine/Endocrinology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Cardiovascular Research Institute, Wayne State University School of Medicine, Detroit, MI 48201 (United States)

    2013-07-12

    Highlights: •CARP-1 is identified for the first time in bone cells. •PTH downregulates CARP-1 expression in differentiated osteoblasts. •PTH displaces CARP-1 from nucleus to the cytoplasm in differentiated osteoblasts. •Downregulation of CARP-1 by PTH involves PKA, PKC and P-p38 MAPK pathways. -- Abstract: Bone mass is dependent on osteoblast proliferation, differentiation and life-span of osteoblasts. Parathyroid hormone (PTH) controls osteoblast cell cycle regulatory proteins and suppresses mature osteoblasts apoptosis. Intermittent administration of PTH increases bone mass but the mechanism of action are complex and incompletely understood. Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 (aka CCAR1) is a novel transducer of signaling by diverse agents including cell growth and differentiation factors. To gain further insight into the molecular mechanism, we investigated involvement of CARP-1 in PTH signaling in osteoblasts. Immunostaining studies revealed presence of CARP-1 in osteoblasts and osteocytes, while a minimal to absent levels were noted in the chondrocytes of femora from 10 to 12-week old mice. Treatment of 7-day differentiated MC3T3-E1 clone-4 (MC-4) mouse osteoblastic cells and primary calvarial osteoblasts with PTH for 30 min to 5 h followed by Western blot analysis showed 2- to 3-fold down-regulation of CARP-1 protein expression in a dose- and time-dependent manner compared to the respective vehicle treated control cells. H-89, a Protein Kinase A (PKA) inhibitor, suppressed PTH action on CARP-1 protein expression indicating PKA-dependent mechanism. PMA, a Protein Kinase C (PKC) agonist, mimicked PTH action, and the PKC inhibitor, GF109203X, partially blocked PTH-dependent downregulation of CARP-1, implying involvement of PKC. U0126, a Mitogen-Activated Protein Kinase (MAPK) Kinase (MEK) inhibitor, failed to interfere with CARP-1 suppression by PTH. In contrast, SB203580, p38 inhibitor, attenuated PTH down-regulation of CARP-1

  5. Overexpression of Bcl2 in osteoblasts inhibits osteoblast differentiation and induces osteocyte apoptosis.

    Directory of Open Access Journals (Sweden)

    Takeshi Moriishi

    Full Text Available Bcl2 subfamily proteins, including Bcl2 and Bcl-X(L, inhibit apoptosis. As osteoblast apoptosis is in part responsible for osteoporosis in sex steroid deficiency, glucocorticoid excess, and aging, bone loss might be inhibited by the upregulation of Bcl2; however, the effects of Bcl2 overexpression on osteoblast differentiation and bone development and maintenance have not been fully investigated. To investigate these issues, we established two lines of osteoblast-specific BCL2 transgenic mice. In BCL2 transgenic mice, bone volume was increased at 6 weeks of age but not at 10 weeks of age compared with wild-type mice. The numbers of osteoblasts and osteocytes increased, but osteoid thickness and the bone formation rate were reduced in BCL2 transgenic mice with high expression at 10 weeks of age. The number of BrdU-positive cells was increased but that of TUNEL-positive cells was unaltered at 2 and 6 weeks of age. Osteoblast differentiation was inhibited, as shown by reduced Col1a1 and osteocalcin expression. Osteoblast differentiation of calvarial cells from BCL2 transgenic mice also fell in vitro. Overexpression of BCL2 in primary osteoblasts had no effect on osteoclastogenesis in co-culture with bone marrow cells. Unexpectedly, overexpression of BCL2 in osteoblasts eventually caused osteocyte apoptosis. Osteocytes, which had a reduced number of processes, gradually died with apoptotic structural alterations and the expression of apoptosis-related molecules, and dead osteocytes accumulated in cortical bone. These findings indicate that overexpression of BCL2 in osteoblasts inhibits osteoblast differentiation, reduces osteocyte processes, and causes osteocyte apoptosis.

  6. MiR-214 regulates the function of osteoblast under simulated microgravity by targeting ATF4

    Science.gov (United States)

    Li, Yingxian; Wang, Xiaogang; Li, Qi; Lv, Ke; Wan, Yumin; Li, Yinghui; Bai, Yanqiang

    Background: MicroRNAs (miRNAs) are small fragments of single-stranded RNA containing 18-24 nucleotides, and are generated from endogenous transcripts. MicroRNAs function in post-transcriptional gene silencing by targeting the 3'-untranslated region (UTR) of mRNAs, resulting in translational repression. Growing evidence shows that microRNAs (miRNAs) regu-late various developmental and homeostatic events in vertebrates and invertebrates. Osteoblast differentiation is a key step in proper skeletal development and acquisition of bone mass; How-ever, the physiological role of non-coding small RNAs, especially miRNAs, in osteoblast dif-ferentiation remains elusive. Methods: To study the potential involvement of miRNAs in osteoblast differentiation under stimulated microgravity, we analyzed the expression of 20 bone relative miRNAs using real time PCR platform to find particularly miRNAs whose expression is altered during osteoblast differentiation. TargetScan, miRBase and Miranda were used to predict the target gene of candidate miRNA. To investigate whether ATF4 can be directly targeted by miR-214, we engineered luciferase reporters that have either the wild-type 3'UTRs of these genes, or the mutant UTRs with a 6 base pair (bp) deletion in the target sites. Lastly, to address the in vivo role of miR-214 in bone formation, tail suspension mice model was used to simulate the change of osteoblast function and bone loss. Results: Recent studies have sug-gested that miRNAs might play a role in osteoblast differentiation and bone formation. Here, we identify miR-214 in MC3T3-E1 cells, which is a primary mouse osteoblasts cell line, to promote osteoblast differentiation by repressing Activating Transcription Factor4 (ATF4) ex-pression at the posttranscriptional level. What is more, miR-214 was found to be transcribed in C2C12 cells during bone morphogenetic protein 2-induced (BMP2-induced) osteogenesis, and overexpression of miR-214 attenuated BMP2-induced osteoblastogenesis

  7. Regulation of drugs affecting striatal cholinergic activity by corticostriatal projections

    International Nuclear Information System (INIS)

    Research demonstrates that the chronic degeneration of the corticostriatal excitatory pathway makes the cholinergic neurons of the striatum insensitive to the neuropharmacological action of a number of different drugs. Female rats were used; they were killed and after the i.v. infusion of tritium-choline precursor, choline acetyltransferase activity was measured. Striatal noradrenaline, dopamine and serotonin content was measured by electrochemical detection coupled with high pressure liquid chromatography. Uptake of tritium-glutamic acid was estimated. The data were analyzed statistically. It is shown that there is evidence that the effects of a number of drugs capable of depressing cholinergic activity through receptor-mediated responses are operative only if the corticostriatal pathway is integral. Neuropharmacological responses in the brain appear to be the result of an interaction between several major neurotransmitter systems

  8. Seasonal Pacing - Match Importance Affects Activity in Professional Soccer

    Science.gov (United States)

    Link, Daniel; de Lorenzo, Michael F.

    2016-01-01

    This research explores the influence of match importance on player activity in professional soccer. Therefore, we used an observational approach and analyzed 1,211 matches of German Bundesliga and 2nd Bundesliga. The importance measurement employed is based on post season consequences of teams involved in a match. This means, if a match result could potentially influence the final rank, and this rank would lead to different consequences for a team, such as qualification for Champions League opposed to qualification for Europe League, then this match is classified as important; otherwise not. Activity was quantified by TOTAL DISTANCE COVERED, SPRINTS, FAST RUNS, DUELS, FOULS and ATTEMPTS. Running parameters were recorded using a semi-automatic optical tracking system, while technical variables were collected by professional data loggers. Based on our importance classification, low important matches occurred at the beginning of round 29. A two-way ANOVA indicates significantly increased FAST RUNS (+4%, d = 0.3), DUELS (+16%, d = 1.0) and FOULS (+36%, d = 1.2) in important matches compared to low important ones. For FAST RUNS and FOULS, this effect only exists in Bundesliga. A comparison of the two leagues show that TOTAL DISTANCE COVERED (+3%, d = 0.9), SPRINTS (+25%, d = 1.4) and FAST RUNS (+15%, d = 1.4) are higher compared to 2nd Bundesliga, whilst FOULS is less in Bundesliga (-7%, d = 0.3). No difference in player activity was found between matches at the beginning of a season (round 1–6) and at the end of a season (round 29–34). We conclude that match importance influences player activity in German professional soccer. The most reasonable explanation is a conscious or unconscious pacing strategy, motivated by preserving abilities or preventing injury. Since this tendency mainly exists in Bundesliga, this may suggest that more skilled players show a higher awareness for the need of pacing. PMID:27281051

  9. New thiazolidinediones affect endothelial cell activation and angiogenesis.

    Science.gov (United States)

    Rudnicki, Martina; Tripodi, Gustavo L; Ferrer, Renila; Boscá, Lisardo; Pitta, Marina G R; Pitta, Ivan R; Abdalla, Dulcineia S P

    2016-07-01

    Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor-γ (PPARγ) agonists used in treating type 2 diabetes that may exhibit beneficial pleiotropic effects on endothelial cells. In this study, we characterized the effects of three new TZDs [GQ-32 (3-biphenyl-4-ylmethyl-5-(4-nitro-benzylidene)-thiazolidine-2,4-dione), GQ-169 (5-(4-chloro-benzylidene)-3-(2,6-dichloro-benzyl)-thiazolidine-2,4-dione), and LYSO-7 (5-(5-bromo-1H-indol-3-ylmethylene)-3-(4-chlorobenzyl)-thiazolidine-2,4-dione)] on endothelial cells. The effects of the new TZDs were evaluated on the production of nitric oxide (NO) and reactive oxygen species (ROS), cell migration, tube formation and the gene expression of adhesion molecules and angiogenic mediators in human umbilical vein endothelial cells (HUVECs). PPARγ activation by new TZDs was addressed with a reporter gene assay. The three new TZDs activated PPARγ and suppressed the tumor necrosis factor α-induced expression of vascular cell adhesion molecule 1 and intercellular adhesion molecule 1. GQ-169 and LYSO-7 also inhibited the glucose-induced ROS production. Although NO production assessed with 4-amino-5-methylamino-2',7'-difluorofluorescein-FM probe indicated that all tested TZDs enhanced intracellular levels of NO, only LYSO-7 treatment significantly increased the release of NO from HUVEC measured by chemiluminescence analysis of culture media. Additionally, GQ-32 and GQ-169 induced endothelial cell migration and tube formation by the up-regulation of angiogenic molecules expression, such as vascular endothelial growth factor A and interleukin 8. GQ-169 also increased the mRNA levels of basic fibroblast growth factor, and GQ-32 enhanced transforming growth factor-β expression. Together, the results of this study reveal that these new TZDs act as partial agonists of PPARγ and modulate endothelial cell activation and endothelial dysfunction besides to stimulate migration and tube formation. PMID:27108791

  10. Disturbances of electrodynamic activity affect abortion in human

    Czech Academy of Sciences Publication Activity Database

    Jandová, Anna; Nedbalová, M.; Kobilková, J.; Čoček, A.; Dohnalová, A.; Cifra, Michal; Pokorný, Jiří

    Vol. 329. Bristol: IOP, 2011 - (Cifra, M.; Pokorny, J.; Kučera, O.), 012030 ISSN 1742-6588. [9th International Frohlich's Symposium on Electrodynamic Activity of Living Cells - Including Microtubule Coherent Modes and Cancer Cell Physics. Praha (CZ), 01.07.2011-03.07.2011] Institutional research plan: CEZ:AV0Z2067918 Keywords : Biochemical research * Cellular structure * Control groups Subject RIV: JA - Electronics ; Optoelectronics, Electrical Engineering

  11. Disturbances of electrodynamic activity affect abortion in animals

    Czech Academy of Sciences Publication Activity Database

    Nedbalová, M.; Jandová, Anna; Dohnalová, A.

    Vol. 329. Bristol: IOP, 2011 - (Cifra, M.; Pokorny, J.; Kučera, O.), 012036 ISSN 1742-6588. [9th International Frohlich's Symposium on Electrodynamic Activity of Living Cells - Including Microtubule Coherent Modes and Cancer Cell Physics. Praha (CZ), 01.07.2011-03.07.2011] Institutional research plan: CEZ:AV0Z20670512 Keywords : Energy supplies * Genetic process * Information transfers Subject RIV: JA - Electronics ; Optoelectronics, Electrical Engineering

  12. Seasonal Pacing - Match Importance Affects Activity in Professional Soccer.

    Science.gov (United States)

    Link, Daniel; de Lorenzo, Michael F

    2016-01-01

    This research explores the influence of match importance on player activity in professional soccer. Therefore, we used an observational approach and analyzed 1,211 matches of German Bundesliga and 2nd Bundesliga. The importance measurement employed is based on post season consequences of teams involved in a match. This means, if a match result could potentially influence the final rank, and this rank would lead to different consequences for a team, such as qualification for Champions League opposed to qualification for Europe League, then this match is classified as important; otherwise not. Activity was quantified by TOTAL DISTANCE COVERED, SPRINTS, FAST RUNS, DUELS, FOULS and ATTEMPTS. Running parameters were recorded using a semi-automatic optical tracking system, while technical variables were collected by professional data loggers. Based on our importance classification, low important matches occurred at the beginning of round 29. A two-way ANOVA indicates significantly increased FAST RUNS (+4%, d = 0.3), DUELS (+16%, d = 1.0) and FOULS (+36%, d = 1.2) in important matches compared to low important ones. For FAST RUNS and FOULS, this effect only exists in Bundesliga. A comparison of the two leagues show that TOTAL DISTANCE COVERED (+3%, d = 0.9), SPRINTS (+25%, d = 1.4) and FAST RUNS (+15%, d = 1.4) are higher compared to 2nd Bundesliga, whilst FOULS is less in Bundesliga (-7%, d = 0.3). No difference in player activity was found between matches at the beginning of a season (round 1-6) and at the end of a season (round 29-34). We conclude that match importance influences player activity in German professional soccer. The most reasonable explanation is a conscious or unconscious pacing strategy, motivated by preserving abilities or preventing injury. Since this tendency mainly exists in Bundesliga, this may suggest that more skilled players show a higher awareness for the need of pacing. PMID:27281051

  13. Early life stress affects limited regional brain activity in depression

    OpenAIRE

    Du, Lian; Wang, Jingjie; Meng, Ben; Yong, Na; Yang, Xiangying; Huang, Qingling; Zhang, Yan; Yang, Lingling; Qu, Yuan; Chen, Zhu; Li, Yongmei; Lv, Fajin; Hu, Hua

    2016-01-01

    Early life stress (ELS) can alter brain function and increases the risk of major depressive disorder (MDD) in later life. This study investigated whether ELS contributes to differences in regional brain activity between MDD patients and healthy controls (HC), as measured by amplitude of low-frequency fluctuation (ALFF)/fractional (f)ALFF. Eighteen first-episode, treatment-naïve MDD patients and HC were assessed with the Childhood Trauma Questionnaire and resting-state functional magnetic reso...

  14. Disturbances of electrodynamic activity affect abortion in human

    International Nuclear Information System (INIS)

    Biochemical research of biological systems is highly developed, and it has disclosed a spectrum of chemical reactions, genetic processes, and the pathological development of various diseases. The fundamental hypothesis of physical processes in biological systems, in particular of coherent electrically polar vibrations and electromagnetic activity, was formulated by H. Fröhlich; he assumed connection of cancer process with degradation of coherent electromagnetic activity. But the questions of cellular structures capable of the coherent electrical polar oscillation, mechanisms of energy supply, and the specific role of the endogenous electromagnetic fields in transport, organisation, interactions, and information transfer remained open. The nature of physical disturbances caused by some diseases (including the recurrent abortion in humans and the cancer) was unknown. We have studied the reasons of recurrent abortions in humans by means of the cell mediated immunity (using immunologic active RNA prepared from blood of inbred laboratory mice strain C3H/H2K, infected with the lactate dehydrogenase elevating virus-LD V) and the cytogenetic examination from karyotype pictures. The recurrent abortion group contained women with dg. spontaneous abortion (n = 24) and the control group was composed of 30 healthy pregnant women. Our hypothesis was related to quality of endometrium in relation to nidation of the blastocyst. The energetic insufficiency (ATP) inhibits normal development of fetus and placenta. We hope that these ideas might have impact on further research, which could provide background for effective interdisciplinary cooperation of malignant and non-malignant diseases.

  15. Disturbances of electrodynamic activity affect abortion in human

    Science.gov (United States)

    Jandová, A.; Nedbalová, M.; Kobilková, J.; Čoček, A.; Dohnalová, A.; Cifra, M.; Pokorný, J.

    2011-12-01

    Biochemical research of biological systems is highly developed, and it has disclosed a spectrum of chemical reactions, genetic processes, and the pathological development of various diseases. The fundamental hypothesis of physical processes in biological systems, in particular of coherent electrically polar vibrations and electromagnetic activity, was formulated by H. Fröhlich he assumed connection of cancer process with degradation of coherent electromagnetic activity. But the questions of cellular structures capable of the coherent electrical polar oscillation, mechanisms of energy supply, and the specific role of the endogenous electromagnetic fields in transport, organisation, interactions, and information transfer remained open. The nature of physical disturbances caused by some diseases (including the recurrent abortion in humans and the cancer) was unknown. We have studied the reasons of recurrent abortions in humans by means of the cell mediated immunity (using immunologic active RNA prepared from blood of inbred laboratory mice strain C3H/H2K, infected with the lactate dehydrogenase elevating virus-LD V) and the cytogenetic examination from karyotype pictures. The recurrent abortion group contained women with dg. spontaneous abortion (n = 24) and the control group was composed of 30 healthy pregnant women. Our hypothesis was related to quality of endometrium in relation to nidation of the blastocyst. The energetic insufficiency (ATP) inhibits normal development of fetus and placenta. We hope that these ideas might have impact on further research, which could provide background for effective interdisciplinary cooperation of malignant and non-malignant diseases.

  16. Zirconium ions up-regulate the BMP/SMAD signaling pathway and promote the proliferation and differentiation of human osteoblasts.

    Directory of Open Access Journals (Sweden)

    Yongjuan Chen

    Full Text Available Zirconium (Zr is an element commonly used in dental and orthopedic implants either as zirconia (ZrO2 or in metal alloys. It can also be incorporated into calcium silicate-based ceramics. However, the effects of in vitro culture of human osteoblasts (HOBs with soluble ionic forms of Zr have not been determined. In this study, primary culture of human osteoblasts was conducted in the presence of medium containing either ZrCl4 or Zirconium (IV oxynitrate (ZrO(NO32 at concentrations of 0, 5, 50 and 500 µM, and osteoblast proliferation, differentiation and calcium deposition were assessed. Incubation of human osteoblast cultures with Zr ions increased the proliferation of human osteoblasts and also gene expression of genetic markers of osteoblast differentiation. In 21 and 28 day cultures, Zr ions at concentrations of 50 and 500 µM increased the deposition of calcium phosphate. In addition, the gene expression of BMP2 and BMP receptors was increased in response to culture with Zr ions and this was associated with increased phosphorylation of SMAD1/5. Moreover, Noggin suppressed osteogenic gene expression in HOBs co-treated with Zr ions. In conclusion, Zr ions appear able to induce both the proliferation and the differentiation of primary human osteoblasts. This is associated with up-regulation of BMP2 expression and activation of BMP signaling suggesting this action is, at least in part, mediated by BMP signaling.

  17. Disturbances of electrodynamic activity affect abortion in animals

    International Nuclear Information System (INIS)

    A specific kind of intracellular organelles, the mitochondria, is the place of metabolic energy production by oxidative mechanism. We used cell mediated immunity method for verification of the energy metabolism (ATP production). The antigen (immunological functional RNA) was obtained from blood of inbred laboratory mice strain C3H/H2K, infected with the lactate dehydrogenase elevating virus (LDV) and prepared by the high pressure gel chromatography (HPGC). We have studied the immunological adaptability of LDH viral antigen in 62 pigs (12 parents and 50 piglings). Exitus of piglings was in case of positive imunological response on LDV. The statement results from a comparison of the relative frequency of an incidence of identical findings in male piglets and sows and from identical findings in female piglets and pigs. The efficient elaboration and utilization of energy in cell may be damaged by the changes of energy production systems and also by long-term parasitary depletion of ATP energy. Biological activity is based not only on biochemical but also on biophysical mechanisms. Biophysical processes are also involved in the transfer of information and its processing for making decisions and providing control, which are important parts of biological activity. These experimental results were used for the same study in human.

  18. Toxicity of iron oxide nanoparticles against osteoblasts

    International Nuclear Information System (INIS)

    Magnetic nanoparticles have been widely used for tissue repair, magnetic resonance imaging, immunoassays and drug delivery. They are very promising in orthopaedic applications and several magnetic nanoparticles have been exploited for the treatment of orthopaedic disease. Here, we conducted an in vitro study to examine the interaction of magnetic iron oxide nanoparticles with human osteoblasts to evaluate the dose-related toxicity of the nanoparticles on osteoblasts. A transmission electron microscope was used to visualise the internalised magnetic nanoparticles in osteoblasts. The CCK-8 results revealed increased cell viability (107.5 % vitality compared with the control group) when co-cultured at a low concentration (20 μg/mL) and decreased cell viability (59.5 % vitality in a concentration of 300 μg/mL and 25.9 % in 500 μg/mL) when co-cultured in high concentrations. The flow cytometric detection revealed similar results with 5.48 % of apoptosis in a concentration of 20 μg/mL, 23.40 % of apoptosis in a concentration of 300 μg/mL and 28.49 % in a concentration of 500 μg/mL. The disrupted cytoskeleton of osteoblasts was also revealed using a laser scanning confocal microscope. We concluded that use of a low concentration of magnetic iron oxide nanoparticles is important to avoid damage to osteoblasts.

  19. Toxicity of iron oxide nanoparticles against osteoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Shi Sifeng [Shanghai Jiao Tong University, Department of Orthopaedic Surgery, Shanghai Sixth People' s Hospital (China); Jia Jingfu [Shanghai Jiao Tong University, School of Chemistry and Chemical Technology (China); Guo Xiaokui [Shanghai Jiao Tong University School of Medicine, Department of Medical Microbiology and Parasitology, Institutes of Medical Sciences (China); Zhao Yaping [Shanghai Jiao Tong University, School of Chemistry and Chemical Technology (China); Liu Boyu [Shanghai Jiao Tong University School of Medicine, Department of Medical Microbiology and Parasitology, Institutes of Medical Sciences (China); Chen Desheng; Guo Yongyuan; Zhang Xianlong, E-mail: zhangxianlong20101@163.com [Shanghai Jiao Tong University, Department of Orthopaedic Surgery, Shanghai Sixth People' s Hospital (China)

    2012-09-15

    Magnetic nanoparticles have been widely used for tissue repair, magnetic resonance imaging, immunoassays and drug delivery. They are very promising in orthopaedic applications and several magnetic nanoparticles have been exploited for the treatment of orthopaedic disease. Here, we conducted an in vitro study to examine the interaction of magnetic iron oxide nanoparticles with human osteoblasts to evaluate the dose-related toxicity of the nanoparticles on osteoblasts. A transmission electron microscope was used to visualise the internalised magnetic nanoparticles in osteoblasts. The CCK-8 results revealed increased cell viability (107.5 % vitality compared with the control group) when co-cultured at a low concentration (20 {mu}g/mL) and decreased cell viability (59.5 % vitality in a concentration of 300 {mu}g/mL and 25.9 % in 500 {mu}g/mL) when co-cultured in high concentrations. The flow cytometric detection revealed similar results with 5.48 % of apoptosis in a concentration of 20 {mu}g/mL, 23.40 % of apoptosis in a concentration of 300 {mu}g/mL and 28.49 % in a concentration of 500 {mu}g/mL. The disrupted cytoskeleton of osteoblasts was also revealed using a laser scanning confocal microscope. We concluded that use of a low concentration of magnetic iron oxide nanoparticles is important to avoid damage to osteoblasts.

  20. Synergistic effects of tributyltin and 2,3,7,8-tetrachlorodibenzo-p-dioxin on differentiating osteoblasts and osteoclasts

    International Nuclear Information System (INIS)

    The purpose of this study was to examine the effects of the persistent and accumulative environmental pollutants tributyltin (TBT) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) individually and in combination on differentiating bone cells. TBT and TCDD are chemically distinct compounds with different mechanisms of toxicity, but they typically have the same sources of exposure and both have been shown to affect bone development at low exposure levels. Bone marrow stem cells were isolated from femurs and tibias of C57BL/6 J mice, differentiated in culture into osteoblasts or osteoclasts and exposed to 0.1–10 nM TBT, 0.01–1 nM TCDD or 10 nM TBT + 1 nM TCDD. In osteoblasts, the combined exposure to TBT and TCDD significantly decreased the mRNA expression of alkaline phosphatase and osteocalcin more than TBT or TCDD alone. PCR array showed different gene expression profiles for TBT and TCDD individually, and the combination evoked several additional alterations in gene expression. Expression of aryl hydrocarbon receptor repressor (AHRR) was increased by TCDD as expected, but simultaneous exposure to TBT prevented the increase thus potentially strengthening AHR-mediated effects of TCDD. The number of osteoclasts was reduced by TCDD alone and in combination with TBT, but TBT alone had no effect. However, the total area of resorbed bone was remarkably lower after combined exposure than after TBT or TCDD alone. In conclusion, very low concentrations of TBT and TCDD have synergistic deleterious effects on bone formation and additive effects on bone resorption. -- Highlights: ► Combined exposure to TCDD and TBT evoked a unique gene expression profile. ► Osteoblast differentiation was synergistically disturbed after combined exposure. ► Bone resorbing activity was additively decreased after combined exposure.

  1. Synergistic effects of tributyltin and 2,3,7,8-tetrachlorodibenzo-p-dioxin on differentiating osteoblasts and osteoclasts

    Energy Technology Data Exchange (ETDEWEB)

    Koskela, Antti, E-mail: antti.koskela@oulu.fi [University of Oulu, Department of Anatomy and Cell Biology, Oulu (Finland); Viluksela, Matti [National Institute for Health and Welfare, Department of Environmental Health, Kuopio (Finland); Keinänen, Meeri; Tuukkanen, Juha [University of Oulu, Department of Anatomy and Cell Biology, Oulu (Finland); Korkalainen, Merja [National Institute for Health and Welfare, Department of Environmental Health, Kuopio (Finland)

    2012-09-01

    The purpose of this study was to examine the effects of the persistent and accumulative environmental pollutants tributyltin (TBT) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) individually and in combination on differentiating bone cells. TBT and TCDD are chemically distinct compounds with different mechanisms of toxicity, but they typically have the same sources of exposure and both have been shown to affect bone development at low exposure levels. Bone marrow stem cells were isolated from femurs and tibias of C57BL/6 J mice, differentiated in culture into osteoblasts or osteoclasts and exposed to 0.1–10 nM TBT, 0.01–1 nM TCDD or 10 nM TBT + 1 nM TCDD. In osteoblasts, the combined exposure to TBT and TCDD significantly decreased the mRNA expression of alkaline phosphatase and osteocalcin more than TBT or TCDD alone. PCR array showed different gene expression profiles for TBT and TCDD individually, and the combination evoked several additional alterations in gene expression. Expression of aryl hydrocarbon receptor repressor (AHRR) was increased by TCDD as expected, but simultaneous exposure to TBT prevented the increase thus potentially strengthening AHR-mediated effects of TCDD. The number of osteoclasts was reduced by TCDD alone and in combination with TBT, but TBT alone had no effect. However, the total area of resorbed bone was remarkably lower after combined exposure than after TBT or TCDD alone. In conclusion, very low concentrations of TBT and TCDD have synergistic deleterious effects on bone formation and additive effects on bone resorption. -- Highlights: ► Combined exposure to TCDD and TBT evoked a unique gene expression profile. ► Osteoblast differentiation was synergistically disturbed after combined exposure. ► Bone resorbing activity was additively decreased after combined exposure.

  2. Root activity, some crops as affected by soil strength

    International Nuclear Information System (INIS)

    To find out the relationship between soil strength and root activity of different crops, the experiment was conducted on Haryana Agricultural University Farm, Hissar. Open drums were placed one foot deep. 5 cm thick densities 1.4 (Control), 1.6 and 1.8 g/cc were placed at 25 cm depth in various drums. Test crops taken were pea, gram, wheat and barley. Bulk density of higher order in combination with low moisture levels resulted in more detrimental effects on root penetration of the crop in general but at some stages significant interaction between bulk density and moisture was observed where low moisture favoured the root entry through the compacted layers of soil. (author)

  3. Human osteoblastic cells propagate intercellular calcium signals by two different mechanisms

    DEFF Research Database (Denmark)

    Jørgensen, Niklas Rye; Henriksen, Z; Brot, C;

    2000-01-01

    . After the fast intercellular calcium waves were blocked, we observed slower calcium waves that were dependent on gap junctional communication and influx of extracellular calcium. These results show that human osteoblastic cells can propagate calcium signals from cell to cell by two markedly different...... cells: autocrine activation of P2 (purinergic) receptors leading to release of intracellular calcium stores, and gap junction-mediated communication resulting in influx of extracellular calcium. In the current work we asked whether human osteoblastic cells (HOB) were capable of mechanically induced...... intercellular calcium signaling, and if so, by which mechanisms. Upon mechanical stimulation, human osteoblasts propagated fast intercellular calcium waves, which required activation of P2 receptors and release of intracellular calcium stores but did not require calcium influx or gap junctional communication...

  4. Cobalt in alluvial Egyptian soils as affected by industrial activities

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Twenty-five surface (0-20 cm) soil samples were collected from different locations in Egypt representing non-polluted,moderately and highly polluted soils. The aim of this study was to evaluate total Co content in alluvial soils of Delta in Egypt using the delayed Neturen activation analysis technique (DNAA). The two prominent gamma ray lines at 1173.2 and 1332.5 keV was efficiently used for 60Co determination. Co content in non-polluted soil samples ranged between 13.12 to 23.20 ppm Co with an average of 18.16*4.38 ppm. Cobalt content in moderately polluted soils ranged between 26.5 to 30.00 ppm with an average of 28.3*1.3 ppm. The highest Co levels (ranged from 36 to 64.69 ppm with an average of 51.9*9.5); were observed in soil samples collected from, either highly polluted agricultural soils due to prolonged irrigation with industrial wastewater or surface soil samples from industrial sites.

  5. Sampling frequency affects ActiGraph activity counts

    DEFF Research Database (Denmark)

    Brønd, Jan Christian; Arvidsson, Daniel

    Introduction: The ActiLife Data Analysis Software processes ActiGraph accelerometer data and has a bandpass filter attenuating accelerations falling outside the normal human frequency passband considered to be 0.25-2.5 Hz. This frequency passband disfavour vigorous physical activity that is...... normally performed at frequencies higher than 2.5 Hz. With the ActiGraph model GT3X one has the option to select sample frequency from 30 to 100 Hz. This study investigated the effect of the sampling frequency on the ouput of the bandpass filter.Methods: A synthetic frequency sweep of 0-15 Hz was generated...... in Matlab and sampled at frequencies of 30-100 Hz. Also, acceleration signals during indoor walking and running were sampled at 30 Hz using the ActiGraph GT3X and resampled in Matlab to frequencies of 40-100 Hz. All data was processed with the ActiLife software.Results: Acceleration frequencies...

  6. Tasting calories differentially affects brain activation during hunger and satiety.

    Science.gov (United States)

    van Rijn, Inge; de Graaf, Cees; Smeets, Paul A M

    2015-02-15

    An important function of eating is ingesting energy. Our objectives were to assess whether oral exposure to caloric and non-caloric stimuli elicits discriminable responses in the brain and to determine in how far these responses are modulated by hunger state and sweetness. Thirty women tasted three stimuli in two motivational states (hunger and satiety) while their brain responses were measured using functional magnetic resonance imaging in a randomized crossover design. Stimuli were solutions of sucralose (sweet, no energy), maltodextrin (non-sweet, energy) and sucralose+maltodextrin (sweet, energy). We found no main effect of energy content and no interaction between energy content and sweetness. However, there was an interaction between hunger state and energy content in the median cingulate (bilaterally), ventrolateral prefrontal cortex, anterior insula and thalamus. This indicates that the anterior insula and thalamus, areas in which hunger state and taste of a stimulus are integrated, also integrate hunger state with caloric content of a taste stimulus. Furthermore, in the median cingulate and ventrolateral prefrontal cortex, tasting energy resulted in more activation during satiety compared to hunger. This finding indicates that these areas, which are known to be involved in processes that require approach and avoidance, are also involved in guiding ingestive behavior. In conclusion, our results suggest that energy sensing is a hunger state dependent process, in which the median cingulate, ventrolateral prefrontal cortex, anterior insula and thalamus play a central role by integrating hunger state with stimulus relevance. PMID:25449847

  7. Temperature affects microbial abundance, activity and interactions in anaerobic digestion.

    Science.gov (United States)

    Lin, Qiang; De Vrieze, Jo; Li, Jiabao; Li, Xiangzhen

    2016-06-01

    Temperature is a major factor determining the performance of the anaerobic digestion process. The microbial abundance, activity and interactional networks were investigated under a temperature gradient from 25°C to 55°C through amplicon sequencing, using 16S ribosomal RNA and 16S rRNA gene-based approaches. Comparative analysis of past accumulative elements presented by 16S rRNA gene-based analysis, and the in-situ conditions presented by 16S rRNA-based analysis, provided new insights concerning the identification of microbial functional roles and interactions. The daily methane production and total biogas production increased with temperature up to 50°C, but decreased at 55°C. Increased methanogenesis and hydrolysis at 50°C were main factors causing higher methane production which was also closely related with more well-defined methanogenic and/or related modules with comprehensive interactions and increased functional orderliness referred to more microorganisms participating in interactions. This research demonstrated the importance of evaluating functional roles and interactions of microbial community. PMID:26970926

  8. Pigment Epithelium-Derived Factor (PEDF) Protects Osteoblastic Cell Line from Glucocorticoid-Induced Apoptosis via PEDF-R.

    Science.gov (United States)

    Yao, Shengcheng; Zhang, Yingnan; Wang, Xiaoyu; Zhao, Fengchao; Sun, Maji; Zheng, Xin; Dong, Hongyan; Guo, Kaijin

    2016-01-01

    Pigment epithelial-derived factor (PEDF) is known as a widely expressed multifunctional secreted glycoprotein whose biological actions are cell-type dependent. Recent studies demonstrated that PEDF displays cytoprotective activity in several cell types. However, it remains unknown whether PEDF is involved in glucocorticoid-induced osteoblast death. The aim of this study was to examine the role of PEDF in osteoblast survival in response to dexamethasone, an active glucocorticoid analogue, and explore the underlying mechanism. In the present study, dexamethasone (DEX) was used to induce MC3T3-E1 pre-osteoblast apoptosis. PEDF mRNA and protein levels and cell apoptosis were determined respectively. Then PEDF receptor (PEDF-R)- and lysophosphatidic acid (LPA)-related signal transductions were assessed. Here we show that DEX down-regulates PEDF expression, which contributes to osteoblast apoptosis. As a result, exogenous recombinant PEDF (rPEDF) inhibited DEX-induced cell apoptosis. We confirmed that PEDF-R was expressed on MC3T3-E1 pre-osteoblast membrane and could bind to PEDF which increased the level of LPA and activated the phosphorylation of Akt. Our results suggest that PEDF attenuated DEX-induced apoptosis in MC3T3-E1 pre-osteoblasts through LPA-dependent Akt activation via PEDF-R. PMID:27187377

  9. Nanostructured niobium oxide coatings influence osteoblast adhesion.

    Science.gov (United States)

    Eisenbarth, E; Velten, D; Müller, M; Thull, R; Breme, J

    2006-10-01

    The interaction of osteoblasts was correlated to the roughness of nanosized surface structures of Nb(2)O(5) coatings on polished CP titanium grade 2. Nb(2)O(5) sol-gel coatings were selected as a model surface to study the interaction of osteoblasts with nanosized surface structures. The surface roughness was quantified by determination of the average surface finish (Ra number) by means of atomic force microscopy. Surface topographies with Ra = 7, 15, and 40 nm were adjusted by means of the annealing process parameters (time and temperature) within a sol-gel coating procedure. The observed osteoblast migration was fastest on smooth surfaces with Ra = 7 nm. The adhesion strength, spreading area, and collagen-I synthesis showed the best results on an intermediate roughness of Ra = 15 nm. The surface roughness of Ra = 40 nm was rather peaked and reduced the speed of cell reactions belonging to the adhesion process. PMID:16788971

  10. Paracrine sonic hedgehog signalling by prostate cancer cells induces osteoblast differentiation

    Directory of Open Access Journals (Sweden)

    Iannaccone Philip

    2009-03-01

    Full Text Available Abstract Background Sonic hedgehog (Shh and components of its signalling pathway have been identified in human prostate carcinoma and increased levels of their expression appear to correlate with disease progression and metastasis. The mechanism through which Shh signalling could promote metastasis in bone, the most common site for prostate carcinoma metastasis, has not yet been investigated. The present study determined the effect of Shh signalling between prostate cancer cells and pre-osteoblasts on osteoblast differentiation, a requisite process for new bone formation that characterizes prostate carcinoma metastasis. Results LNCaP human prostate cancer cells modified to overexpress Shh (designated LNShh cells and MC3T3 mouse pre-osteoblasts were maintained as mixed populations within the same culture chamber. In this non-conventional mixed culture system, LNShh cells upregulated the expression of Shh target genes Gli1 and Patched 1 (Ptc1 in MC3T3 cells and this was inhibited by cyclopamine, a specific chemical inhibitor of hedgehog signalling. Concomitantly, MC3T3 cells exhibited time-dependent decreased cell proliferation, upregulated alkaline phosphatase Akp2 gene expression, and increased alkaline phosphatase activity indicative of early phase osteoblast differentiation. LNShh cell-induced differentiation was inhibited in MC3T3 cells stably transfected with a dominant negative form of Gli1, a transcription factor that mediates Shh signalling. Interestingly, LNShh cells did not significantly increase the endogenous expression of the osteoblast differentiation transcription factor Runx2 and its target genes osteocalcin and osteopontin. Consistent with these results, exogenous Shh peptide did not upregulate Runx2 expression in MC3T3 cells. However, Runx2 levels were increased in MC3T3 cells by ascorbic acid, a known stimulator of osteoblast differentiation. Conclusion Altogether, these data demonstrate that Shh-expressing prostate cancer

  11. Palmitate attenuates osteoblast differentiation of fetal rat calvarial cells

    Energy Technology Data Exchange (ETDEWEB)

    Yeh, Lee-Chuan C.; Ford, Jeffery J. [Department of Biochemistry, The University of Texas Health Science Center at San Antonio, TX (United States); Lee, John C. [Department of Biochemistry, The University of Texas Health Science Center at San Antonio, TX (United States); The Sam and Ann Barshop Institute for Longevity and Aging Studies, The University of Texas Health Science Center at San Antonio, TX (United States); Adamo, Martin L., E-mail: adamo@biochem.uthscsa.edu [Department of Biochemistry, The University of Texas Health Science Center at San Antonio, TX (United States); The Sam and Ann Barshop Institute for Longevity and Aging Studies, The University of Texas Health Science Center at San Antonio, TX (United States)

    2014-07-18

    Highlights: • Palmitate inhibits osteoblast differentiation. • Fatty acid synthase. • PPARγ. • Acetyl Co-A carboxylase inhibitor TOFA. • Fetal rat calvarial cell culture. - Abstract: Aging is associated with the accumulation of ectopic lipid resulting in the inhibition of normal organ function, a phenomenon known as lipotoxicity. Within the bone marrow microenvironment, elevation in fatty acid levels may produce an increase in osteoclast activity and a decrease in osteoblast number and function, thus contributing to age-related osteoporosis. However, little is known about lipotoxic mechanisms in intramembraneous bone. Previously we reported that the long chain saturated fatty acid palmitate inhibited the expression of the osteogenic markers RUNX2 and osteocalcin in fetal rat calvarial cell (FRC) cultures. Moreover, the acetyl CoA carboxylase inhibitor TOFA blocked the inhibitory effect of palmitate on expression of these two markers. In the current study we have extended these observations to show that palmitate inhibits spontaneous mineralized bone formation in FRC cultures in association with reduced mRNA expression of RUNX2, alkaline phosphatase, osteocalcin, and bone sialoprotein and reduced alkaline phosphatase activity. The effects of palmitate on osteogenic marker expression were inhibited by TOFA. Palmitate also inhibited the mRNA expression of fatty acid synthase and PPARγ in FRC cultures, and as with osteogenic markers, this effect was inhibited by TOFA. Palmitate had no effect on FRC cell proliferation or apoptosis, but inhibited BMP-7-induced alkaline phosphatase activity. We conclude that palmitate accumulation may lead to lipotoxic effects on osteoblast differentiation and mineralization and that increases in fatty acid oxidation may help to prevent these lipotoxic effects.

  12. Palmitate attenuates osteoblast differentiation of fetal rat calvarial cells

    International Nuclear Information System (INIS)

    Highlights: • Palmitate inhibits osteoblast differentiation. • Fatty acid synthase. • PPARγ. • Acetyl Co-A carboxylase inhibitor TOFA. • Fetal rat calvarial cell culture. - Abstract: Aging is associated with the accumulation of ectopic lipid resulting in the inhibition of normal organ function, a phenomenon known as lipotoxicity. Within the bone marrow microenvironment, elevation in fatty acid levels may produce an increase in osteoclast activity and a decrease in osteoblast number and function, thus contributing to age-related osteoporosis. However, little is known about lipotoxic mechanisms in intramembraneous bone. Previously we reported that the long chain saturated fatty acid palmitate inhibited the expression of the osteogenic markers RUNX2 and osteocalcin in fetal rat calvarial cell (FRC) cultures. Moreover, the acetyl CoA carboxylase inhibitor TOFA blocked the inhibitory effect of palmitate on expression of these two markers. In the current study we have extended these observations to show that palmitate inhibits spontaneous mineralized bone formation in FRC cultures in association with reduced mRNA expression of RUNX2, alkaline phosphatase, osteocalcin, and bone sialoprotein and reduced alkaline phosphatase activity. The effects of palmitate on osteogenic marker expression were inhibited by TOFA. Palmitate also inhibited the mRNA expression of fatty acid synthase and PPARγ in FRC cultures, and as with osteogenic markers, this effect was inhibited by TOFA. Palmitate had no effect on FRC cell proliferation or apoptosis, but inhibited BMP-7-induced alkaline phosphatase activity. We conclude that palmitate accumulation may lead to lipotoxic effects on osteoblast differentiation and mineralization and that increases in fatty acid oxidation may help to prevent these lipotoxic effects

  13. Pharmacological induction of ferritin prevents osteoblastic transformation of smooth muscle cells.

    Science.gov (United States)

    Becs, Gergely; Zarjou, Abolfazl; Agarwal, Anupam; Kovács, Katalin Éva; Becs, Ádám; Nyitrai, Mónika; Balogh, Enikő; Bányai, Emese; Eaton, John W; Arosio, Paolo; Poli, Maura; Jeney, Viktória; Balla, József; Balla, György

    2016-02-01

    Vascular calcification is a frequent complication of atherosclerosis, diabetes and chronic kidney disease. In the latter group of patients, calcification is commonly seen in tunica media where smooth muscle cells (SMC) undergo osteoblastic transformation. Risk factors such as elevated phosphorus levels and vitamin D3 analogues have been identified. In the light of earlier observations by our group and others, we sought to inhibit SMC calcification via induction of ferritin. Human aortic SMC were cultured using β-glycerophosphate with activated vitamin D3 , or inorganic phosphate with calcium, and induction of alkaline phosphatase (ALP) and osteocalcin as well as accumulation of calcium were used to monitor osteoblastic transformation. In addition, to examine the role of vitamin D3 analogues, plasma samples from patients on haemodialysis who had received calcitriol or paricalcitol were tested for their tendency to induce calcification of SMC. Addition of exogenous ferritin mitigates the transformation of SMC into osteoblast-like cells. Importantly, pharmacological induction of heavy chain ferritin by 3H-1,2-Dithiole-3-thione was able to inhibit the SMC transition into osteoblast-like cells and calcification of extracellular matrix. Plasma samples collected from patients after the administration of activated vitamin D3 caused significantly increased ALP activity in SMC compared to the samples drawn prior to activated vitamin D3 and here, again induction of ferritin diminished the osteoblastic transformation. Our data suggests that pharmacological induction of ferritin prevents osteoblastic transformation of SMC. Hence, utilization of such agents that will cause enhanced ferritin synthesis may have important clinical applications in prevention of vascular calcification. PMID:26499096

  14. Src is a major signaling component for CTGF induction by TGF-β1 in osteoblasts

    Science.gov (United States)

    X, Zhang; JA, Arnott; S, Rehman; WG, DeLong; A, Sanjay; FF, Safadi; SN, Popoff

    2010-01-01

    Connective tissue growth factor (CTGF/CCN2) is induced by transforming growth factor beta 1(TGF-β1) where it acts as a downstream mediator of TGF-β1 induced matrix production in osteoblasts. We have shown the requirement of Src, Erk and Smad signaling for CTGF induction by TGF-β1 in osteoblasts, however the potential interaction among these signaling pathways remains undetermined. In this study we demonstrate that TGF-β1 activates Src kinase in ROS17/2.8 cells and that treatment with the Src family kinase inhibitor PP2 prevents Src activation and CTGF induction by TGF-β1. Additionally, inhibiting Src activation prevented Erk activation, Smad 2 & 3 activation and nuclear translocation by TGF-β1, demonstrating that Src is an essential upstream signaling partner of both Erk and Smads in osteoblasts. MAPKs such as Erk can modulate the Smad pathway through directly mediating the phosphorylation of Smads or indirectly through activation/inactivation of required nuclear co-activators that mediate Smad DNA binding. When we treated cells with the Erk inhibitor, PD98059 it inhibited TGF-β1-induced CTGF protein expression but had no effect on Src activation, Smad activation or Smad nuclear translocation. However PD98059 impaired transcriptional complex formation on the Smad binding element (SBE) on the CTGF promoter, demonstrating that Erk activation was required for SBE transactivation. This data demonstrates that Src is an essential upstream signaling transducer of Erk and Smad signaling with respect to TGF-β1 in osteoblasts and that Smads and Erk function independently but are both essential for forming a transcriptionally active complex on the CTGF promoter in osteoblasts. PMID:20432467

  15. The Role of KV7.3 in Regulating Osteoblast Maturation and Mineralization.

    Science.gov (United States)

    Yang, Ji Eun; Song, Min Seok; Shen, Yiming; Ryu, Pan Dong; Lee, So Yeong

    2016-01-01

    KCNQ (KV7) channels are voltage-gated potassium (KV) channels, and the function of KV7 channels in muscles, neurons, and sensory cells is well established. We confirmed that overall blockade of KV channels with tetraethylammonium augmented the mineralization of bone-marrow-derived human mesenchymal stem cells during osteogenic differentiation, and we determined that KV7.3 was expressed in MG-63 and Saos-2 cells at the mRNA and protein levels. In addition, functional KV7 currents were detected in MG-63 cells. Inhibition of KV7.3 by linopirdine or XE991 increased the matrix mineralization during osteoblast differentiation. This was confirmed by alkaline phosphatase, osteocalcin, and osterix in MG-63 cells, whereas the expression of Runx2 showed no significant change. The extracellular glutamate secreted by osteoblasts was also measured to investigate its effect on MG-63 osteoblast differentiation. Blockade of KV7.3 promoted the release of glutamate via the phosphorylation of extracellular signal-regulated kinase 1/2-mediated upregulation of synapsin, and induced the deposition of type 1 collagen. However, activation of KV7.3 by flupirtine did not produce notable changes in matrix mineralization during osteoblast differentiation. These results suggest that KV7.3 could be a novel regulator in osteoblast differentiation. PMID:26999128

  16. The Role of KV7.3 in Regulating Osteoblast Maturation and Mineralization

    Directory of Open Access Journals (Sweden)

    Ji Eun Yang

    2016-03-01

    Full Text Available KCNQ (KV7 channels are voltage-gated potassium (KV channels, and the function of KV7 channels in muscles, neurons, and sensory cells is well established. We confirmed that overall blockade of KV channels with tetraethylammonium augmented the mineralization of bone-marrow-derived human mesenchymal stem cells during osteogenic differentiation, and we determined that KV7.3 was expressed in MG-63 and Saos-2 cells at the mRNA and protein levels. In addition, functional KV7 currents were detected in MG-63 cells. Inhibition of KV7.3 by linopirdine or XE991 increased the matrix mineralization during osteoblast differentiation. This was confirmed by alkaline phosphatase, osteocalcin, and osterix in MG-63 cells, whereas the expression of Runx2 showed no significant change. The extracellular glutamate secreted by osteoblasts was also measured to investigate its effect on MG-63 osteoblast differentiation. Blockade of KV7.3 promoted the release of glutamate via the phosphorylation of extracellular signal-regulated kinase 1/2-mediated upregulation of synapsin, and induced the deposition of type 1 collagen. However, activation of KV7.3 by flupirtine did not produce notable changes in matrix mineralization during osteoblast differentiation. These results suggest that KV7.3 could be a novel regulator in osteoblast differentiation.

  17. The importance of physical activity and sleep for affect on stressful days: Two intensive longitudinal studies.

    Science.gov (United States)

    Flueckiger, Lavinia; Lieb, Roselind; Meyer, Andrea H; Witthauer, Cornelia; Mata, Jutta

    2016-06-01

    We investigated the potential stress-buffering effect of 3 health behaviors-physical activity, sleep quality, and snacking-on affect in the context of everyday life in young adults. In 2 intensive longitudinal studies with up to 65 assessment days over an entire academic year, students (Study 1, N = 292; Study 2, N = 304) reported stress intensity, sleep quality, physical activity, snacking, and positive and negative affect. Data were analyzed using multilevel regression analyses. Stress and positive affect were negatively associated; stress and negative affect were positively associated. The more physically active than usual a person was on a given day, the weaker the association between stress and positive affect (Study 1) and negative affect (Studies 1 and 2). The better than usual a person's sleep quality had been during the previous night, the weaker the association between stress and positive affect (Studies 1 and 2) and negative affect (Study 2). The association between daily stress and positive or negative affect did not differ as a function of daily snacking (Studies 1 and 2). On stressful days, increasing physical activity or ensuring high sleep quality may buffer adverse effects of stress on affect in young adults. These findings suggest potential targets for health-promotion and stress-prevention programs, which could help reduce the negative impact of stress in young adults. (PsycINFO Database Record PMID:26709860

  18. Wnt Protein Signaling Reduces Nuclear Acetyl-CoA Levels to Suppress Gene Expression during Osteoblast Differentiation.

    Science.gov (United States)

    Karner, Courtney M; Esen, Emel; Chen, Jiakun; Hsu, Fong-Fu; Turk, John; Long, Fanxin

    2016-06-17

    Developmental signals in metazoans play critical roles in inducing cell differentiation from multipotent progenitors. The existing paradigm posits that the signals operate directly through their downstream transcription factors to activate expression of cell type-specific genes, which are the hallmark of cell identity. We have investigated the mechanism through which Wnt signaling induces osteoblast differentiation in an osteoblast-adipocyte bipotent progenitor cell line. Unexpectedly, Wnt3a acutely suppresses the expression of a large number of genes while inducing osteoblast differentiation. The suppressed genes include Pparg and Cebpa, which encode adipocyte-specifying transcription factors and suppression of which is sufficient to induce osteoblast differentiation. The large scale gene suppression induced by Wnt3a corresponds to a global decrease in histone acetylation, an epigenetic modification that is associated with gene activation. Mechanistically, Wnt3a does not alter histone acetyltransferase or deacetylase activities but, rather, decreases the level of acetyl-CoA in the nucleus. The Wnt-induced decrease in histone acetylation is independent of β-catenin signaling but, rather, correlates with suppression of glucose metabolism in the tricarboxylic acid cycle. Functionally, preventing histone deacetylation by increasing nucleocytoplasmic acetyl-CoA levels impairs Wnt3a-induced osteoblast differentiation. Thus, Wnt signaling induces osteoblast differentiation in part through histone deacetylation and epigenetic suppression of an alternative cell fate. PMID:27129247

  19. Involvement of the G-protein-coupled receptor 4 in RANKL expression by osteoblasts in an acidic environment.

    Science.gov (United States)

    Okito, Asuka; Nakahama, Ken-Ichi; Akiyama, Masako; Ono, Takashi; Morita, Ikuo

    2015-03-01

    Osteoclast activity is enhanced in acidic environments following systemic or local inflammation. However, the regulatory mechanism of receptor activator of NF-κB ligand (RANKL) expression in osteoblasts under acidic conditions is not fully understood. In the present paper, we detected the mRNA expression of the G-protein-coupled receptor (GPR) proton sensors GPR4 and GPR65 (T-cell death-associated gene 8, TDAG8), in osteoblasts. RANKL expression and the cyclic AMP (cAMP) level in osteoblasts were up-regulated under acidic culture conditions. Acidosis-induced up-regulation of RANKL was abolished by the protein kinase A inhibitor H89. To clarify the role of GPR4 in RANKL expression, GPR4 gain and loss of function experiments were performed. Gene knockdown and forced expression of GPR4 caused reduction and induction of RANKL expression, respectively. These results suggested that, at least in part, RANKL expression by osteoblasts in an acidic environment was mediated by cAMP/PKA signaling resulting from GPR4 activation. A comprehensive microarray analysis of gene expression of osteoblasts revealed that, under acidic conditions, the phenotype of osteoblasts was that of an osteoclast supporting cell rather than that of a mineralizing cell. These findings will contribute to a molecular understanding of bone disruption in an acidic environment. PMID:25668130

  20. Rapid oriented fibril formation of fish scale collagen facilitates early osteoblastic differentiation of human mesenchymal stem cells.

    Science.gov (United States)

    Matsumoto, Rena; Uemura, Toshimasa; Xu, Zhefeng; Yamaguchi, Isamu; Ikoma, Toshiyuki; Tanaka, Junzo

    2015-08-01

    We studied the effect of fibril formation of fish scale collagen on the osteoblastic differentiation of human mesenchymal stem cells (hMSCs). We found that hMSCs adhered easily to tilapia scale collagen, which remarkably accelerated the early stage of osteoblastic differentiation in hMSCs during in vitro cell culture. Osteoblastic markers such as ALP activity, osteopontin, and bone morphogenetic protein 2 were markedly upregulated when the hMSCs were cultured on a tilapia collagen surface, especially in the early osteoblastic differentiation stage. We hypothesized that this phenomenon occurs due to specific fibril formation of tilapia collagen. Thus, we examined the time course of collagen fibril formation using high-speed atomic force microscopy. Moreover, to elucidate the effect of the orientation of fibril formation on the differentiation of hMSCs, we measured ALP activity of hMSCs cultured on two types of tilapia scale collagen membranes with different degrees of fibril formation. The ALP activity in hMSCs cultured on a fibrous collagen membrane was significantly higher than on a non-fibrous collagen membrane even before adding osteoblastic differentiation medium. These results showed that the degree of the fibril formation of tilapia collagen was essential for the osteoblastic differentiation of hMSCs. PMID:25546439

  1. Youth perceptions of how neighborhood physical environment and peers affect physical activity: a focus group study

    OpenAIRE

    Smith, Alan L.; Troped, Philip J; McDonough, Meghan H; DeFreese, J.D.

    2015-01-01

    Objective There is need for a youth-informed conceptualization of how environmental and social neighborhood contexts influence physical activity. We assessed youths’ perceptions of their neighborhood physical and peer environments as affecting physical activity. Methods Thirty-three students (20 girls; ages 12-14 years) participated in focus groups about the physical environment and peers within their neighborhoods, and their understanding of how they affect physical activity. Results Inducti...

  2. Involvement of the G-protein-coupled receptor 4 in RANKL expression by osteoblasts in an acidic environment

    International Nuclear Information System (INIS)

    Osteoclast activity is enhanced in acidic environments following systemic or local inflammation. However, the regulatory mechanism of receptor activator of NF-κB ligand (RANKL) expression in osteoblasts under acidic conditions is not fully understood. In the present paper, we detected the mRNA expression of the G-protein-coupled receptor (GPR) proton sensors GPR4 and GPR65 (T-cell death-associated gene 8, TDAG8), in osteoblasts. RANKL expression and the cyclic AMP (cAMP) level in osteoblasts were up-regulated under acidic culture conditions. Acidosis-induced up-regulation of RANKL was abolished by the protein kinase A inhibitor H89. To clarify the role of GPR4 in RANKL expression, GPR4 gain and loss of function experiments were performed. Gene knockdown and forced expression of GPR4 caused reduction and induction of RANKL expression, respectively. These results suggested that, at least in part, RANKL expression by osteoblasts in an acidic environment was mediated by cAMP/PKA signaling resulting from GPR4 activation. A comprehensive microarray analysis of gene expression of osteoblasts revealed that, under acidic conditions, the phenotype of osteoblasts was that of an osteoclast supporting cell rather than that of a mineralizing cell. These findings will contribute to a molecular understanding of bone disruption in an acidic environment. - Highlights: • RANKL expression was increased in osteoblasts under acidosis via cAMP/PKA pathway. • GRP4 knockdown resulted in decrease of RANKL expression. • GRP4 overexpression resulted in increase of RANKL expression. • Osteoblast mineralization was reduced under acidic condition

  3. Involvement of the G-protein-coupled receptor 4 in RANKL expression by osteoblasts in an acidic environment

    Energy Technology Data Exchange (ETDEWEB)

    Okito, Asuka [Department of Cellular Physiological Chemistry, Tokyo Medical and Dental University, Tokyo (Japan); Department of Orthodontic Science, Tokyo Medical and Dental University, Tokyo (Japan); Nakahama, Ken-ichi, E-mail: nakacell@tmd.ac.jp [Department of Cellular Physiological Chemistry, Tokyo Medical and Dental University, Tokyo (Japan); Akiyama, Masako [Department of Cellular Physiological Chemistry, Tokyo Medical and Dental University, Tokyo (Japan); Ono, Takashi [Department of Orthodontic Science, Tokyo Medical and Dental University, Tokyo (Japan); Morita, Ikuo [Department of Cellular Physiological Chemistry, Tokyo Medical and Dental University, Tokyo (Japan)

    2015-03-06

    Osteoclast activity is enhanced in acidic environments following systemic or local inflammation. However, the regulatory mechanism of receptor activator of NF-κB ligand (RANKL) expression in osteoblasts under acidic conditions is not fully understood. In the present paper, we detected the mRNA expression of the G-protein-coupled receptor (GPR) proton sensors GPR4 and GPR65 (T-cell death-associated gene 8, TDAG8), in osteoblasts. RANKL expression and the cyclic AMP (cAMP) level in osteoblasts were up-regulated under acidic culture conditions. Acidosis-induced up-regulation of RANKL was abolished by the protein kinase A inhibitor H89. To clarify the role of GPR4 in RANKL expression, GPR4 gain and loss of function experiments were performed. Gene knockdown and forced expression of GPR4 caused reduction and induction of RANKL expression, respectively. These results suggested that, at least in part, RANKL expression by osteoblasts in an acidic environment was mediated by cAMP/PKA signaling resulting from GPR4 activation. A comprehensive microarray analysis of gene expression of osteoblasts revealed that, under acidic conditions, the phenotype of osteoblasts was that of an osteoclast supporting cell rather than that of a mineralizing cell. These findings will contribute to a molecular understanding of bone disruption in an acidic environment. - Highlights: • RANKL expression was increased in osteoblasts under acidosis via cAMP/PKA pathway. • GRP4 knockdown resulted in decrease of RANKL expression. • GRP4 overexpression resulted in increase of RANKL expression. • Osteoblast mineralization was reduced under acidic condition.

  4. Chronic skin inflammation leads to bone loss by IL-17-mediated inhibition of Wnt signaling in osteoblasts.

    Science.gov (United States)

    Uluçkan, Özge; Jimenez, Maria; Karbach, Susanne; Jeschke, Anke; Graña, Osvaldo; Keller, Johannes; Busse, Björn; Croxford, Andrew L; Finzel, Stephanie; Koenders, Marije; van den Berg, Wim; Schinke, Thorsten; Amling, Michael; Waisman, Ari; Schett, Georg; Wagner, Erwin F

    2016-03-16

    Inflammation has important roles in tissue regeneration, autoimmunity, and cancer. Different inflammatory stimuli can lead to bone loss by mechanisms that are not well understood. We show that skin inflammation induces bone loss in mice and humans. In psoriasis, one of the prototypic IL-17A-mediated inflammatory human skin diseases, low bone formation and bone loss correlated with increased serum IL-17A levels. Similarly, in two mouse models with chronic IL-17A-mediated skin inflammation,K14-IL17A(ind)andJunB(Δep), strong inhibition of bone formation was observed, different from classical inflammatory bone loss where osteoclast activation leads to bone degradation. We show that under inflammatory conditions, skin-resident cells such as keratinocytes, γδ T cells, and innate lymphoid cells were able to express IL-17A, which acted systemically to inhibit osteoblast and osteocyte function by a mechanism involving Wnt signaling. IL-17A led to decreased Wnt signaling in vitro, and importantly, pharmacological blockade of IL-17A rescued Wnt target gene expression and bone formation in vivo. These data provide a mechanism where IL-17A affects bone formation by regulating Wnt signaling in osteoblasts and osteocytes. This study suggests that using IL-17A blocking agents in psoriasis could be beneficial against bone loss in these patients. PMID:27089206

  5. ATF4 mediation of NF1 functions in osteoblast reveals a nutritional basis for congenital skeletal dysplasiae.

    Science.gov (United States)

    Elefteriou, Florent; Benson, M Douglas; Sowa, Hideaki; Starbuck, Michael; Liu, Xiuyun; Ron, David; Parada, Luis F; Karsenty, Gerard

    2006-12-01

    The transcription factor ATF4 enhances bone formation by favoring amino acid import and collagen synthesis in osteoblasts, a function requiring its phosphorylation by RSK2, the kinase inactivated in Coffin-Lowry Syndrome. Here, we show that in contrast, RSK2 activity, ATF4-dependent collagen synthesis, and bone formation are increased in mice lacking neurofibromin in osteoblasts (Nf1(ob)(-/-) mice). Independently of RSK2, ATF4 phosphorylation by PKA is enhanced in Nf1(ob)(-/-) mice, thereby increasing Rankl expression, osteoclast differentiation, and bone resorption. In agreement with ATF4 function in amino acid transport, a low-protein diet decreased bone protein synthesis and normalized bone formation and bone mass in Nf1(ob)(-/-) mice without affecting other organ weight, while a high-protein diet overcame Atf4(-/-) and Rsk2(-/-) mice developmental defects, perinatal lethality, and low bone mass. By showing that ATF4-dependent skeletal dysplasiae are treatable by dietary manipulations, this study reveals a molecular connection between nutrition and skeletal development. PMID:17141628

  6. Osteogenic gene expression of murine osteoblastic (MC3T3-E1) cells under cyclic tension

    Science.gov (United States)

    Kao, C. T.; Chen, C. C.; Cheong, U.-I.; Liu, S. L.; Huang, T. H.

    2014-08-01

    Low-level laser therapy (LLLT) can promote cell proliferation. The remodeling ability of the tension side of orthodontic teeth affects post-orthodontic stability. The purpose of the present study was to investigate the osteogenic effects of LLLT on osteoblast-like cells treated with a simulated tension system that provides a mechanical tension regimen. Murine osteoblastic (MC3T3-E1) cells were cultured in a Flexcell strain unit with programmed loads of 12% elongation at a frequency of 0.5 Hz for 24 and 48 h. The cultured cells were treated with a low-level diode laser using powers of 5 J and 10 J. The proliferation of MC3T3-E1 cells was determined using the Alamar Blue assay. The expression of osteogenic genes (type I collagen (Col-1), osteopontin (OPN), osteocalcin (OC), osteoprotegerin (OPG), receptor activator of nuclear factor kappa B ligand (RANKL), bone morphologic protein (BMP-2), and bone morphologic protein (BMP-4)) in MC3T3-E1 cells was analyzed using reverse transcription polymerase chain reaction (RT-PCR). The data were analyzed using one-way analysis of variance. The proliferation rate of tension-cultured MC3T3-E1 cells under 5 J and 10 J LLLT increased compared with that of the control group (p LLLT group. Osteogenic genes (Col-1, OC, OPG and BMP-2) were significantly expressed in the MC3T3-E1 cells treated with 5 J and 10 J LLLT (p LLLT in tension-cultured MC3T3-E1 cells showed synergistic osteogenic effects, including increases in cell proliferation and Col-1, OPN, OC, OPG and BMP-2 gene expression. LLLT might be beneficial for bone remodeling on the tension side of orthodontics.

  7. Evaluation of epigallocatechin-3-gallate (EGCG) cross-linked collagen membranes and concerns on osteoblasts.

    Science.gov (United States)

    Chu, Chenyu; Deng, Jia; Xiang, Lin; Wu, Yingying; Wei, Xiawei; Qu, Yili; Man, Yi

    2016-10-01

    Collagen membranes have ideal biological and mechanical properties for supporting infiltration and proliferation of osteoblasts and play a vital role in guided bone regeneration (GBR). However, pure collagen can lead to inflammation, resulting in progressive bone resorption. Therefore, a method for regulating the level of inflammatory cytokines at surgical sites is paramount for the healing process. Epigallocatechin-3-gallate (EGCG) is a component extracted from green tea with numerous biological activities including an anti-inflammatory effect. Herein, we present a novel cross-linked collagen membrane containing different concentrations of EGCG (0.0064%, 0.064%, and 0.64%) to regulate the level of inflammatory factors secreted by pre-osteoblast cells; improve cell proliferation; and increase the tensile strength, wettability, and thermal stability of collagen membranes. Scanning electron microscope images show that the surfaces of collagen membranes became smoother and the collagen fiber diameters became larger with EGCG treatment. Measurement of the water contact angle demonstrated that introducing EGCG improved membrane wettability. Fourier transform infrared spectroscopy analyses indicated that the backbone of collagen was intact, and the thermal stability was significant improved in differential scanning calorimetry. The mechanical properties of 0.064% and 0.64% EGCG-treated collagen membranes were 1.5-fold greater than those of the control. The extent of cross-linking was significantly increased, as determined by a 2,4,6-trinitrobenzenesulfonic acid solution assay. The Cell Counting Kit-8 (CCK-8) and live/dead assays revealed that collagen membrane cross-linked by 0.0064% EGCG induced greater cell proliferation than pure collagen membranes. Additionally, real-time polymerase chain reaction and enzyme-linked immunosorbent assay results showed that EGCG significantly affected the production of inflammatory factors secreted by MC3T3-E1 cells. Taken together, our

  8. Momentary Affective States Are Associated with Momentary Volume, Prospective Trends, and Fluctuation of Daily Physical Activity

    Science.gov (United States)

    Kanning, Martina K.; Schoebi, Dominik

    2016-01-01

    Several interventions aiming to enhance physical activity in everyday life showed mixed effects. Affective constructs are thought to potentially support health behavior change. However, little is known about within-subject associations between momentary affect and subsequent physical activity in everyday life. This study analyzed the extent to which three dimensions of affective states (valence, calmness, and energetic arousal) were associated with different components of daily activity trajectories. Sixty-five undergraduates’ students (Age: M = 24.6; SD = 3.2; females: 57%) participated in this study. Physical activity was assessed objectively through accelerometers during 24 h. Affective states assessments were conducted randomly every 45 min using an e-diary with a six-item mood scale that was especially designed for ambulatory assessment. We conducted three-level multi-level analyses to investigate the extent to which momentary affect accounted for momentary volume, prospective trends, and stability vs. fluctuation of physical activity in everyday life. All three affect dimensions were significantly associated with momentary activity volumes and prospective trends over 45 min periods. Physical activity didn’t fluctuate freely, but featured significant autocorrelation across repeated measurements, suggesting some stability of physical activity across 5-min assessments. After adjusting for the autoregressive structure in physical activity assessments, only energetic arousal remained a significant predictor. Feeling energized and awake was associated with an increased momentary volume of activity and initially smaller but gradually growing decreases in subsequent activity within the subsequent 45 min. Although not related to trends in physical activity, higher valence predicted lower stability in physical activity across subsequent 45 min, suggesting more short-term fluctuations in daily activity the more participants reported positive affective valence. The

  9. Porous hydroxyapatite and biphasic calcium phosphate ceramics promote ectopic osteoblast differentiation from mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Lingli Zhang, Nobutaka Hanagata, Megumi Maeda, Takashi Minowa, Toshiyuki Ikoma, Hongsong Fan and Xingdong Zhang

    2009-01-01

    Full Text Available Because calcium phosphate (Ca–P ceramics have been used as bone substitutes, it is necessary to investigate what effects the ceramics have on osteoblast maturation. We prepared three types of Ca–P ceramics with different Ca–P ratios, i.e. hydroxyapatite (HA, beta-tricalcium phosphate (β-TCP, and biphasic calcium phosphate (BCP ceramics with dense-smooth and porous structures. Comprehensive gene expression microarray analysis of mouse osteoblast-like cells cultured on these ceramics revealed that porous Ca–P ceramics considerably affected the gene expression profiles, having a higher potential for osteoblast maturation. In the in vivo study that followed, porous Ca–P ceramics were implanted into rat skeletal muscle. Sixteen weeks after the implantation, more alkaline-phosphatase-positive cells were observed in the pores of hydroxyapatite and BCP, and the expression of the osteocalcin gene (an osteoblast-specific marker in tissue grown in pores was also higher in hydroxyapatite and BCP than in β-TCP. In the pores of any Ca–P ceramics, 16 weeks after the implantation, we detected the expressions of marker genes of the early differentiation stage of chondrocytes and the complete differentiation stage of adipocytes, which originate from mesenchymal stem cells, as well as osteoblasts. These marker gene expressions were not observed in the muscle tissue surrounding the implanted Ca–P ceramics. These observations indicate that porous hydroxyapatite and BCP had a greater potential for promoting the differentiation of mesenchymal stem cells into osteoblasts than β-TCP.

  10. Asporin competes with decorin for collagen binding, binds calcium and promotes osteoblast collagen mineralization

    DEFF Research Database (Denmark)

    Kalamajski, Sebastian; Aspberg, Anders; Lindblom, Karin;

    2009-01-01

    The interactions of the ECM (extracellular matrix) protein asporin with ECM components have previously not been investigated. Here, we show that asporin binds collagen type I. This binding is inhibited by recombinant asporin fragment LRR (leucine-rich repeat) 10-12 and by full-length decorin, but...... not by biglycan. We demonstrate that the polyaspartate domain binds calcium and regulates hydroxyapatite formation in vitro. In the presence of asporin, the number of collagen nodules, and mRNA of osteoblastic markers Osterix and Runx2, were increased. Moreover, decorin or the collagen-binding asporin...... fragment LRR 10-12 inhibited the pro-osteoblastic activity of full-length asporin. Our results suggest that asporin and decorin compete for binding to collagen and that the polyaspartate in asporin directly regulates collagen mineralization. Therefore asporin has a role in osteoblast-driven collagen...

  11. Cultures of human embryonic osteoblasts. A new in vitro model for biocompatibility studies.

    Science.gov (United States)

    Riccio, V; Della Ragione, F; Marrone, G; Palumbo, R; Guida, G; Oliva, A

    1994-11-01

    Cell populations derived from human embryonic bone were isolated according to the ability of osteoblasts to migrate from bone onto glass fragments. Morphologic and biochemical assays showed (1) osteoblast-like appearance; (2) elevated alkaline phosphatase 1,25(OH)2D3 responsive activity associated with plasma membranes and matrix vesicles; (3) production of a thick extracellular matrix, mainly composed of Type I collagen, which mineralized in the presence of 10 mM beta-glycerophosphate; and (4) higher growth rate and viability when compared with their mature counterpart. Cultures of embryonic cells were challenged with particles of several biomaterials, and their effects on morphology, vitality, and osteogenic capacity of the cultured cells were tested. Stainless steel, titanium alloy, Co-Cr-Mo alloy (vitallium), carbon fiber-reinforced polybutylene terephtalate, ultra-high molecular weight polyethylene, ceramic, calcium phosphate, and hydroxyapatite did not exert any significant deleterious effects on the cultured human osteoblasts. PMID:7955704

  12. Taurine inhibits serum deprivation-induced osteoblast apoptosis via the taurine transporter/ERK signaling pathway

    OpenAIRE

    Lei-Yi Zhang; Yue-Ying Zhou; Fei Chen; Bing Wang; Jing Li; You-Wen Deng; Wei-Dong Liu; Zheng-Guang Wang; Ya-Wei Li; Dong-Zhe Li; Guo-Hua Lv; Bang-Liang Yin

    2011-01-01

    Taurine has positive effects on bone metabolism. However, the effects of taurine on osteoblast apoptosis in vitro have not been reported. The aim of this study was to investigate the activity of taurine on apoptosis of mouse osteoblastic MC3T3-E1 cells. The data showed that 1, 5, 10, or 20 mM taurine resulted in 16.7, 34.2, 66.9, or 63.75% reduction of MC3T3-E1 cell apoptosis induced by the serum deprivation (serum-free α-MEM), respectively. Taurine (1, 5, or 10 mM) also reduced cytochrome c ...

  13. Effect of the neridronate on RANKL release in differentiated primary human osteoblasts

    Directory of Open Access Journals (Sweden)

    Vanessa Nicolin

    2011-03-01

    Full Text Available Bisphosphonates are important inhibitors of bone resorp- tion and widely used clinically to treat osteoporosis, metabolic bone diseases and other orthopaedic disorders . Inhibiting osteoclasts via the mevalonate pathway is recognized as the primary mechanism of its inhibitory action. Recent evidence suggests that bisphosphonates may regulate essential signaling molecules involved in osteoclastogenesis such as RANKL (receptor activator of NF-kB ligand which are synthesized by osteoblasts. In this report we have investigated into the neridronate-osteoblast interactions in modulating essential signaling molecule such as RANKL.

  14. Osteoblast-Specific Transcription Factor Osterix Increases Vitamin D Receptor Gene Expression in Osteoblasts

    OpenAIRE

    Zhang, Chi; Tang, Wanjin; LI Yang; Yang, Fan; Dowd, Diane R.; MacDonald, Paul N.

    2011-01-01

    Osterix (Osx) is an osteoblast-specific transcription factor required for osteoblast differentiation from mesenchymal stem cells. In Osx knock-out mice, no bone formation occurs. The vitamin D receptor (VDR) is a member of the nuclear hormone receptor superfamily that regulates target gene transcription to ensure appropriate control of calcium homeostasis and bone development. Here, we provide several lines of evidence that show that the VDR gene is a target for transcriptional regulation by ...

  15. Bone development and its relation to fracture repair. The role of mesenchymal osteoblasts and surface osteoblasts

    OpenAIRE

    Shapiro, F

    2008-01-01

    Bone development occurs by two mechanisms: intramembranous bone formation and endochondral bone formation. Bone tissue forms by eventual differentiation of osteoprogenitor cells into either mesenchymal osteoblasts (MOBL), which synthesize woven bone in random orientation, or surface osteoblasts (SOBL), which synthesize bone on surfaces in a well oriented lamellar array. Bone repair uses the same formation patterns as bone development but the specific mechanism of repair is determined by the b...

  16. Nanoscale topography of nanocrystalline diamonds promotes differentiation of osteoblasts.

    Science.gov (United States)

    Kalbacova, M; Rezek, B; Baresova, V; Wolf-Brandstetter, C; Kromka, A

    2009-10-01

    The excellent mechanical, tribological and biochemical properties of diamond coatings are promising for improving orthopedic or stomatology implants. A crucial prerequisite for such applications is an understanding and control of the biological response of the diamond coatings. This study concentrates on the correlation of diamond surface properties with osteoblast behavior. Nanocrystalline diamond (NCD) films (grain size up to 200 nm, surface roughness 20 nm) were deposited on silicon substrates of varying roughnesses (1, 270 and 500 nm) and treated by oxygen plasma to generate a hydrophilic surface. Atomic force microscopy was used for topographical characterization of the films. As a reference surface, tissue culture polystyrene (PS) was used. Scanning electron microscopy and immunofluorescence staining was used to visualize cell morphological features as a function of culture time. Metabolic activity, alkaline phosphatase activity, and calcium and phosphate deposition was also monitored. The results show an enhanced osteoblast adhesion as well as increased differentiation (raised alkaline phosphatase activity and mineral deposition) on NCD surfaces (most significantly on RMS 20 nm) compared to PS. This is attributed mainly to the specific surface topography as well as to the biocompatible properties of diamond. Hence the controlled (topographically structured) diamond coating of various substrates is promising for preparation of better implants, which offer faster colonization by specific cells as well as longer-term stability. PMID:19433140

  17. Osteoblastic bone metastases from renal cell carcinoma

    International Nuclear Information System (INIS)

    RCC accounts for only 2–3% of all cancers. Due to its’ non-specific symptoms disease is often diagnosed in advanced stage. Disseminated RCC frequently produces bone metastases that are almost always highly destructive, hyper vascularized and purely osteolytic. In this article we describe a case of a 71-year old male patient with disseminated osteoblastic bone metastases from renal cell carcinoma (RCC), and present a short review of published literature reporting cases of osteoblastic bone metastases from RCC. Our patient presented with thoracic pain aggravated by movement. He was diagnosed with predominantly osteoblastic bone metastases in the skeleton of thoracic and lumbar vertebra along with metastases in iliac bones, ribs, humerus and clavicles. Initially, origin of bone metastases was unknown, but later a small tumor in patient’s right kidney was identified. Microscopic evaluation of the open bone biopsy showed clear cell RCC with sarcomatoid differentiation. Although, due to its’ rarity, RCC is not included in the primary differential diagnosis in patients with osteoblastic metastases, such rare cases suggest that RCC may be considered in the diagnosis when there no other primary tumor is found

  18. Osteoblastic response to pectin nanocoating on titanium surfaces.

    Science.gov (United States)

    Gurzawska, Katarzyna; Svava, Rikke; Yihua, Yu; Haugshøj, Kenneth Brian; Dirscherl, Kai; Levery, Steven B; Byg, Inge; Damager, Iben; Nielsen, Martin W; Jørgensen, Bodil; Jørgensen, Niklas Rye; Gotfredsen, Klaus

    2014-10-01

    Osseointegration of titanium implants can be improved by organic and inorganic nanocoating of the surface. The aim of our study was to evaluate the effect of organic nanocoating of titanium surface with unmodified and modified pectin Rhamnogalacturonan-Is (RG-Is) isolated from potato and apple with respect to surface properties and osteogenic response in osteoblastic cells. Nanocoatings on titanium surfaces were evaluated by scanning electron microscopy, contact angle measurements, atomic force microscopy, and X-ray photoelectron spectroscopy. The effect of coated RG-Is on cell adhesion, cell viability, bone matrix formation and mineralization was tested using SaOS-2 cells. Nanocoating with pectin RG-Is affected surface properties and in consequence changed the environment for cellular response. The cells cultured on surfaces coated with RG-Is from potato with high content of linear 1.4-linked galactose produced higher level of mineralized matrix compared with control surfaces and surfaces coated with RG-I with low content of linear 1.4-linked galactose. The study showed that the pectin RG-Is nanocoating not only changed chemical and physical titanium surface properties, but also specific coating with RG-Is containing high amount of galactan increased mineralized matrix formation of osteoblastic cells in vitro. PMID:25175196

  19. Compressive forces induce osteogenic gene expression in calvarial osteoblasts.

    Science.gov (United States)

    Rath, Bjoern; Nam, Jin; Knobloch, Thomas J; Lannutti, John J; Agarwal, Sudha

    2008-01-01

    Bone cells and their precursors are sensitive to changes in their biomechanical environment. The importance of mechanical stimuli has been observed in bone homeostasis and osteogenesis, but the mechanisms responsible for osteogenic induction in response to mechanical signals are poorly understood. We hypothesized that compressive forces could exert an osteogenic effect on osteoblasts and act in a dose-dependent manner. To test our hypothesis, electrospun poly(epsilon-caprolactone) (PCL) scaffolds were used as a 3-D microenvironment for osteoblast culture. The scaffolds provided a substrate allowing cell exposure to levels of externally applied compressive force. Pre-osteoblasts adhered, proliferated and differentiated in the scaffolds and showed extensive matrix synthesis by scanning electron microscopy (SEM) and increased Young's modulus (136.45+/-9.15 kPa) compared with acellular scaffolds (24.55+/-8.5 kPa). Exposure of cells to 10% compressive strain (11.81+/-0.42 kPa) resulted in a rapid induction of bone morphogenic protein-2 (BMP-2), runt-related transcription factor 2 (Runx2), and MAD homolog 5 (Smad5). These effects further enhanced the expression of genes and proteins required for extracellular matrix (ECM) production, such as alkaline phosphatase (Akp2), collagen type I (Col1a1), osteocalcin/bone gamma carboxyglutamate protein (OC/Bglap), osteonectin/secreted acidic cysteine-rich glycoprotein (ON/Sparc) and osteopontin/secreted phosphoprotein 1 (OPN/Spp1). Exposure of cell-scaffold constructs to 20% compressive strain (30.96+/-2.82 kPa) demonstrated that these signals are not osteogenic. These findings provide the molecular basis for the experimental and clinical observations that appropriate physical activities or microscale compressive loading can enhance fracture healing due in part to the anabolic osteogenic effects. PMID:18191137

  20. Vibrational force alters mRNA expression in osteoblasts

    Science.gov (United States)

    Tjandrawinata, R. R.; Vincent, V. L.; Hughes-Fulford, M.

    1997-01-01

    Serum-deprived mouse osteoblastic (MC3T3E1) cells were subjected to a vibrational force modeled by NASA to simulate a space shuttle launch (7.83 G rms). The mRNA levels for eight genes were investigated to determine the effect of vibrational force on mRNA expression. The mRNA levels of two growth-related protooncogenes, c-fos and c-myc, were up-regulated significantly within 30 min after vibration, whereas those of osteocalcin as well as transforming growth factor-beta1 were decreased significantly within 3 h after vibration. No changes were detected in the levels of beta-actin, histone H4, or cytoplasmic phospholipase A2 after vibration. No basal levels of cyclooxygenase-2 expression were detected. In addition, the extracellular concentrations of prostaglandin E2 (PGE2), a potent autocrine/paracrine growth factor in bone, were not significantly altered after vibration most likely due to the serum deprivation state of the osteoblasts. In comparison with the gravitational launch profile, vibrational-induced changes in gene expression were greater both in magnitude and number of genes activated. Taken together, these data suggest that the changes in mRNA expression are due to a direct mechanical effect of the vibrational force on the osteoblast cells and not to changes in the local PGE2 concentrations. The finding that launch forces induce gene expression is of utmost importance since many of the biological experiments do not dampen vibrational loads on experimental samples. This lack of dampening of vibrational forces may partially explain why 1-G onboard controls sometimes do not reflect 1-G ground controls. These data may also suggest that scientists use extra ground controls that are exposed to launch forces, have these forces dampened on launched samples, or use facilities such as Biorack that provide an onboard 1-G centrufuge in order to control for space shuttle launch forces.

  1. Familial Vulnerability to ADHD Affects Activity in the Cerebellum in Addition to the Prefrontal Systems

    Science.gov (United States)

    Mulder, Martijn J.; Baeyens, Dieter; Davidson, Matthew C.; Casey, B. J.; Van Den Ban, Els; Van Engeland, Herman; Durston, Sarah

    2008-01-01

    The study examines whether cerebellar systems are sensitive to familial risk for ADHD in addition to frontostriatal circuitry. The results conclude that familial vulnerability to ADHD affects activity in both the prefrontal cortex and cerebellum.

  2. Modulation of osteoblast attachment and growth in vitro by inertial forces

    Science.gov (United States)

    Kacena, Melissa Ann

    1999-11-01

    cultures is minimally, if at all, affected by alterations in inertial environments. However, in sparse cultures about half as many cells are found attached initially. The remaining attached cells appear to multiply and function normally. These results suggest that the effects of spaceflight on bone are thus not likely to be caused by direct intrinsic effects of gravity on single osteoblasts that can be simulated in laboratory experiments in vitro experiments.

  3. Resveratrol inhibits myeloma cell growth, prevents osteoclast formation, and promotes osteoblast differentiation

    DEFF Research Database (Denmark)

    Boissy, Patrice; Andersen, Thomas L; Abdallah, Basem M;

    2005-01-01

    challenge for treating multiple myeloma is discovering drugs targeting not only myeloma cells but also osteoclasts and osteoblasts. Because resveratrol (trans-3,4',5-trihydroxystilbene) is reported to display antitumor activities on a variety of human cancer cells, we investigated the effects of this...... attention as potential drugs for treating multiple myeloma....

  4. Two populations of endochondral osteoblasts with differential sensitivity to Hedgehog signalling

    NARCIS (Netherlands)

    Hammond, Christina Lindsey; Schulte-Merker, Stefan

    2009-01-01

    Hedgehog (Hh) signalling has been implicated in the development of osteoblasts and osteoclasts whose balanced activities are critical for proper bone formation. As many mouse mutants in the Hh pathway are embryonic lethal, questions on the exact effects of Hh signalling on osteogenesis remain. Using

  5. The Effects of Farnesol on Staphylococcus aureus Biofilms and Osteoblasts: An in Vitro Study

    OpenAIRE

    Unnanuntana, Aasis; Bonsignore, Lindsay; Shirtliff, Mark E; Greenfield, Edward M.

    2009-01-01

    Background: Bacterial biofilms play a major role in chronic orthopaedic infections. Recently, farnesol (an antifungal agent) has been shown to express antimicrobial activities against Staphylococcus aureus and Streptococcus mutans. However, the effects of farnesol on the formation of bacterial biofilms on orthopaedic biomaterials and its effects on osteoblasts have not been investigated, to our knowledge, and are therefore the focus of this study.

  6. Carriers in mesenchymal stem cell osteoblast mineralization-State-of-the-art

    DEFF Research Database (Denmark)

    Dahl, Morten; Jørgensen, Niklas Rye; Hørberg, Mette;

    2014-01-01

    , on mineralization and gene expression. ß-tricalcium phosphate (ß-TCP) revealed elevated alkaline phosphatase activity, and calcium-deficient hydroxyapatite a greater gene expression of osteocalcin when seeded with induced MSCs. CONCLUSION: No data are published on titanium used as a carrier in MSC osteoblast...

  7. Physical Activity in Adolescents — Barriers and Impact on Depressed Affect

    OpenAIRE

    Langguth, Nadine

    2016-01-01

    Adolescence is a high-risk period for physical inactivity as well as depressed affect, both related to various short-, mid-, and long-term negative consequences for adolescents' physical and mental health. Therefore, this developmental period is ideally suited for studying the association between change processes of everyday physical activity and change processes of depressed affect within person by applying an intensive longitudinal design. Given that physical activity substantially decrease...

  8. Comparative study on the cellular activities of osteoblast-like cells and new bone formation of anorganic bone mineral coated with tetra-cell adhesion molecules and synthetic cell binding peptide

    OpenAIRE

    Yu, Hyeon-Seok; Noh, Woo-Chang; Park, Jin-Woo; Lee, Jae-Mok; Yang, Dong-Jun; Park, Kwang-Bum; Suh, Jo-Young

    2011-01-01

    Purpose We have previously reported that tetra-cell adhesion molecule (T-CAM) markedly enhanced the differentiation of osteoblast-like cells grown on anorganic bone mineral (ABM). T-CAM comprises recombinant peptides containing the Arg-Gly-Asp (RGD) sequence in the tenth type III domain, Pro-His-Ser-Arg-Asn (PHSRN) sequence in the ninth type III domain of fibronectin (FN), and the Glu-Pro-Asp-Ilu-Met (EPDIM) and Tyr-His (YH) sequence in the fourth fas-1 domain of βig-h3. Therefore, the purpos...

  9. Interleukin-1β, lipocalin 2 and nitric oxide synthase 2 are mechano-responsive mediators of mouse and human endothelial cell-osteoblast crosstalk.

    Science.gov (United States)

    Veeriah, Vimal; Zanniti, Angelo; Paone, Riccardo; Chatterjee, Suvro; Rucci, Nadia; Teti, Anna; Capulli, Mattia

    2016-01-01

    Endothelial cells are spatially close to osteoblasts and regulate osteogenesis. Moreover, they are sensitive to mechanical stimuli, therefore we hypothesized that they are implicated in the regulation of bone metabolism during unloading. Conditioned media from endothelial cells (EC-CM) subjected to simulated microgravity (0.08g and 0.008g) increased osteoblast proliferation and decreased their differentiation compared to unit gravity (1g) EC-CM. Microgravity-EC-CM increased the expression of osteoblast Rankl and subsequent osteoclastogenesis, and induced the osteoblast de-differentiating factor, Lipocalin 2 (Lcn2), whose downregulation recovered osteoblast activity, decreased Rankl expression and reduced osteoclastogenesis. Microgravity-EC-CM enhanced osteoblast NO-Synthase2 (NOS2) and CycloOXygenase2 (COX2) expression. Inhibition of NOS2 or NO signaling reduced osteoblast proliferation and rescued their differentiation. Nuclear translocation of the Lcn2/NOS2 transcription factor, NF-κB, occurred in microgravity-EC-CM-treated osteoblasts and in microgravity-treated endothelial cells, alongside high expression of the NF-κB activator, IL-1β. IL-1β depletion and NF-κB inhibition reduced osteoblast proliferation and rescued differentiation. Lcn2 and NOS2 were incremented in ex vivo calvarias cultured in microgravity-EC-CM, and in vivo tibias and calvarias injected with microgravity-EC-CM. Furthermore, tibias of botulin A toxin-treated and tail-suspended mice, which featured unloading and decreased bone mass, showed higher expression of IL-1β, Lcn2 and Nos2, suggesting their pathophysiologic involvement in endothelial cell-osteoblast crosstalk. PMID:27430980

  10. Contribution of human osteoblasts and macrophages to bone matrix degradation and proinflammatory cytokine release after exposure to abrasive endoprosthetic wear particles.

    Science.gov (United States)

    Jonitz-Heincke, Anika; Lochner, Katrin; Schulze, Christoph; Pohle, Diana; Pustlauk, Wera; Hansmann, Doris; Bader, Rainer

    2016-08-01

    One of the major reasons for failure after total joint arthroplasty is aseptic loosening of the implant. At articulating surfaces, defined as the interface between implant and surrounding bone cement, wear particles can be generated and released into the periprosthetic tissue, resulting in inflammation and osteolysis. The aim of the present study was to evaluate the extent to which osteoblasts and macrophages are responsible for the osteolytic and inflammatory reactions following contact with generated wear particles from Ti‑6Al‑7Nb and Co‑28Cr‑6Mo hip stems. To this end, human osteoblasts and THP‑1 monocytic cells were incubated with the experimentally generated wear particles as well as reference particles (0.01 and 0.1 mg/ml) for 48 h under standard culture conditions. To evaluate the impact of these particles on the two cell types, the release of different bone matrix degrading matrix metalloproteinases (MMPs), tissue inhibitors of MMPs (TIMPs), and relevant cytokines were determined by multiplex enzyme‑linked immunosorbent assays. Following incubation with wear particles, human osteoblasts showed a significant upregulation of MMP1 and MMP8, whereas macrophages reacted with enhanced MMP3, MMP8 and MMP10 production. Moreover, the synthesis of TIMPs 1 and 2 was inhibited. The osteoblasts and macrophages also responded with modified expression of the inflammatory mediators interleukin (IL)‑6, IL‑8, monocyte chemoattractant protein‑1 and vascular endothelial growth factor. These results demonstrate that the release of wear particles affects the release of proinflammatory cytokines and has a negative impact on bone matrix formation during the first 48 h of particle exposure. Human osteoblasts are directly involved in the proinflammatory cascade of bone matrix degradation. The simultaneous activation and recruitment of monocytes/macrophages boosted osteolytic processes in the periprosthetic tissue. By the downregulation of TIMP production and the

  11. MG63 osteoblast-like cells exhibit different behavior when grown on electrospun collagen matrix versus electrospun gelatin matrix.

    Directory of Open Access Journals (Sweden)

    Shiao-Wen Tsai

    Full Text Available Electrospinning is a simple and efficient method of fabricating a non-woven polymeric nanofiber matrix. However, using fluorinated alcohols as a solvent for the electrospinning of proteins often results in protein denaturation. TEM and circular dichroism analysis indicated a massive loss of triple-helical collagen from an electrospun collagen (EC matrix, and the random coils were similar to those found in gelatin. Nevertheless, from mechanical testing we found the Young's modulus and ultimate tensile stresses of EC matrices were significantly higher than electrospun gelatin (EG matrices because matrix stiffness can affect many cell behaviors such as cell adhesion, proliferation and differentiation. We hypothesize that the difference of matrix stiffness between EC and EG will affect intracellular signaling through the mechano-transducers Rho kinase (ROCK and focal adhesion kinase (FAK and subsequently regulates the osteogenic phenotype of MG63 osteoblast-like cells. From the results, we found there was no significant difference between the EC and EG matrices with respect to either cell attachment or proliferation rate. However, the gene expression levels of OPN, type I collagen, ALP, and OCN were significantly higher in MG63 osteoblast-like cells grown on the EC than in those grown on the EG. In addition, the phosphorylation levels of Y397-FAK, ERK1/2, BSP, and OPN proteins, as well as ALP activity, were also higher on the EC than on the EG. We further inhibited ROCK activation with Y27632 during differentiation to investigate its effects on matrix-mediated osteogenic differentiation. Results showed the extent of mineralization was decreased with inhibition after induction. Moreover, there is no significant difference between EC and EG. From the results of the protein levels of phosphorylated Y397-FAK, ERK1/2, BSP and OPN, ALP activity and mineral deposition, we speculate that the mechanism that influences the osteogenic differentiation of MG63

  12. Apoptosis Induced by Zinc Deficiency in Rat Osteoblast: Possible Involvement of Protein Kinase C

    Institute of Scientific and Technical Information of China (English)

    CEN XIAO-BO; WANG RUI-SHU; AND WANG HANG

    1999-01-01

    Rat osteoblasts were isolated from the 21-day fetal rat calvarias. The cells were grown in DMEM plus 10% FBS, and were treated for 24 h. With 10 μmol/L TPEN or 10 μmol/L TPEN supplemented with 10 μmol/L Zn2+ . Apoptosis of osteoblasts were measured by flow cytometry, electron microscopy and DNA fragmentation analyzed by gel electrophoresis. In addition, IP3 production and PKC activity were measured in order to show whether they are involved in apoptosis in osteoblast induced by zinc deficiency. The results showed that 10 μmol/L TPEN could induce apoptosis in osteoblast in 24 h. But cells treated with 10 μmol/L TPEN supplemented with 10 μmol/L Zn2+showed no apoptotic changes in 24 h. TPEN significantly reduced the formation of IP3 and PKC activity after 24 h incubation. No differences were observed between the cells treated with TPEN supplemented with Zn2 + simultaneously and the untreated cells. It can be inferred that apoptosis induced by zinc deficiency may be due to the decreased activity of PKC which is impaired by reduced formation of IP3.

  13. The role of adhesion strength in human mesenchymal stem cell osteoblastic differentiation on biodegradable polymers

    Science.gov (United States)

    Krizan, Sylva Jana

    Human mesenchymal stem cells (hMSC) are promising candidates for promoting bone growth on biodegradable polymer scaffolds however little is known about early hMSC-polymer interactions. Adhesion is highly dynamic and during adhesive reinforcement, numerous proteins form adhesion plaques linking the cell's cytoskeleton with the extracellular environment. These proteins are known to affect cellular function but their role in hMSC differentiation is less clear. Adhesion plaques are associated with adhesive force, still a detachment force of hMSC on polycaprolactone (PCL), poly-lactide-co-glycolide (PLGA) or alginate has never been described or shown to affect downstream function. We demonstrate that hMSC attached to PCL, PLGA and alginate exhibit different adhesion strengths (tau50) as determined by both fluid shear and spinning disk systems, with PLGA demonstrating the greatest tau 50. Elastic modulus and hydrophobicity were characterized for these surfaces and correlated positively with tau50 to an optimum. Attachment studies of hMSC showed that adhesion plateau timespans were independent of cell line and surface but both morphology and focal adhesion expression varied by polymer type. Differentiation studies of hMSC on PLGA and PCL showed a strong association between markers of differentiation (alkaline phosphatase activity and mineral content) and tau50 within polymer groups, but a poor relationship was found between tau50 and differentiation across polymer groups, suggesting that other polymer properties may be important for differentiation. Subsequently, we examined the role of focal adhesion kinase (FAK) and Rho-GTPase (RhoA) on hMSC adhesion and differentiation when plated onto PLGA. hMSC were retrovirally transduced with mutant constructs of FAK and RhoA cDNA. Alternatively, hMSC were treated with Rho-kinase inhibitor, Y27632. Both cells transduced with mutant RhoA or FAK constructs, or those treated with Y27632 displayed aberrant cell morphology and changes

  14. Assessing the osteoblast transcriptome in a model of enhanced bone formation due to constitutive Gs–G protein signaling in osteoblasts

    International Nuclear Information System (INIS)

    G protein-coupled receptor (GPCR) signaling in osteoblasts (OBs) is an important regulator of bone formation. We previously described a mouse model expressing Rs1, an engineered constitutively active Gs-coupled GPCR, under the control of the 2.3 kb Col I promoter. These mice showed a dramatic age-dependent increase in trabecular bone of femurs. Here, we further evaluated the effects of enhanced Gs signaling in OBs on intramembranous bone formation by examining calvariae of 1- and 9-week-old Col1(2.3)/Rs1 mice and characterized the in vivo gene expression specifically occurring in osteoblasts with activated Gs G protein-coupled receptor signaling, at the cellular level rather than in a whole bone. Rs1 calvariae displayed a dramatic increase in bone volume with partial loss of cortical structure. By immunohistochemistry, Osterix was detected in cells throughout the inter-trabecular space while Osteocalcin was expressed predominantly in cells along bone surfaces, suggesting the role of paracrine mediators secreted from OBs driven by 2.3 kb Col I promoter could influence early OB commitment, differentiation, and/or proliferation. Gene expression analysis of calvarial OBs revealed that genes affected by Rs1 signaling include those encoding proteins important for cell differentiation, cytokines and growth factors, angiogenesis, coagulation, and energy metabolism. The set of Gs-GPCRs and other GPCRs that may contribute to the observed skeletal phenotype and candidate paracrine mediators of the effect of Gs signaling in OBs were also determined. Our results identify novel detailed in vivo cellular changes of the anabolic response of the skeleton to Gs signaling in mature OBs. - Highlights: • OB expression of an engineered Gs-coupled receptor dramatically increases bone mass. • We investigated the changes in gene expression in vivo in enhanced OB Gs signaling. • Genes in cell cycle and transcription were increased in enhanced OB Gs signaling. • GPCRs and paracrine

  15. Assessing the osteoblast transcriptome in a model of enhanced bone formation due to constitutive G{sub s}–G protein signaling in osteoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Wattanachanya, Lalita, E-mail: lalita_md@yahoo.com [Endocrine Research Unit, Veterans Affairs Medical Center and Departments of Medicine and Physiology, University of California, San Francisco, CA (United States); Division of Endocrinology and Metabolism, Department of Medicine, Faculty of Medicine, Chulalongkorn University and King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Bangkok (Thailand); Wang, Liping, E-mail: lipingwang05@yahoo.com [Endocrine Research Unit, Veterans Affairs Medical Center and Departments of Medicine and Physiology, University of California, San Francisco, CA (United States); Millard, Susan M., E-mail: susan.millard@mater.uq.edu.au [Endocrine Research Unit, Veterans Affairs Medical Center and Departments of Medicine and Physiology, University of California, San Francisco, CA (United States); Lu, Wei-Dar, E-mail: weidar_lu@yahoo.com [Endocrine Research Unit, Veterans Affairs Medical Center and Departments of Medicine and Physiology, University of California, San Francisco, CA (United States); O’Carroll, Dylan, E-mail: dylancocarroll@gmail.com [Endocrine Research Unit, Veterans Affairs Medical Center and Departments of Medicine and Physiology, University of California, San Francisco, CA (United States); Hsiao, Edward C., E-mail: Edward.Hsiao@ucsf.edu [Division of Endocrinology and Metabolism, Department of Medicine, University of California, San Francisco, CA (United States); Conklin, Bruce R., E-mail: bconklin@gladstone.ucsf.edu [Gladstone Institute of Cardiovascular Disease, San Francisco, CA (United States); Department of Medicine, University of California, San Francisco, CA (United States); Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA (United States); Nissenson, Robert A., E-mail: Robert.Nissenson@ucsf.edu [Endocrine Research Unit, Veterans Affairs Medical Center and Departments of Medicine and Physiology, University of California, San Francisco, CA (United States)

    2015-05-01

    G protein-coupled receptor (GPCR) signaling in osteoblasts (OBs) is an important regulator of bone formation. We previously described a mouse model expressing Rs1, an engineered constitutively active G{sub s}-coupled GPCR, under the control of the 2.3 kb Col I promoter. These mice showed a dramatic age-dependent increase in trabecular bone of femurs. Here, we further evaluated the effects of enhanced G{sub s} signaling in OBs on intramembranous bone formation by examining calvariae of 1- and 9-week-old Col1(2.3)/Rs1 mice and characterized the in vivo gene expression specifically occurring in osteoblasts with activated G{sub s} G protein-coupled receptor signaling, at the cellular level rather than in a whole bone. Rs1 calvariae displayed a dramatic increase in bone volume with partial loss of cortical structure. By immunohistochemistry, Osterix was detected in cells throughout the inter-trabecular space while Osteocalcin was expressed predominantly in cells along bone surfaces, suggesting the role of paracrine mediators secreted from OBs driven by 2.3 kb Col I promoter could influence early OB commitment, differentiation, and/or proliferation. Gene expression analysis of calvarial OBs revealed that genes affected by Rs1 signaling include those encoding proteins important for cell differentiation, cytokines and growth factors, angiogenesis, coagulation, and energy metabolism. The set of G{sub s}-GPCRs and other GPCRs that may contribute to the observed skeletal phenotype and candidate paracrine mediators of the effect of G{sub s} signaling in OBs were also determined. Our results identify novel detailed in vivo cellular changes of the anabolic response of the skeleton to G{sub s} signaling in mature OBs. - Highlights: • OB expression of an engineered G{sub s}-coupled receptor dramatically increases bone mass. • We investigated the changes in gene expression in vivo in enhanced OB G{sub s} signaling. • Genes in cell cycle and transcription were increased in

  16. Vitamin D and gene networks in human osteoblasts

    Directory of Open Access Journals (Sweden)

    JohannesVan Leeuwen

    2014-04-01

    Full Text Available Bone formation is indirectly influenced by 1,25-dihydroxyvitamin D3 (1,25D3 through the stimulation of calcium uptake in the intestine and re-absorption in the kidneys. Direct effects on osteoblasts and bone formation have also been established. The vitamin D receptor (VDR is expressed in osteoblasts and 1,25D3 modifies gene expression of various osteoblast differentiation and mineralization-related genes, such as alkaline phosphatase (ALPL, osteocalcin (BGLAP and osteopontin (SPP1. 1,25D3 is known to stimulate mineralization of human osteoblasts in vitro, and recently it was shown that 1,25D3 induces mineralization via effects in the period preceding mineralization during the pre-mineralization period. For a full understanding of the action of 1,25D3 in osteoblasts it is important to get an integrated network view of the 1,25D3-regulated genes during osteoblast differentiation and mineralization. The current data will be presented and discussed alluding to future studies to fully delineate the 1,25D3 action in osteoblast. Describing and understanding the vitamin D regulatory networks and identifying the dominant players in these networks may help develop novel (personalized vitamin D-based treatments. The following topics will be discussed in this overview: 1 Bone metabolism and osteoblasts, 2 Vitamin D, bone metabolism and osteoblast function, 3 Vitamin D induced transcriptional networks in the context of osteoblast differentiation and bone formation.

  17. Effect of peroxisome proliferator activated receptor γ agonist on the proliferation and differentiation of rat osteoblasts in vitro%过氧化物酶体增殖物活化受体激动剂对大鼠成骨细胞增殖分化的影响

    Institute of Scientific and Technical Information of China (English)

    吴艳; 夏秦

    2011-01-01

    Objective To study the effect of rosiglitazone (RSG), the agonist of peroxisome proliferator activated receptor γ (PPARγ), on the proliferation and differentiation of rat osteoblasts and the related mechanisms. Methods The identification of rat primary osteoblasts was performed by alkaline phosphatase (ALP) staining and mineralized nodules. The 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay and p-nitrophenyl phosphate (PNPP) assay were used to observe the effects of different concentrations of RSG on proliferation and differentiation of the osteoblasts. The reverse transcription polymerase chain reaction (RT-PCR) were used to detect the expression of connective tissue growth factor (CTGF) mRNA. The effects of the different concentrations (0,1,2,5,10 and 20 μmol/L) of RSG on TGF-β1-induced CTGF mRNA expression in osteoblasts were detected. Results (1)Different concentrations of RSG could not change the proliferation of osteoblasts (P>0. 05). (2)Compared with control group, all different concentrations of RSG could suppress ALP activity in osteoblasts (P<0. 01 ). (3) RSG suppressed the osteoblats CTGF mRNA expression induced by TGF-β1 in a dose-dependent manner (P<0. 01). Conclusions In vitro, RSG can inhibit the TGF-β1 induced rat osteoblasts CTGF mRNA expression. RSG may play a potential role in preventing the differentiation of the rat osteoblasts.%目的 研究过氧化物酶体增殖物激活受体(peroxisome proliferator activated receptor,PPARγ)激动剂罗格列酮(RSG)对大鼠成骨细胞增殖分化能力的影响,并探讨其对成骨细胞作用的可能路径.方法 取新生SD大鼠颅骨进行原代培养,经碱性磷酸酶染色和茜素红矿化结节染色检测鉴定为成骨细胞.应用比色法(MTT)法和4-硝基苯基磷酸二钠盐偶氮(PNPP)法检测不同浓度RSG刺激对大鼠成骨细胞增殖和碱性磷酸酶活性的影响.运用荧光定量反转录聚合酶链反应(RT-PCR)检测6

  18. Osteoblast response on co-modified titanium surfaces via anodization and electrospinning

    Energy Technology Data Exchange (ETDEWEB)

    Bayram, Cem [Nanotechnology and Nanomedicine Division, Hacettepe University, Ankara, Beytepe, 06800 (Turkey); Chemistry Department, Aksaray University, Aksaray, 68100 (Turkey); Demirbilek, Murat; Yalçın, Eda [Nanotechnology and Nanomedicine Division, Hacettepe University, Ankara, Beytepe, 06800 (Turkey); Bozkurt, Murat; Doğan, Metin [Orthopaedics and Traumatology Division, Yıldırım Beyazıt University, School of Medicine, Cankaya, 06550 (Turkey); Denkbaş, Emir Baki, E-mail: denkbas@hacettepe.edu.tr [Chemistry Department, Hacettepe University, Ankara, Beytepe, 06800 (Turkey)

    2014-01-01

    Topography plays a key role in osseointegration and surface modifications at the subcellular level, increasing initial cell attachment in the early period. In the past decade, nanosized texture on metal like a nanotube layer and also more recently extracellular matrix like surface modifications – such as polymeric nanofibrils – have been proposed for a better osseointegration in the literature. Here, we investigate two types of nanoscaled modifications alone and together for the first time. We characterized different types of surface modifications morphologically and investigated how they affected osteoblast cells in vitro, in terms of cell adhesion, proliferation, alkaline phosphatase activity and calcium content. We anodized titanium samples with a thickness of 0.127 mm to obtain a nanotubular titania layer and the silk fibroin (SF), as a biocompatible polymeric material, was electrospun onto both anodized and unanodized samples to acquire 4 sample groups. We analyzed the resulting samples morphologically by scanning electron microscopy (SEM). Cell adhesion, proliferation, alkaline phosphatase (ALP) activity and calcium content were evaluated at 3, 7 and 14 days. We found that cell proliferation increased by 70% on the groups having two modifications respect to unmodified titanium and after 7 days, ALP activity and calcium content were 110% and 150%, respectively, higher on surfaces having both surface treatments than that of unmodified group. In conclusion, a nanotube layer and SF nanofibers on a titanium surface enhanced cell attachment and proliferation most. Comodification of titanium surfaces by anodization and SF electrospinning may be useful to enhance osseointegration but it requires in vivo confirmation.

  19. Osteoblast response on co-modified titanium surfaces via anodization and electrospinning

    International Nuclear Information System (INIS)

    Topography plays a key role in osseointegration and surface modifications at the subcellular level, increasing initial cell attachment in the early period. In the past decade, nanosized texture on metal like a nanotube layer and also more recently extracellular matrix like surface modifications – such as polymeric nanofibrils – have been proposed for a better osseointegration in the literature. Here, we investigate two types of nanoscaled modifications alone and together for the first time. We characterized different types of surface modifications morphologically and investigated how they affected osteoblast cells in vitro, in terms of cell adhesion, proliferation, alkaline phosphatase activity and calcium content. We anodized titanium samples with a thickness of 0.127 mm to obtain a nanotubular titania layer and the silk fibroin (SF), as a biocompatible polymeric material, was electrospun onto both anodized and unanodized samples to acquire 4 sample groups. We analyzed the resulting samples morphologically by scanning electron microscopy (SEM). Cell adhesion, proliferation, alkaline phosphatase (ALP) activity and calcium content were evaluated at 3, 7 and 14 days. We found that cell proliferation increased by 70% on the groups having two modifications respect to unmodified titanium and after 7 days, ALP activity and calcium content were 110% and 150%, respectively, higher on surfaces having both surface treatments than that of unmodified group. In conclusion, a nanotube layer and SF nanofibers on a titanium surface enhanced cell attachment and proliferation most. Comodification of titanium surfaces by anodization and SF electrospinning may be useful to enhance osseointegration but it requires in vivo confirmation.

  20. Insights into the Alteration of Osteoblast Mechanical Properties upon Adhesion on Chitosan

    Directory of Open Access Journals (Sweden)

    Antonia G. Moutzouri

    2014-01-01

    Full Text Available Cell adhesion on substrates is accompanied by significant changes in shape and cytoskeleton organization, which affect subsequent cellular and tissue responses, determining the long-term success of an implant. Alterations in osteoblast stiffness upon adhesion on orthopaedic implants with different surface chemical composition and topography are, thus, of central interest in the field of bone implant research. This work aimed to study the mechanical response of osteoblasts upon adhesion on chitosan-coated glass surfaces and to investigate possible correlations with the level of adhesion, spreading, and cytoskeleton reorganization. Using the micropipette aspiration technique, the osteoblast elastic modulus was found higher on chitosan-coated than on uncoated control substrates, and it was found to increase in the course of spreading for both substrates. The cell-surface contact area was measured throughout several time points of adhesion to quantify cell spreading kinetics. Significant differences were found between chitosan and control surfaces regarding the response of cell spreading, while both groups displayed a sigmoidal kinetical behavior with an initially elevated spreading rate which stabilizes in the second hour of attachment. Actin filament structural changes were confirmed after observation with confocal microscope. Biomaterial surface modification can enhance osteoblast mechanical response and induce favorable structural organization for the implant integration.

  1. Pre-adsorbed type-I collagen structure-dependent changes in osteoblastic phenotype

    International Nuclear Information System (INIS)

    Type-I collagen is the most abundant extracellular matrix in bones and modulates various functions of osteoblasts. We prepared two different structures of type-I collagen on tissue culture grade polystylene (TCPS) surfaces, one is feltwork structure of filamentous molecules from acid solutions (ACs) and the other is network structure of fibrils from neutral solutions (NCs), to examine effects of the structures on the maturation process of osteoblast-like cells. No significant differences of cell proliferation were observed between TCPS and ACs, but NCs delayed the proliferation. In initial cell attachment, the cells on ACs had tense lamellipodia with sharp tips, while those on NCs had loose lamellipodia. No detectable differences in levels of expressed integrin α2- and α5-subunits were observed between the structures. Although the matrix mineralization in NCs was also delayed in comparison with TCPS and ACs, fully mineralized levels in NCs were the same as those of TCPS and ACs. In addition, although we examined the effects of densities of pre-adsorbed collagen molecules on osteoblast maturation, the effects were less serious than those of the structures. This study suggests that the structures of collagen affect proliferation and mineralization of osteoblast-like cells

  2. Puerarin Suppress Apoptosis of Human Osteoblasts via ERK Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Ling-juan Liu

    2013-01-01

    Full Text Available Puerarin, the main isoflavone glycoside extracted from Radix Puerariae, is an isoflavone traditional Chinese herb. Previous studies have demonstrated that puerarin could regulate osteoblast proliferation and differentiation to promote bone formation. However, the effect of puerarin on the process of human osteoblasts (hOBs apoptosis is still unclear. In this study, we detected the function of puerarin on serum-free-induced cell apoptosis using ELISA and TUNEL arrays and then found that the mortality of hOBs was significantly decreased after exposure to 10−10–10−6 M puerarin and reached the maximal antiapoptotic effect at the concentration of 10−8 M. In addition, compared with the control group, puerarin notably increased the Bcl-2 protein levels while it decreased the Bax protein levels in the hOBs in a dose-dependent way. 10−7 M puerarin decreased the Bax/Bcl-2 ratio with a maximal decrease to 0.08. Moreover, puerarin activated ERK signaling pathways in hOBs, and the antiapoptotic effect induced by puerarin was abolished by incubation of ERK inhibitor PD98059. Similarly, the estrogen receptor antagonist ICI182780 also suppressed the inhibitory effect of puerarin on hOBs apoptosis. In conclusion, puerarin could prevent hOBs apoptosis via ERK signaling pathway, which might be effective in providing protection against bone loss and bone remolding associated with osteoporosis.

  3. Different titanium surfaces modulate the bone phenotype of SaOS-2 osteoblast-like cells

    Directory of Open Access Journals (Sweden)

    L Postiglione

    2009-06-01

    Full Text Available Commercially pure titanium implants presenting a relatively smooth, machined surface or a roughened endosseous surface show a large percentage of clinical success. Surface properties of dental implants seem to affect bone cells response. Implant topography appears to modulate cell growth and differentiation of osteoblasts affecting the bone healing around the titanium implant. The aim of the present study was to examine the effects of 1cm diameter and 1mm thick titanium disks on cellular morphology, adhesion and bone phenotypic expression of human osteoblast-like cells, SaOS-2. SaOS-2 cells were cultured on commercially 1 cm pure titanium disks with three different surface roughness: smooth (S, sandblasted (SB and titanium plasma sprayed (TPS. Differences in the cellular morphology were found when they were grown on the three different surfaces. An uniform monolayer of cells recovered the S surface, while clusters of multilayered irregularly shaped cells were distributed on the rough SB and TPS surfaces. The adhesion of SaOS-2 cells, as measured after 3h of culture, was not affected by surface roughness. ECM components such as Collagen I (CoI, Fibronectin (FN, Vitronectin (VN and Tenascin (TN were secreted and organized only on the SB and TPS surfaces while they remained into the cytoplasm on the S surfaces. Osteopontin and BSP-II were largely detected on the SB and TPS surfaces, while only minimal production was observed on the S ones. These data show that titanium surface roughness affects bone differentiation of osteoblast like-cells, SaOS-2, indicating that surface properties may be able to modulate the osteoblast phenotype. These observations also suggest that the bone healing response around dental implants can be affected by surface topography.

  4. Effects of bioglass powders with and without mesoporous structures on fibroblast and osteoblast responses

    Energy Technology Data Exchange (ETDEWEB)

    Shih, Chi-Jen, E-mail: cjshih@kmu.edu.tw [Department of Fragrance and Cosmetic Science, College of Pharmacy, Kaohsiung Medical University, 100 Shi-Chuan 1st Road, Kaohsiung 80708, Taiwan (China); Lu, Pei-Shan [Department of Fragrance and Cosmetic Science, College of Pharmacy, Kaohsiung Medical University, 100 Shi-Chuan 1st Road, Kaohsiung 80708, Taiwan (China); Hsieh, Chih-Hsin [Department of Orthopaedics, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China); Chen, Wen-Cheng [Advanced Medical Devices and Composites Laboratory, Department of Fiber and Composite Materials, College of Engineering, Feng Chia University, Taichung 40724, Taiwan (China); Chen, Jian-Chih [Department of Orthopaedics, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China); Department of Orthopaedics, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China)

    2014-09-30

    Highlights: • Fluorescent microscopy images show that BG-M has excellent cellular affinity. • Both the BG and BG-M substrates had positive effects on the proliferation of the osteoblastic cells. • Cells cultured on BG-M had approximately 1.4 times higher proliferation activity. - Abstract: The main objective of this study was to compare the responses of fibroblasts and osteoblasts to bioglass (BG) and bioglass-containing mesoporous structure (BG-M) powders. The BG-M powders exhibited specific surface areas approximately three times larger than those of the BG powders. The formation of a hysteresis loop also signified the presence of mesoporous structures in the BG-M samples; however, a hysteresis loop was not observed for the BG samples, resulting in 1/5 the pore volume of the BG-M samples. The viabilities of the fibroblasts and osteoblasts cultured in media containing the BG-M powders for 1, 2, and 3 days were greater than 90%. Importantly, the results of fluorescent microscopy images show that BG-M has excellent cellular affinity. Both the BG and BG-M substrates had positive effects on the proliferation of the osteoblastic cells. However, cells cultured on BG-M had approximately 1.4 times higher proliferation activity.

  5. Low-intensity pulsed ultrasound regulates proliferation and differentiation of osteoblasts through osteocytes

    International Nuclear Information System (INIS)

    Highlights: ► CM from LIPUS-stimulated osteocytes inhibits proliferation of osteoblasts. ► CM from LIPUS-stimulated osteocytes enhances differentiation of osteoblasts. ► LIPUS stimulates MLO-Y4 cells to secrete PGE2 and NO. -- Abstract: Low-intensity pulsed ultrasound (LIPUS) has been used as a safe and effective modality to enhance fracture healing. As the most abundant cells in bone, osteocytes orchestrate biological activities of effector cells via direct cell-to-cell contacts and by soluble factors. In this study, we have used the osteocytic MLO-Y4 cells to study the effects of conditioned medium from LIPUS-stimulated MLO-Y4 cells on proliferation and differentiation of osteoblastic MC3T3-E1 cells. Conditioned media from LIPUS-stimulated MLO-Y4 cells (LIPUS-Osteocyte-CM) were collected and added on MC3T3-E1 cell cultures. MC3T3-E1 cells cultured in LIPUS-Osteocyte-CM demonstrated a significant inhibition of proliferation and an increased alkaline phosphatase activity. The results of PGE2 and NO assay showed that LIPUS could enhance PGE2 and NO secretion from MLO-Y4 cells at all time points within 24 h after LIPUS stimulation. We conclude that LIPUS regulates proliferation and differentiation of osteoblasts through osteocytes in vitro. Increased secretion of PGE2 from osteocytes may play a role in this effect.

  6. Effects of bioglass powders with and without mesoporous structures on fibroblast and osteoblast responses

    International Nuclear Information System (INIS)

    Highlights: • Fluorescent microscopy images show that BG-M has excellent cellular affinity. • Both the BG and BG-M substrates had positive effects on the proliferation of the osteoblastic cells. • Cells cultured on BG-M had approximately 1.4 times higher proliferation activity. - Abstract: The main objective of this study was to compare the responses of fibroblasts and osteoblasts to bioglass (BG) and bioglass-containing mesoporous structure (BG-M) powders. The BG-M powders exhibited specific surface areas approximately three times larger than those of the BG powders. The formation of a hysteresis loop also signified the presence of mesoporous structures in the BG-M samples; however, a hysteresis loop was not observed for the BG samples, resulting in 1/5 the pore volume of the BG-M samples. The viabilities of the fibroblasts and osteoblasts cultured in media containing the BG-M powders for 1, 2, and 3 days were greater than 90%. Importantly, the results of fluorescent microscopy images show that BG-M has excellent cellular affinity. Both the BG and BG-M substrates had positive effects on the proliferation of the osteoblastic cells. However, cells cultured on BG-M had approximately 1.4 times higher proliferation activity

  7. Osteoblastic response to pectin nanocoating on titanium surfaces

    DEFF Research Database (Denmark)

    Gurzawska, Katarzyna; Svava, Rikke; Yihua, Yu;

    2014-01-01

    Osseointegration of titanium implants can be improved by organic and inorganic nanocoating of the surface. The aim of our study was to evaluate the effect of organic nanocoating of titanium surface with unmodified and modified pectin Rhamnogalacturonan-Is (RG-Is) isolated from potato and apple with...... respect to surface properties and osteogenic response in osteoblastic cells. Nanocoatings on titanium surfaces were evaluated by scanning electron microscopy, contact angle measurements, atomic force microscopy, and X-ray photoelectron spectroscopy. The effect of coated RG-Is on cell adhesion, cell...... viability, bone matrix formation and mineralization was tested using SaOS-2 cells. Nanocoating with pectin RG-Is affected surface properties and in consequence changed the environment for cellular response. The cells cultured on surfaces coated with RG-Is from potato with high content of linear 1.4-linked...

  8. Influence of crystallite size of nanophased hydroxyapatite on fibronectin and osteonectin adsorption and on MC3T3-E1 osteoblast adhesion and morphology.

    Science.gov (United States)

    Ribeiro, N; Sousa, S R; Monteiro, F J

    2010-11-15

    The characteristic topographical features (crystallite dimensions, surface morphology and roughness) of bioceramics may influence the adsorption of proteins relevant to bone regeneration. This work aims at analyzing the influence of two distinct nanophased hydroxyapatite (HA) ceramics, HA725 and HA1000 on fibronectin (FN) and osteonectin (ON) adsorption and MC3T3-E1 osteoblast adhesion and morphology. Both substrates were obtained using the same hydroxyapatite nanocrystals aggregates and applying the sintering temperatures of 725°C and 1000°C, respectively. The two proteins used in this work, FN as an adhesive glycoprotein and ON as a counter-adhesive protein, are known to be involved in the early stages of osteogenesis (cell adhesion, mobility and proliferation). The properties of the nanoHA substrates had an important role in the adsorption behavior of the two studied proteins and clearly affected the MC3T3-E1 morphology, distribution and metabolic activity. HA1000 surfaces presenting slightly larger grain size, higher root-mean-square roughness (Rq), lower surface area and porosity, allowed for higher amounts of both proteins adsorbed. These substrates also revealed increased number of exposed FN cell-binding domains as well as higher affinity for osteonectin. Regarding the osteoblast adhesion results, improved viability and cell number were found for HA1000 surfaces as compared to HA725 ones, independently of the presence or type of adsorbed protein. Therefore the osteoblast adhesion and metabolic activity seemed to be more sensitive to surfaces morphology and roughness than to the type of adsorbed proteins. PMID:20810127

  9. Proliferation of osteoblast cells on nanotubes

    Institute of Scientific and Technical Information of China (English)

    F.WATARI; T.AKASAKA; Xiaoming LI; M.UO; A.YOKOYAMA

    2009-01-01

    Carbon nanotubes (CNT) have a unique structme and feature. In the present study, cell proliferation was performed on the scaffolds of single-walled CNTs (SWCNT), multiwalled CNTs (MWCNT), and on gra-phita, one of the representative isomorphs of pure carbon,for the sake of comparison. Scanning electron microscopy observation of the growth of osteoblast-like cells (Saps2) cultttred on CNTs showed the morphology fully developed for the whole direction, which is different from that extended to one direction on the usual scaffold. Numerous filopodia were grown from cell edge, extended far long and combined with the CNT meshwork. CNTs showed the affinity for collagen and proteins. Proliferated cell numbers are largest on SWCNTs, followed by MWCNTs, and are very low on graphite. This is in good agreement with the sequence in the results of the adsorbed amount of proteins and expression of alkaline phosphatase activity for these scaffolds. The adsorption of protains would be one of the most influential factors to make a contrast difference in cell attachment and proliferation between graphite and CNTs,both of which are isomorphs of carbon and composed of similar graphene sheet crystal structure. In addition, the nanosize meshwork structure with large porosity is another properly responsible for the excellent cell adhesion and growth on CNTs. CNTs could be the favorable materials for biomedical applications.CNTs with different structures and compositions have been synthesized and discovered [3]. Nanomaterials [2-9] and nanocomposites [10-15] may have various effects onliving organisms. In this study, a fundamental study for biomedical application, cell proliferation was performed on various nanotubes (biT), including (1) single-walled CNTs (SWCNT), (2) multiwalled CNTs (MWCNT), and on graphite, an isomorph of CNT, as a comparison.Figure 1 shows the schematic figures of two different crystal structures of carbon: graphite and CNT. Graphite has the layer-by-layer laminated

  10. THE STUDY OF FACTORS AFFECTING THE ACTIVITY OF MEAT ANTIOXIDANT SYSTEM

    OpenAIRE

    Patrakova, I.; Gurinovich, G.

    2015-01-01

    Oxidation of lipids and myoglobin in raw meat are interrelated processes that affect the overall meat quality. The intensity of oxidation processes in meat raw material is regulated by its own antioxidant system (catalase, peroxidase, glutathione, etc.), the activity of which should be considered in the development of new technological solutions. Oxidation of lipids and myoglobin, directly affect the quality and safety of meat products, and reducing of the intensity of these processes contrib...

  11. Curcumin increases rat mesenchymal stem cell osteoblast differentiation but inhibits adipocyte differentiation

    Directory of Open Access Journals (Sweden)

    Qiaoli Gu

    2012-01-01

    Full Text Available Background: Curcumin is a phenolic natural product isolated from the rhizome of Curcuma longa (turmeric and has effects on bone health and fat formation. The bone marrow mesenchymal stem cells (MSCs are multipotent cells capable of differentiating into osteoblasts and adipocytes. Osteoblast differentiation of MSCs can be a result of upregulation of heme oxygenase (HO-1 expression. Curcumin can potently induce HO-1 expression. Objective: The present study describes the effects of curcumin on rat MSC (rMSCs differentiation into osteoblasts and adipocytes. Materials and Methods: Rat bone marrow MSCs were isolated and treated with or without curcumin. Osteoblast differentiation was confirmed and determined by alkaline phosphatase (ALP activity, mineralized nodule formation, the expression of Runx2 (runt-related transcription factor 2 and osteocalcin. Adipocyte differentiation was determined by Oil red O staining and the expression of peroxisome proliferator-activated receptor-γ 2 (PPARγ2 and CCAAT/enhancer-binding protein (C/EBP α. Results: Curcumin increased ALP activity and osteoblast-specific mRNA expression of Runx2 and osteocalcin when rMSCs were cultured in osteogenic medium. In contrast, curcumin decreased adipocyte differentiation and inhibited adipocyte-specific mRNA expression of PPARγ2 and C/EBPα when rMSCs were cultured in adipogenic medium. HO-1 expression was increased during osteogenic differentiation of rMSCs. Conclusions: These findings demonstrate that curcumin can promote osteogenic differentiation of rMSCs and inhibit adipocyte formation. The effect of curcumin on osteogenic differentiation of rMSCs is correlated with HO-1 expression.

  12. Sphingosine kinase-1 mediates androgen-induced osteoblast cell growth.

    Science.gov (United States)

    Martin, Claire; Lafosse, Jean-Michel; Malavaud, Bernard; Cuvillier, Olivier

    2010-01-01

    Herein we report that the lipid kinase sphingosine kinase-1 (SphK1) is instrumental in mediating androgen-induced cell proliferation in osteoblasts. Dihydrotestosterone (DHT) triggered cell growth in steroid-deprived MC3T3 cells, which was associated with a rapid stimulation of SphK1 and activation of both Akt and ERK signaling pathways. This mechanism relied on functional androgen receptor/PI3K/Akt nongenotropic signaling as pharmacological antagonists could block SphK1 stimulation by DHT and its consequences. Finally, SphK1 inhibition not only abrogated DHT-induced ERK activation but also blocked cell proliferation, while ERK inhibition had no impact, suggesting that SphK1 was critical for DHT signaling yet independently of the ERK. PMID:19932089

  13. Sphingosine kinase-1 mediates androgen-induced osteoblast cell growth

    International Nuclear Information System (INIS)

    Herein we report that the lipid kinase sphingosine kinase-1 (SphK1) is instrumental in mediating androgen-induced cell proliferation in osteoblasts. Dihydrotestosterone (DHT) triggered cell growth in steroid-deprived MC3T3 cells, which was associated with a rapid stimulation of SphK1 and activation of both Akt and ERK signaling pathways. This mechanism relied on functional androgen receptor/PI3K/Akt nongenotropic signaling as pharmacological antagonists could block SphK1 stimulation by DHT and its consequences. Finally, SphK1 inhibition not only abrogated DHT-induced ERK activation but also blocked cell proliferation, while ERK inhibition had no impact, suggesting that SphK1 was critical for DHT signaling yet independently of the ERK.

  14. Sphingosine kinase-1 mediates androgen-induced osteoblast cell growth

    Energy Technology Data Exchange (ETDEWEB)

    Martin, Claire [CNRS, Institut de Pharmacologie et de Biologie Structurale, Toulouse F-31000 (France); Universite de Toulouse, UPS, IPBS, Toulouse F-31000 (France); Lafosse, Jean-Michel [CHU Toulouse, Hopital Rangueil, Service d' orthopedie et Traumatologie, Toulouse F-31000 (France); Malavaud, Bernard [CNRS, Institut de Pharmacologie et de Biologie Structurale, Toulouse F-31000 (France); Universite de Toulouse, UPS, IPBS, Toulouse F-31000 (France); CHU Toulouse, Hopital Rangueil, Service d' Urologie et de Transplantation Renale, Toulouse F-31000 (France); Cuvillier, Olivier, E-mail: olivier.cuvillier@ipbs.fr [CNRS, Institut de Pharmacologie et de Biologie Structurale, Toulouse F-31000 (France); Universite de Toulouse, UPS, IPBS, Toulouse F-31000 (France)

    2010-01-01

    Herein we report that the lipid kinase sphingosine kinase-1 (SphK1) is instrumental in mediating androgen-induced cell proliferation in osteoblasts. Dihydrotestosterone (DHT) triggered cell growth in steroid-deprived MC3T3 cells, which was associated with a rapid stimulation of SphK1 and activation of both Akt and ERK signaling pathways. This mechanism relied on functional androgen receptor/PI3K/Akt nongenotropic signaling as pharmacological antagonists could block SphK1 stimulation by DHT and its consequences. Finally, SphK1 inhibition not only abrogated DHT-induced ERK activation but also blocked cell proliferation, while ERK inhibition had no impact, suggesting that SphK1 was critical for DHT signaling yet independently of the ERK.

  15. Dioscin promotes osteoblastic proliferation and differentiation via Lrp5 and ER pathway in mouse and human osteoblast-like cell lines

    OpenAIRE

    Zhang, Chunfang; Peng, Jinyong; Wu, Shan; Jin, Yue; Xia, Fan; Wang, Changyuan; Liu, Kexin; Sun, Huijun; Liu, Mozhen

    2014-01-01

    Background Dioscin, a typical steroid saponin, is isolated from Dioscorea nipponica Makino and Dioscorea zingiberensis Wright. It has estrogenic activity and many studies have also reported that dioscorea plants have an effect in preventing and treating osteoporosis. However, the molecular mechanisms underlying their effect on osteoporosis treatment are poorly understood. Therefore, the present study aims to investigate the mechanism (s) by which dioscin promotes osteoblastic proliferation an...

  16. Affective response to a loved one's pain: insula activity as a function of individual differences.

    Directory of Open Access Journals (Sweden)

    Viridiana Mazzola

    Full Text Available Individual variability in emotion processing may be associated with genetic variation as well as with psychological predispositions such as dispositional affect styles. Our previous fMRI study demonstrated that amygdala reactivity was independently predicted by affective-cognitive styles (phobic prone or eating disorders prone and genotype of the serotonin transporter in a discrimination task of fearful facial expressions. Since the insula is associated with the subjective evaluation of bodily states and is involved in human feelings, we explored whether its activity could also vary in function of individual differences. In the present fMRI study, the association between dispositional affects and insula reactivity has been examined in two groups of healthy participants categorized according to affective-cognitive styles (phobic prone or eating disorders prone. Images of the faces of partners and strangers, in both painful and neutral situations, were used as visual stimuli. Interaction analyses indicate significantly different activations in the two groups in reaction to a loved one's pain: the phobic prone group exhibited greater activation in the left posterior insula. These results demonstrate that affective-cognitive style is associated with insula activity in pain empathy processing, suggesting a greater involvement of the insula in feelings for a certain cohort of people. In the mapping of individual differences, these results shed new light on variability in neural networks of emotion.

  17. Serotonin regulates osteoblast proliferation and function in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Dai, S.Q.; Yu, L.P. [Department of Orthopedic Surgery, The First Affiliated Hospital, Nanjing Medical University, Nanjing, Jiangsu (China); Shi, X. [Department of Obstetrics and Gynecology, The First Affiliated Hospital, Nanjing Medical University, Nanjing, Jiangsu (China); Wu, H. [Emergency Department, The First Affiliated Hospital, Soochow University, Suzhou (China); Shao, P.; Yin, G.Y.; Wei, Y.Z. [Department of Orthopedic Surgery, The First Affiliated Hospital, Nanjing Medical University, Nanjing, Jiangsu (China)

    2014-08-01

    The monoamine serotonin (5-hydroxytryptamine, 5-HT), a well-known neurotransmitter, also has important functions outside the central nervous system. The objective of this study was to investigate the role of 5-HT in the proliferation, differentiation, and function of osteoblasts in vitro. We treated rat primary calvarial osteoblasts with various concentrations of 5-HT (1 nM to 10 µM) and assessed the rate of osteoblast proliferation, expression levels of osteoblast-specific proteins and genes, and the ability to form mineralized nodules. Next, we detected which 5-HT receptor subtypes were expressed in rat osteoblasts at different stages of osteoblast differentiation. We found that 5-HT could inhibit osteoblast proliferation, differentiation, and mineralization at low concentrations, but this inhibitory effect was mitigated at relatively high concentrations. Six of the 5-HT receptor subtypes (5-HT{sub 1A}, 5-HT{sub 1B}, 5-HT{sub 1D}, 5-HT{sub 2A}, 5-HT{sub 2B}, and 5-HT{sub 2C}) were found to exist in rat osteoblasts. Of these, 5-HT{sub 2A} and 5-HT{sub 1B} receptors had the highest expression levels, at both early and late stages of differentiation. Our results indicated that 5-HT can regulate osteoblast proliferation and function in vitro.

  18. Azanitrile Cathepsin K Inhibitors: Effects on Cell Toxicity, Osteoblast-Induced Mineralization and Osteoclast-Mediated Bone Resorption.

    Directory of Open Access Journals (Sweden)

    Zhong-Yuan Ren

    Full Text Available The cysteine protease cathepsin K (CatK, abundantly expressed in osteoclasts, is responsible for the degradation of bone matrix proteins, including collagen type 1. Thus, CatK is an attractive target for new anti-resorptive osteoporosis therapies, but the wider effects of CatK inhibitors on bone cells also need to be evaluated to assess their effects on bone. Therefore, we selected, among a series of synthetized isothiosemicarbazides, two molecules which are highly selective CatK inhibitors (CKIs to test their effects on osteoblasts and osteoclasts.Cell viability upon treatment of CKIs were was assayed on human osteoblast-like Saos-2, mouse monocyte cell line RAW 264.7 and mature mouse osteoclasts differentiated from bone marrow. Osteoblast-induced mineralization in Saos-2 cells and in mouse primary osteoblasts from calvaria, with or without CKIs,; were was monitored by Alizarin Red staining and alkaline phosphatase activity, while osteoclast-induced bone resorption was performed on bovine slices.Treatments with two CKIs, CKI-8 and CKI-13 in human osteoblast-like Saos-2, murine RAW 264.7 macrophages stimulated with RANKL and mouse osteoclasts differentiated from bone marrow stimulated with RANKL and MCSF were found not to be toxic at doses of up to 100 nM. As probed by Alizarin Red staining, CKI-8 did not inhibit osteoblast-induced mineralization in mouse primary osteoblasts as well as in osteoblast-like Saos-2 cells. However, CKI-13 led to a reduction in mineralization of around 40% at 10-100 nM concentrations in osteoblast-like Saos-2 cells while it did not in primary cells. After a 48-hour incubation, both CKI-8 and CKI-13 decreased bone resorption on bovine bone slices. CKI-13 was more efficient than the commercial inhibitor E-64 in inhibiting bone resorption induced by osteoclasts on bovine bone slices. Both CKI-8 and CKI-13 created smaller bone resorption pits on bovine bone slices, suggesting that the mobility of osteoclasts was slowed

  19. Osteoblasts generate an osteogenic microenvironment when grown on surfaces with rough microtopographies

    Directory of Open Access Journals (Sweden)

    Boyan B. D.

    2003-10-01

    Full Text Available Osteoblasts respond to microarchitectural features of their substrate. On smooth surfaces (tissue culture plastic, tissue culture glass, and titanium, the cells attach and proliferate but they exhibit relatively low expression of differentiation markers in monolayer cultures, even when confluent. When grown on microrough Ti surfaces with an average roughness (Ra of 4-7 µm, proliferation is reduced but differentiation is enhanced and in some cases, is synergistic with the effects of surface microtopography. In addition, cells on microrough Ti substrates form hydroxyapatite in a manner that is more typical of bone than do cells cultured on smooth surfaces. Osteoblasts also respond to growth factors and cytokines in a surface-dependent manner. On rougher surfaces, the effects of regulatory factors like 1alpha,25(OH2D3 or 17beta-estradiol are enhanced. The response to the surface is mediated by integrins, which signal to the cell through many of the same mechanisms used by growth factors and hormones. Studies using PEG-modified surfaces indicate that increased differentiation may be related to altered attachment to the surface. When osteoblasts are grown on surfaces with chemistries or microarchitectures that reduce cell attachment and proliferation, and enhance differentiation, the cells tend to increase production of factors like TGF-beta1 that promote osteogenesis while decreasing osteoclastic activity. Thus, on microrough Ti surface, osteoblasts create a microenvironment conducive to new bone formation.

  20. In vitro culture and characterization of alveolar bone osteoblasts isolated from type 2 diabetics

    International Nuclear Information System (INIS)

    In order to understand the mechanisms of poor osseointegration following dental implants in type 2 diabetics, it is important to study the biological properties of alveolar bone osteoblasts isolated from these patients. We collected alveolar bone chips under aseptic conditions and cultured them in vitro using the tissue explants adherent method. The biological properties of these cells were characterized using the following methods: alkaline phosphatase (ALP) chemical staining for cell viability, Alizarin red staining for osteogenic characteristics, MTT test for cell proliferation, enzyme dynamics for ALP contents, radio-immunoassay for bone gla protein (BGP) concentration, and ELISA for the concentration of type I collagen (COL-I) in the supernatant. Furthermore, we detected the adhesion ability of two types of cells from titanium slices using non-specific immunofluorescence staining and cell count. The two cell forms showed no significant difference in morphology under the same culture conditions. However, the alveolar bone osteoblasts received from type 2 diabetic patients had slower growth, lower cell activity and calcium nodule formation than the normal ones. The concentration of ALP, BGP and COL-I was lower in the supernatant of alveolar bone osteoblasts received from type 2 diabetic patients than in that received from normal subjects (P < 0.05). The alveolar bone osteoblasts obtained from type 2 diabetic patients can be successfully cultured in vitro with the same morphology and biological characteristics as those from normal patients, but with slower growth and lower concentration of specific secretion and lower combining ability with titanium than normal ones

  1. Zinc-modified titanium surface enhances osteoblast differentiation of dental pulp stem cells in vitro.

    Science.gov (United States)

    Yusa, Kazuyuki; Yamamoto, Osamu; Takano, Hiroshi; Fukuda, Masayuki; Iino, Mitsuyoshi

    2016-01-01

    Zinc is an essential trace element that plays an important role in differentiation of osteoblasts and bone modeling. This in vitro study aimed to evaluate the osteoblast differentiation of human dental pulp stem cells (DPSCs) on zinc-modified titanium (Zn-Ti) that releases zinc ions from its surface. Based on real-time PCR, alkaline phosphatase (ALP) activity and Western blot analysis data, we investigated osteoblast differentiation of DPSCs cultured on Zn-Ti and controls. DPSCs cultured on Zn-Ti exhibited significantly up-regulated gene expression levels of osteoblast-related genes of type I collagen (Col I), bone morphogenetic protein 2 (BMP2), ALP, runt-related transcription factor 2 (Runx2), osteopontin (OPN), and vascular endothelial growth factor A (VEGF A), as compared with controls. We also investigated extracellular matrix (ECM) mineralization by Alizarin Red S (ARS) staining and found that Zn-Ti significantly promoted ECM mineralization when compared with controls. These findings suggest that the combination of Zn-Ti and DPSCs provides a novel approach for bone regeneration therapy. PMID:27387130

  2. A Receptor Tyrosine Kinase Inhibitor, Dovitinib (TKI-258), Enhances BMP-2-Induced Osteoblast Differentiation In Vitro.

    Science.gov (United States)

    Lee, Yura; Bae, Kyoung Jun; Chon, Hae Jung; Kim, Seong Hwan; Kim, Soon Ae; Kim, Jiyeon

    2016-05-31

    Dovitinib (TKI258) is a small molecule multi-kinase inhibitor currently in clinical phase I/II/III development for the treatment of various types of cancers. This drug has a safe and effective pharmacokinetic/pharmacodynamic profile. Although dovitinib can bind several kinases at nanomolar concentrations, there are no reports relating to osteoporosis or osteoblast differentiation. Herein, we investigated the effect of dovitinib on human recombinant bone morphogenetic protein (BMP)-2-induced osteoblast differentiation in a cell culture model. Dovitinib enhanced the BMP-2-induced alkaline phosphatase (ALP) induction, which is a representative marker of osteoblast differentiation. Dovitinib also stimulated the translocation of phosphorylated Smad1/5/8 into the nucleus and phosphorylation of mitogen-activated protein kinases, including ERK1/2 and p38. In addition, the mRNA expression of BMP-4, BMP-7, ALP, and OCN increased with dovitinib treatment. Our results suggest that dovitinib has a potent stimulating effect on BMP-2-induced osteoblast differentiation and this existing drug has potential for repositioning in the treatment of bone-related disorders. PMID:27025387

  3. Porphyromonas gingivalis decreases osteoblast proliferation through IL-6-RANKL/OPG and MMP-9/TIMPs pathways

    Directory of Open Access Journals (Sweden)

    Le Xuan

    2009-01-01

    Full Text Available Background: Porphyromonas gingivalis, an important periodontal pathogen, is closely associated with inflammatory alveolar bone resorption. This bacterium exerts its pathogenic effect indirectly through multiple virulence factors, such as lipopolysaccharides, fimbriae, and proteases. Another possible pathogenic path may be through a direct interaction with the host′s soft and hard tissues (e.g., alveolar bone, which could lead to periodontitis. Aims and Objectives: The aim of the present study was to investigate the direct effect of live and heat-inactivated P gingivalis on bone resorption, using an in vitro osteoblast culture model. Results: Optical microscopy and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide MTT assay revealed that live P gingivalis induced osteoblast detachment and reduced their proliferation. This effect was specific to live bacteria and was dependent on their concentration. Live P gingivalis increased IL-6 mRNA expression and protein production and downregulated RANKL and OPG mRNA expression. The effect of live P gingivalis on bone resorption was strengthened by an increase in MMP-9 expression and its activity. This increase was accompanied by an increase in TIMP-1 and TIMP-2 mRNA expression and protein production by osteoblasts infected with live P gingivalis. Conclusion: Overall, the results suggest that direct contact of P gingivalis with osteoblasts induces bone resorption through an inflammatory pathway that involves IL-6, RANKL/OPG, and MMP-9/TIMPs.

  4. In vitro culture and characterization of alveolar bone osteoblasts isolated from type 2 diabetics

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Dao-Cai [Department of Implant Dentistry, School of Stomatology, Fourth Military Medical University, Xi' an (China); Department of Stomatology, The 291st Hospital of P.L.A, Baotou (China); Li, De-Hua [Department of Implant Dentistry, School of Stomatology, Fourth Military Medical University, Xi' an (China); Ji, Hui-Cang [Military Sanatorium of Retired Cadres, Baotou (China); Rao, Guo-Zhou [Center of Laboratory, School of Stomatology, Xi' an Jiaotong University, Xi' an (China); Liang, Li-Hua [Department of Implant Dentistry, School of Stomatology, Fourth Military Medical University, Xi' an (China); Ma, Ai-Jie [Xi' an Technology University, Xi' an (China); Xie, Chao; Zou, Gui-Ke; Song, Ying-Liang [Department of Implant Dentistry, School of Stomatology, Fourth Military Medical University, Xi' an (China)

    2012-04-05

    In order to understand the mechanisms of poor osseointegration following dental implants in type 2 diabetics, it is important to study the biological properties of alveolar bone osteoblasts isolated from these patients. We collected alveolar bone chips under aseptic conditions and cultured them in vitro using the tissue explants adherent method. The biological properties of these cells were characterized using the following methods: alkaline phosphatase (ALP) chemical staining for cell viability, Alizarin red staining for osteogenic characteristics, MTT test for cell proliferation, enzyme dynamics for ALP contents, radio-immunoassay for bone gla protein (BGP) concentration, and ELISA for the concentration of type I collagen (COL-I) in the supernatant. Furthermore, we detected the adhesion ability of two types of cells from titanium slices using non-specific immunofluorescence staining and cell count. The two cell forms showed no significant difference in morphology under the same culture conditions. However, the alveolar bone osteoblasts received from type 2 diabetic patients had slower growth, lower cell activity and calcium nodule formation than the normal ones. The concentration of ALP, BGP and COL-I was lower in the supernatant of alveolar bone osteoblasts received from type 2 diabetic patients than in that received from normal subjects (P < 0.05). The alveolar bone osteoblasts obtained from type 2 diabetic patients can be successfully cultured in vitro with the same morphology and biological characteristics as those from normal patients, but with slower growth and lower concentration of specific secretion and lower combining ability with titanium than normal ones.

  5. Osteoblast adhesion to orthopaedic implant alloys: effects of cell adhesion molecules and diamond-like carbon coating.

    Science.gov (United States)

    Kornu, R; Maloney, W J; Kelly, M A; Smith, R L

    1996-11-01

    In total joint arthroplasty, long-term outcomes depend in part on the biocompatibility of implant alloys. This study analyzed effects of surface finish and diamond-like carbon coating on osteoblast cell adhesion to polished titanium-aluminum-vanadium and polished or grit-blasted cobalt-chromium-molybdenum alloys. Osteoblast binding was tested in the presence and absence of the cell adhesion proteins fibronectin, laminin, fibrinogen, and vitronectin and was quantified by measurement of DNA content. Although adherence occurred in serum-free medium, maximal osteoblast binding required serum and was similar for titanium and cobalt alloys at 2 and 12 hours. With the grit-blasted cobalt alloy, cell binding was reduced 48% (p Coating the alloys with diamond-like carbon did not alter osteoblast adhesion, whereas fibronectin pretreatment increased cell binding 2.6-fold (p enhance cell adhesion. These results support the hypothesis that cell adhesion proteins can modify cell binding to orthopaedic alloys. Although osteoblast binding was not affected by the presence of diamond-like carbon, this coating substance may influence other longer term processes, such as bone formation, and deserves further study. PMID:8982128

  6. Aberrant gene expression profiles, during in vitro osteoblast differentiation, of telomerase deficient mouse bone marrow stromal stem cells (mBMSCs)

    DEFF Research Database (Denmark)

    Saeed, H.; Iqtedar, M.

    2015-01-01

    culture. Osteogenic super array at day 10 of osteoblast differentiation revealed that telomerase deficiency strongly affected the osteoblast commitment by down-regulating Runx2, Twist and Vdr - known transcription regulators of osteogenesis. Similarly, in Terc(-/-) BMSCs a marked reduction in other genes...... engaged in various phases of osteoblast differentiation were observed, such as Fgfr2 involved in bone mineralization, Phex and Dmp1 engaged in ossification, and Col11a1 and Col2a1 involved in cartilage condensation. A similar trend was observed for genes involved in osteoblast proliferation (Tgfb1, Fgfr2...... and Pdgfa) and bone mineral metabolism (Col1a1, Col2a1, Col1a2 and Col11a1). More profound changes were observed in genes engaged in extracellular matrix production: Col1a1, Col1a2, Mmp10, Serpinh1 and Col4a1. Conclusion: Taken together, these data suggest that telomerase deficiency causes impairment...

  7. Alteration of membrane phospholipid methylation by adenosine analogs does not affect T lymphocyte activation

    International Nuclear Information System (INIS)

    Membrane phospholipid methylation has been described during activation of various immune cells. Moreover recent data indicated modulation of immune cells functions by adenosine. As S-adenosyl-methionine and S-adenosyl-homocysteine are adenosine analogs and modulators of transmethylation reactions, the effects of SAH and SAM were investigated on membrane phospholipid methylation and lymphocyte activation. SAM was shown to induce the membrane phospholipid methylation as assessed by the 3Hmethyl-incorporation in membrane extract. This effect was inhibited by SAH. In contrast SAM and SAH did not affect the phytohemagglutinin-induced proliferative response of peripheral blood mononuclear cells. SAH neither modified the early internalization of membrane CD3 antigens nor did it prevent the late expression of HLA-DR antigens on lymphocytes activated by phytohemagglutinin. These results indicate that in vitro alteration of phospholipid methylation does not affect subsequent steps of human T lymphocyte activation and proliferation

  8. Factors affecting uptake of an education and physical activity programme for newly diagnosed type 2 diabetes.

    OpenAIRE

    Visram, S.; Bremner, A.S.; Harrington, B.E.; Hawthorne, G

    2008-01-01

    Background: Intensive lifestyle intervention involving weight reduction and moderate physical activity has been shown to help regulate, and even prevent, type 2 diabetes. Aim: This study sought to explore factors affecting uptake of an education and physical activity programme for those diagnosed with type 2 diabetes. Method: Focus group discussions were conducted with individuals who completed the programme and semi-structured interviews were conducted with those who decline...

  9. CES1 genetic variation affects the activation of angiotensin-converting enzyme inhibitors.

    Science.gov (United States)

    Wang, X; Wang, G; Shi, J; Aa, J; Comas, R; Liang, Y; Zhu, H-J

    2016-06-01

    The aim of the study was to determine the effect of carboxylesterase 1 (CES1) genetic variation on the activation of angiotensin-converting enzyme inhibitor (ACEI) prodrugs. In vitro incubation study of human liver, intestine and kidney s9 fractions demonstrated that the ACEI prodrugs enalapril, ramipril, perindopril, moexipril and fosinopril are selectively activated by CES1 in the liver. The impact of CES1/CES1VAR and CES1P1/CES1P1VAR genotypes and diplotypes on CES1 expression and activity on enalapril activation was investigated in 102 normal human liver samples. Neither the genotypes nor the diplotypes affected hepatic CES1 expression and activity. Moreover, among several CES1 nonsynonymous variants studied in transfected cell lines, the G143E (rs71647871) was a loss-of-function variant for the activation of all ACEIs tested. The CES1 activity on enalapril activation in human livers with the 143G/E genotype was approximately one-third of that carrying the 143G/G. Thus, some functional CES1 genetic variants (for example, G143E) may impair ACEI activation, and consequently affect therapeutic outcomes of ACEI prodrugs. PMID:26076923

  10. Identification of microRNAs involved in osteoblast differentiation of murine embryonic stem cells

    OpenAIRE

    Kaniowska, Dorota

    2012-01-01

    Skeletal development requires stringent control of programs for gene activation and suppression in response to physiological cues. There has been a principal focus on the identification of the mechanisms by which a particular cell phenotype is activated. MicroRNAs (miRNAs, miRs) have emerged as key negative regulators of diverse biological and pathological processes, including developmental timing, organogenesis, apoptosis, cell proliferation and differentiation; how they regulate osteoblast ...

  11. Biocompatibility of Poly-ε-caprolactone-hydroxyapatite composite on mouse bone marrow-derived osteoblasts and endothelial cells

    Directory of Open Access Journals (Sweden)

    Wooley Paul H

    2009-02-01

    Full Text Available Abstract Background Tissue-engineered bone may be developed by seeding the cells capable of both osteogenesis and vascularization on biocompatible composite scaffolds. The current study investigated the performance of mice bone marrow-derived osteogenic cells and endothelial cells as seeded on hydroxyapatite (HA and poly-ε-caprolactone (PCL composite scaffolds. Methods Mononuclear cells were induced to osteoblasts and endothelial cells respectively, which were defined by the expression of osteocalcin, alkaline phosphatase (ALP, and deposits of calcium-containing crystal for osteoblasts, or by the expression of vascular endothelial growth factor receptor-2 (VEGFR-2 and von Willebrand factor (vWF, and the formation of a capillary network in Matrigel™ for endothelial cells. Both types of cell were seeded respectively on PCL-HA scaffolds at HA to PCL weight ratio of 1:1, 1:4, or 0:1 and were evaluated using scanning electron microscopy, ALP activity (of osteoblasts and nitric oxide production (of endothelial cells plus the assessment of cell viability. Results The results indicated that HA led to a positive stimulation of osteoblasts viability and ALP activity, while HA showed less influence on endothelial cells viability. An elevated nitric oxide production of endothelial cells was observed in HA-containing group. Conclusion Supplement of HA into PCL improved biocompatible for bone marrow-derived osteoblasts and endothelial cells. The PCL-HA composite integrating with two types of cells may provide a useful system for tissue-engineered bone grafts with vascularization.

  12. Elements of Design-Based Science Activities That Affect Students' Motivation

    Science.gov (United States)

    Jones, Brett D.; Chittum, Jessica R.; Akalin, Sehmuz; Schram, Asta B.; Fink, Jonathan; Schnittka, Christine; Evans, Michael A.; Brandt, Carol

    2015-01-01

    The primary purpose of this study was to examine the ways in which a 12-week after-school science and engineering program affected middle school students' motivation to engage in science and engineering activities. We used current motivation research and theory as a conceptual framework to assess 14 students' motivation through questionnaires,…

  13. Watered depressions as ecological phenomena in regions affected by mining activities

    International Nuclear Information System (INIS)

    This paper presents the results of the importance of mine watered depressions in a landscape affected by mining activities (model localities - Louky nad Olsi, Orlova and Horni Sucha, Karvina district) from an ecological point of view - conservation and formation of wetland and water ecosystems, genetic resources and biodiversity conservation

  14. Osteoblastic response to pectin nanocoating on titanium surfaces

    International Nuclear Information System (INIS)

    Osseointegration of titanium implants can be improved by organic and inorganic nanocoating of the surface. The aim of our study was to evaluate the effect of organic nanocoating of titanium surface with unmodified and modified pectin Rhamnogalacturonan-Is (RG-Is) isolated from potato and apple with respect to surface properties and osteogenic response in osteoblastic cells. Nanocoatings on titanium surfaces were evaluated by scanning electron microscopy, contact angle measurements, atomic force microscopy, and X-ray photoelectron spectroscopy. The effect of coated RG-Is on cell adhesion, cell viability, bone matrix formation and mineralization was tested using SaOS-2 cells. Nanocoating with pectin RG-Is affected surface properties and in consequence changed the environment for cellular response. The cells cultured on surfaces coated with RG-Is from potato with high content of linear 1.4-linked galactose produced higher level of mineralized matrix compared with control surfaces and surfaces coated with RG-I with low content of linear 1.4-linked galactose. The study showed that the pectin RG-Is nanocoating not only changed chemical and physical titanium surface properties, but also specific coating with RG-Is containing high amount of galactan increased mineralized matrix formation of osteoblastic cells in vitro. - Highlights: • Surface nanocoating with plant-derived Rhamnogalacturonan-I (RG-I) is proposed. • Titanium surface became more hydrophilic after RG-Is nanocoating. • RG-Is with high galactose content resulted in high level of mineralized matrix. • RG-I is a new candidate for improvement of bone healing and osseointegration

  15. Osteoblastic response to pectin nanocoating on titanium surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Gurzawska, Katarzyna, E-mail: kagu@sund.ku.dk [Research Center for Ageing and Osteoporosis, Departments of Medicine and Diagnostics, Copenhagen University Hospital Glostrup, Ndr. Ringvej 57, 2600 Glostrup (Denmark); Institute of Odontology, Faculty of Health and Medical Sciences, University of Copenhagen, Nørre Allé 20, 2200 Copenhagen N (Denmark); Svava, Rikke [Department of Plant Environment Sciences, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C (Denmark); Copenhagen Center for Glycomics, Institute for Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen N (Denmark); Yihua, Yu; Haugshøj, Kenneth Brian [Microtechnology and Surface Analysis, Danish Technological Institute, Gregersensvej 8, 2630 Taastrup (Denmark); Dirscherl, Kai [Dansk Fundamental Metrologi A/S, Matematiktorvet 307, 2800 Lyngby (Denmark); Levery, Steven B. [Copenhagen Center for Glycomics, Institute for Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen N (Denmark); Byg, Inge [Department of Plant Environment Sciences, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C (Denmark); Damager, Iben [Novozymes A/S, Krogshoejvej 36, 2880 Bagsvaerd (Denmark); Nielsen, Martin W. [Department of Systems Biology, Technical University of Denmark, Matematiktorvet, Building 301, Kgs. Lyngby DK-2800 (Denmark); Jørgensen, Bodil [Department of Plant Environment Sciences, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C (Denmark); Jørgensen, Niklas Rye [Research Center for Ageing and Osteoporosis, Departments of Medicine and Diagnostics, Copenhagen University Hospital Glostrup, Ndr. Ringvej 57, 2600 Glostrup (Denmark); and others

    2014-10-01

    Osseointegration of titanium implants can be improved by organic and inorganic nanocoating of the surface. The aim of our study was to evaluate the effect of organic nanocoating of titanium surface with unmodified and modified pectin Rhamnogalacturonan-Is (RG-Is) isolated from potato and apple with respect to surface properties and osteogenic response in osteoblastic cells. Nanocoatings on titanium surfaces were evaluated by scanning electron microscopy, contact angle measurements, atomic force microscopy, and X-ray photoelectron spectroscopy. The effect of coated RG-Is on cell adhesion, cell viability, bone matrix formation and mineralization was tested using SaOS-2 cells. Nanocoating with pectin RG-Is affected surface properties and in consequence changed the environment for cellular response. The cells cultured on surfaces coated with RG-Is from potato with high content of linear 1.4-linked galactose produced higher level of mineralized matrix compared with control surfaces and surfaces coated with RG-I with low content of linear 1.4-linked galactose. The study showed that the pectin RG-Is nanocoating not only changed chemical and physical titanium surface properties, but also specific coating with RG-Is containing high amount of galactan increased mineralized matrix formation of osteoblastic cells in vitro. - Highlights: • Surface nanocoating with plant-derived Rhamnogalacturonan-I (RG-I) is proposed. • Titanium surface became more hydrophilic after RG-Is nanocoating. • RG-Is with high galactose content resulted in high level of mineralized matrix. • RG-I is a new candidate for improvement of bone healing and osseointegration.

  16. Dose-dependent effect of estrogen suppresses the osteo-adipogenic transdifferentiation of osteoblasts via canonical Wnt signaling pathway.

    Directory of Open Access Journals (Sweden)

    Bo Gao

    Full Text Available Fat infiltration within marrow cavity is one of multitudinous features of estrogen deficiency, which leads to a decline in bone formation functionality. The origin of this fat is unclear, but one possibility is that it is derived from osteoblasts, which transdifferentiate into adipocytes that produce bone marrow fat. We examined the dose-dependent effect of 17β-estradiol on the ability of MC3T3-E1 cells and murine bone marrow-derived mesenchymal stem cell (BMMSC-derived osteoblasts to undergo osteo-adipogenic transdifferentiation. We found that 17β-estradiol significantly increased alkaline phosphatase activity (P<0.05; calcium deposition; and Alp, Col1a1, Runx2, and Ocn expression levels dose-dependently. By contrast, 17β-estradiol significantly decreased the number and size of lipid droplets, and Fabp4 and PPARγ expression levels during osteo-adipogenic transdifferentiation (P<0.05. Moreover, the expression levels of brown adipocyte markers (Myf5, Elovl3, and Cidea and undifferentiated adipocyte markers (Dlk1, Gata2, and Wnt10b were also affected by 17β-estradiol during osteo-adipogenic transdifferentiation. Western blotting and immunostaining further showed that canonical Wnt signaling can be activated by estrogen to exert its inhibitory effect of osteo-adipogenesis. This is the first study to demonstrate the dose-dependent effect of 17β-estradiol on the osteo-adipogenic transdifferentiation of MC3T3-E1 cells and BMMSCs likely via canonical Wnt signaling. In summary, our results indicate that osteo-adipogenic transdifferentiation modulated by canonical Wnt signaling pathway in bone metabolism may be a new explanation for the gradually increased bone marrow fat in estrogen-inefficient condition.

  17. The Src inhibitor dasatinib accelerates the differentiation of human bone marrow-derived mesenchymal stromal cells into osteoblasts

    International Nuclear Information System (INIS)

    The proto-oncogene Src is an important non-receptor protein tyrosine kinase involved in signaling pathways that control cell adhesion, growth, migration and differentiation. It negatively regulates osteoblast activity, and, as such, its inhibition is a potential means to prevent bone loss. Dasatinib is a new dual Src/Bcr-Abl tyrosine kinase inhibitor initially developed for the treatment of chronic myeloid leukemia. It has also shown promising results in preclinical studies in various solid tumors. However, its effects on the differentiation of human osteoblasts have never been examined. We evaluated the effects of dasatinib on bone marrow-derived mesenchymal stromal cells (MSC) differentiation into osteoblasts, in the presence or absence of a mixture of dexamethasone, ascorbic acid and β-glycerophosphate (DAG) for up to 21 days. The differentiation kinetics was assessed by evaluating mineralization of the extracellular matrix, alkaline phosphatase (ALP) activity, and expression of osteoblastic markers (receptor activator of nuclear factor kappa B ligand [RANKL], bone sialoprotein [BSP], osteopontin [OPN]). Dasatinib significantly increased the activity of ALP and the level of calcium deposition in MSC cultured with DAG after, respectively, 7 and 14 days; it upregulated the expression of BSP and OPN genes independently of DAG; and it markedly downregulated the expression of RANKL gene and protein (decrease in RANKL/OPG ratio), the key factor that stimulates osteoclast differentiation and activity. Our results suggest a dual role for dasatinib in both (i) stimulating osteoblast differentiation leading to a direct increase in bone formation, and (ii) downregulating RANKL synthesis by osteoblasts leading to an indirect inhibition of osteoclastogenesis. Thus, dasatinib is a potentially interesting candidate drug for the treatment of osteolysis through its dual effect on bone metabolism

  18. Sonme Factors that Affect the Free Radical-scavenging Activity of Tea Extracts

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    Some factors that affect the free radical-scavenging activety of two tea extracts were studied in vitro. It was found that concentration of tea extract or heating tea extract or treating with activated carbon and diatomite all had obvious effect on the scavenging activety of green tea extract ,but heating or treating with diaomite had less effect on the scavenging activity of black tea extract. Ascorbic acid, for having synergic effect with tea extracts, could enhance the scavenging activity of tea extracts markedly, and the contrary was cupric ion. Reducing sugars such as fructose and glucose also had some syncrgic effect to tea extracts.

  19. Antifungal activity of different natural dyes against traditional products affected fungal pathogens

    Institute of Scientific and Technical Information of China (English)

    R Mari selvam; AJA Ranjit Singh; K Kalirajan

    2012-01-01

    Objective: In the present study to evaluate the anti fungal activity of natural dyes against traditional products affected fungal pathogens. Methods: Many traditional craft products affected fungal pathogens were isolated using potato dextrose agar medium. The isolated fungus were identified by morphological and microscopically characterization using Alexopolus manual. 50μl of Turmeric, Terminalli, Guava and Henna natural dyes were poured into the wells of the culture plates. If antifungal activity was present on the plates, it was indicated by an inhibition zone surrounding the well containing the natural dye. Result: At a dose level of 50μl of terminalli dye was able to inhibit the growth of all the fungi tested. The absorbance rate of natural dyes analyzed by UV Spectrophotometer. The absorbance rate is high in terminalli (2.266) and turmeric (2.255). Conclusions: Natural dyes were bound with traditional products to give good colour and good antimicrobial activity against isolated fungal pathogens.

  20. Physical activity and affect in elementary school children’s daily lives

    Directory of Open Access Journals (Sweden)

    JanKühnhausen

    2013-07-01

    Full Text Available A positive influence of physical activity (PA on affect has been shown in numerous studies. However, this relationship has not yet been studied in the daily life of children. We present a part of the FLUX study that attempts to contribute to filling that gap. To this end, a proper way to measure PA and affect in the daily life of children is needed. In pre-studies of the FLUX study, we were able to show that affect can be measured in children with self-report items that are answered using smartphones. In the current article, we show that it is feasible to objectively measure children’s PA with accelerometers for a period of several weeks and report descriptive information on the amount of activity of 51 children from 3rd and 4th grade. Additionally, we investigate the influence of daily PA on daily affect in children. Mixed effects models show no effect of PA on any of the four measured dimensions of affect. We discuss that this might be due to effects taking place at shorter time intervals, which can be investigated in future analyses.

  1. Cattle activities affect abundance and activity of nitrifying and denitrifying microbial communities in upland soil

    Czech Academy of Sciences Publication Activity Database

    Chroňáková, Alica; Radl, V.; Čuhel, Jiří; Gattinger, A.; Šimek, Miloslav; Elhottová, Dana; Schloter, M.

    Uppsala : Swedish University of Agriculture Sciences, 2007. [Achievements of COST 856. Denitrification and related aspects. Final meeting of the ESF COST Action 856 /14./. 05.12.2007-08.12.2007, Uppsala] Institutional research plan: CEZ:AV0Z60660521 Keywords : cattle activities * nitrifying and denitrifying microbial communities * upland soil Subject RIV: EH - Ecology, Behaviour

  2. Does cypermethrin affect enzyme activity, respiration rate and walking behavior of the maize weevil (Sitophilus zeamais)?

    Institute of Scientific and Technical Information of China (English)

    Ronnie Von Santos Veloso; Eliseu José G.Pereira; Raul Narciso C.Guedes; Maria Goreti A.Oliveira

    2013-01-01

    Insecticides cause a range of sub-lethal effects on targeted insects,which are frequently detrimental to them.However,targeted insects are able to cope with insecticides within sub-lethal ranges,which vary with their susceptibility.Here we assessed the response of three strains of the maize weevil Sitophilus zeamais Motschulsky (Coleoptera:Curculionidae) to sub-lethal exposure to the pyrethoid insecticide cypermethrin.We expected enzyme induction associated with cypermethrin resistance since it would aid the resistant insects in surviving such exposure.Lower respiration rate and lower activity were also expected in insecticide-resistant insects since these traits are also likely to favor survivorship under insecticide exposure.Curiously though,cypermethrin did not affect activity of digestive and energy metabolism enzymes,and even reduced the activity of some enzymes (particularly for cellulase and cysteine-proteinase activity in this case).There was strain variation in response,which may be (partially) related to insecticide resistance in some strains.Sub-lethal exposure to cypermethrin depressed proteolytic and mainly cellulolytic activity in the exposed insects,which is likely to impair their fitness.However,such exposure did not affect respiration rate and walking behavior of the insects (except for the susceptible strain where walking activity was reduced).Walking activity varies with strain and may minimize insecticide exposure,which should be a concern,particularly if associated with (physiological) insecticide resistance.

  3. Studies of Some Parameters Affecting The Efficiency and Accuracy of The Neutron Activation Analysis Technique

    International Nuclear Information System (INIS)

    The present studies deal with the optimum physical conditions which seriously affect the neutron activation analysis technique efficiency. An experimental work for the efficiency calibration of hyper pure germanium detectors especially for environmental studies is presented. This work showed that the tested parameters, under consideration, distance, mass and measured time, reveal a significant effect on the obtained data. These results, intern, affect the accuracy of the measurements. Further work on the test of other parameters is planned in our laboratory using special treatments and applying special computer programs

  4. Does methamphetamine affect bone metabolism?

    International Nuclear Information System (INIS)

    There is a close relationship between the central nervous system activity and bone metabolism. Therefore, methamphetamine (METH), which stimulates the central nervous system, is expected to affect bone turnover. The aim of this study was to investigate the role of METH in bone metabolism. Mice were divided into 3 groups, the control group receiving saline injections, and the 5 and 10 mg/kg METH groups (n = 6 in each group). All groups received an injection of saline or METH every other day for 8 weeks. Bone mineral density (BMD) was assessed by X-ray computed tomography. We examined biochemical markers and histomorphometric changes in the second cancellous bone of the left femoral distal end. The animals that were administered 5 mg/kg METH showed an increased locomotor activity, whereas those receiving 10 mg/kg displayed an abnormal and stereotyped behavior. Serum calcium and phosphorus concentrations were normal compared to the controls, whereas the serum protein concentration was lower in the METH groups. BMD was unchanged in all groups. Bone formation markers such as alkaline phosphatase and osteocalcin significantly increased in the 5 mg/kg METH group, but not in the 10 mg/kg METH group. In contrast, bone resorption markers such as C-terminal telopeptides of type I collagen and tartrate-resistant acid phosphatase 5b did not change in any of the METH groups. Histomorphometric analyses were consistent with the biochemical markers data. A significant increase in osteoblasts, especially in type III osteoblasts, was observed in the 5 mg/kg METH group, whereas other parameters of bone resorption and mineralization remained unchanged. These results indicate that bone remodeling in this group was unbalanced. In contrast, in the 10 mg/kg METH group, some parameters of bone formation were significantly or slightly decreased, suggesting a low turnover metabolism. Taken together, our results suggest that METH had distinct dose-dependent effects on bone turnover and that

  5. Multiplicative and Additive Modulation of Neuronal Tuning with Population Activity Affects Encoded Information.

    Science.gov (United States)

    Arandia-Romero, Iñigo; Tanabe, Seiji; Drugowitsch, Jan; Kohn, Adam; Moreno-Bote, Rubén

    2016-03-16

    Numerous studies have shown that neuronal responses are modulated by stimulus properties and also by the state of the local network. However, little is known about how activity fluctuations of neuronal populations modulate the sensory tuning of cells and affect their encoded information. We found that fluctuations in ongoing and stimulus-evoked population activity in primate visual cortex modulate the tuning of neurons in a multiplicative and additive manner. While distributed on a continuum, neurons with stronger multiplicative effects tended to have less additive modulation and vice versa. The information encoded by multiplicatively modulated neurons increased with greater population activity, while that of additively modulated neurons decreased. These effects offset each other so that population activity had little effect on total information. Our results thus suggest that intrinsic activity fluctuations may act as a "traffic light" that determines which subset of neurons is most informative. PMID:26924437

  6. Osteoblast-specific deletion of Pkd2 leads to low-turnover osteopenia and reduced bone marrow adiposity.

    Directory of Open Access Journals (Sweden)

    Zhousheng Xiao

    Full Text Available Polycystin-1 (Pkd1 interacts with polycystin-2 (Pkd2 to form an interdependent signaling complex. Selective deletion of Pkd1 in the osteoblast lineage reciprocally regulates osteoblastogenesis and adipogenesis. The role of Pkd2 in skeletal development has not been defined. To this end, we conditionally inactivated Pkd2 in mature osteoblasts by crossing Osteocalcin (Oc-Cre;Pkd2+/null mice with floxed Pkd2 (Pkd2flox/flox mice. Oc-Cre;Pkd2flox/null (Pkd2Oc-cKO mice exhibited decreased bone mineral density, trabecular bone volume, cortical thickness, mineral apposition rate and impaired biomechanical properties of bone. Pkd2 deficiency resulted in diminished Runt-related transcription factor 2 (Runx2 expressions in bone and impaired osteoblastic differentiation ex vivo. Expression of osteoblast-related genes, including, Osteocalcin, Osteopontin, Bone sialoprotein (Bsp, Phosphate-regulating gene with homologies to endopeptidases on the X chromosome (Phex, Dentin matrix protein 1 (Dmp1, Sclerostin (Sost, and Fibroblast growth factor 23 (FGF23 were reduced proportionate to the reduction of Pkd2 gene dose in bone of Oc-Cre;Pkd2flox/+ and Oc-Cre;Pkd2flox/null mice. Loss of Pkd2 also resulted in diminished peroxisome proliferator-activated receptor γ (PPARγ expression and reduced bone marrow fat in vivo and reduced adipogenesis in osteoblast culture ex vivo. Transcriptional co-activator with PDZ-binding motif (TAZ and Yes-associated protein (YAP, reciprocally acting as co-activators and co-repressors of Runx2 and PPARγ, were decreased in bone of Oc-Cre;Pkd2flox/null mice. Thus, Pkd1 and Pkd2 have coordinate effects on osteoblast differentiation and opposite effects on adipogenesis, suggesting that Pkd1 and Pkd2 signaling pathways can have independent effects on mesenchymal lineage commitment in bone.

  7. Fluid flow induced calcium response in osteoblasts: mathematical modeling.

    Science.gov (United States)

    Su, J H; Xu, F; Lu, X L; Lu, T J

    2011-07-28

    Fluid flow in the bone lacuno-canalicular network can induce dynamic fluctuation of intracellular calcium concentration ([Ca(2+)](i)) in osteoblasts, which plays an important role in bone remodeling. There has been limited progress in the mathematical modeling of this process probably due to its complexity, which is controlled by various factors such as Ca(2+) channels and extracellular messengers. In this study we developed a mathematical model to describe [Ca(2+)](i) response induced by fluid shear stress (SS) by integrating the major factors involved and analyzed the effects of different experimental setups (e.g. [Ca(2+)](i) baseline, pretreatment with ATP). In this model we considered the ATP release process and the activities of multiple ion channels and purinergic receptors. The model was further verified quantitatively by comparing the simulation results with experimental data reported in literature. The results showed that: (i) extracellular ATP concentration has more significant effect on [Ca(2+)](i) baseline (73% increase in [Ca(2+)](i) with extracellular ATP concentration varying between 0 and 10 μM), as compared to that induced by SS (25% variation in [Ca(2+)](i) with SS varying from 0 to 3.5 Pa); (ii) Pretreatment with ATP-medium results in different [Ca(2+)](i) response as compared to the control group (ATP-free medium) under SS; (iii) Relative [Ca(2+)](i) fluctuation over baseline is more reliable to show the [Ca(2+)](i) response process than the absolute [Ca(2+)](i) response peak. The developed model may improve the experimental design and facilitate our understanding of the mechanotransduction process in osteoblasts. PMID:21665208

  8. Effect of dual delivery of antibiotics (vancomycin and cefazolin) and BMP-7 from chitosan microparticles on Staphylococcus epidermidis and pre-osteoblasts in vitro.

    Science.gov (United States)

    Mantripragada, Venkata P; Jayasuriya, Ambalangodage C

    2016-10-01

    The main aims of this manuscript are to: i) determine the effect of commonly used antibiotics to treat osteoarticular infections on osteoblast viability, ii) study the dual release of the growth factor (BMP-7) and antibiotics (vancomycin and cefazolin) from chitosan microparticles iii) demonstrate the bioactivity of the antibiotics released in vitro on Staphylococcus epidermidis. The novelty of this work is dual delivery of growth factor and antibiotic from the chitosan microparticles in a controlled manner without affecting their bioactivity. Cefazolin and vancomycin have different therapeutic concentrations for their action in vivo and therefore, two different concentrations of the drugs were used. Osteoblast cytotoxicity test concluded that cefazolin concentrations of 50 and 100μg/ml were found to have positive influence on osteoblast proliferation. A significant increase in osteoblast proliferation was observed in the presence of cefazolin and BMP-7 in comparison with BMP-7 alone group; indicating cefazolin might play a role in osteoblast proliferation. On the other hand, vancomycin concentration of 1000μg/ml was found to significantly reduce (p<0.01) osteoblast proliferation in comparison with controls. The microbial study indicated that cefazolin at a minimum concentration of 21.5μg/ml could inhibit ~85% growth of S. epidermidis, whereas vancomycin at a concentration of 30μg/ml was found to inhibit ~80% bacterial growth. PMID:27287137

  9. Nanotopography Directs Mesenchymal Stem Cells to Osteoblast Lineage through Regulation of microRNA-SMAD-BMP-2 Circuit

    Science.gov (United States)

    KATO, ROGERIO B.; ROY, BHASKAR; DE OLIVEIRA, FABIOLA S.; FERRAZ, EMANUELA P.; DE OLIVEIRA, PAULO T.; KEMPER, AUSTIN G.; HASSAN, MOHAMMAD Q.; ROSA, ADALBERTO L.; BELOTI, MARCIO M.

    2016-01-01

    The aim of this study was to investigate if chemically produced nanotopography on titanium (Ti) surface induces osteoblast differentiation of cultured human bone marrow mesenchymal stem cells (hMSCs) by regulating the expression of microRNAs (miRs). It was demonstrated that Ti with nanotopography induces osteoblast differentiation of hMSCs as evidenced by upregulation of osteoblast specific markers compared with untreated (control) Ti at day 4. At this time-point, miR-sequencing analysis revealed that 20 miRs were upregulated (>2 fold) while 20 miRs were downregulated (>3 fold) in hMSCs grown on Ti with nanotopography compared with control Ti. Three miRs, namely miR-4448, -4708 and -4773, which were significantly downregulated (>5 fold) by Ti with nanotopography affect osteoblast differentiation of hMSCs. These miRs that directly target SMAD1 and SMAD4, both key transducers of the bone morphogenetic protein 2 (BMP-2) osteogenic signal, were upregulated by Ti with nanotopography. Overexpression of miR-4448, -4708 and 4773 in MC3T3-E1 pre-osteoblasts noticeably inhibited gene and protein expression of SMAD1 and SMAD4 and therefore repressed the gene expression of key bone markers. Additionally, it was observed that the treatment with BMP-2 displayed a higher osteogenic effect on MC3T3-E1 cells grown on Ti with nanotopography compared with control Ti, suggesting that the BMP-2 signaling pathway was more effective on this surface. Taken together, these results indicate that a complex regulatory network involving a miR-SMAD-BMP-2 circuit governs the osteoblast differentiation induced by Ti with nanotopography. PMID:24619927

  10. Role of gangliosides in the differentiation of human mesenchymal-derived stem cells into osteoblasts and neuronal cells

    Directory of Open Access Journals (Sweden)

    Ghislain Moussavou

    2013-11-01

    Full Text Available Gangliosides are complex glycosphingolipids that are the majorcomponent of cytoplasmic cell membranes, and play a rolein the control of biological processes. Human mesenchymalstem cells (hMSCs have received considerable attention as alternativesources of adult stem cells because of their potentialto differentiate into multiple cell lineages. In this study, we focuson various functional roles of gangliosides in the differentiationof hMSCs into osteoblasts or neuronal cells. A relationshipbetween gangliosides and epidermal growth factor receptor(EGFR activation during osteoblastic differentiation ofhMSCs was observed, and the gangliosides may play a majorrole in the regulation of the differentiation. The roles of gangliosidesin osteoblast differentiation are dependent on the originof hMSCs. The reduction of ganglioside biosynthesis inhibitedthe neuronal differentiation of hMSCs during an earlystage of the differentiation process, and the ganglioside expressioncan be used as a marker for the identification of neuronaldifferentiation from hMSCs. [BMB Reports 2013; 46(11:527-532

  11. SOME IMPORTANT FACTORS AFFECTING EVOLUTION OF ACTIVITY BASED COSTING (ABC SYSTEM IN EGYPTIAN MANUFACTURING FIRMS

    Directory of Open Access Journals (Sweden)

    Karim MAMDOUH ABBAS

    2014-04-01

    Full Text Available The present investigation aims to determine the factors affecting evolution of Activity Based Costing (ABC system in Egyptian case. The study used the survey method to describe and analyze these factors in some Egyptian firms. The population of the study is Egyptian manufacturing firms. Accordingly, the number of received questionnaires was 392 (23 Egyptian manufacturing firms in the first half of 2013. Finally, the study stated some influencing factors for evolution this system (ABC in Egyptian manufacturing firms.

  12. TAM receptors affect adult brain neurogenesis by negative regulation of microglial cell activation1

    OpenAIRE

    Ji, Rui; Tian, Shifu; Lu, Helen J.; LU, QINGJUN; Yan ZHENG; Wang, Xiaomin; Ding, Jixiang; Li, Qiutang; Lu, Qingxian

    2013-01-01

    TAM tyrosine kinases play multiple functional roles including regulation of the target genes important in homeostatic regulation of cytokine receptors or Toll-like receptor-mediated signal transduction pathways. Here, we show that TAM receptors affect adult hippocampal neurogenesis and loss of TAM receptors impair hippocampal neurogenesis, largely attributed to exaggerated inflammatory responses by microglia characterized by increased MAP kinase and NF-κB activation and elevated production of...

  13. Critical Success Factors Affecting e-commerce Activities of Small and Medium Enterprises

    OpenAIRE

    Ozgur Dogerlioglu; Volkan Cosgun

    2012-01-01

    Electronic commerce is redefining business and customer relationships, business processes, even sometimes restructuring the whole industry by providing new distribution channel, new delivery methods, new payment methods and new medium for communication. The aim of this study was to explore the critical success factors that affect e-commerce activities of Small and Medium -sized Enterprises (SMEs). In this study, a model has been developed based on previous researches and a questionnaire...

  14. Compensatory premotor activity during affective face processing in subclinical carriers of a single mutant Parkin allele

    OpenAIRE

    Anders, Silke; Sack, Benjamin; Pohl, Anna; Münte, Thomas; Pramstaller, Peter; Klein, Christine; Binkofski, Ferdinand

    2012-01-01

    Patients with Parkinson's disease suffer from significant motor impairments and accompanying cognitive and affective dysfunction due to progressive disturbances of basal ganglia–cortical gating loops. Parkinson's disease has a long presymptomatic stage, which indicates a substantial capacity of the human brain to compensate for dopaminergic nerve degeneration before clinical manifestation of the disease. Neuroimaging studies provide evidence that increased motor-related cortical activity can ...

  15. Factors affecting hospital stay in psychiatric patients: the role of active comorbidity

    OpenAIRE

    Douzenis, Athanassios; Seretis, Dionysios; Nika, Stella; Nikolaidou, Paraskevi; Papadopoulou, Athanassia; Rizos, Emmanouil N; Christodoulou, Christos; Tsopelas, Christos; Mitchell, Dominic; Lykouras, Lefteris

    2012-01-01

    Background Research on length of stay (LOS) of psychiatric inpatients is an under-investigated issue. In this naturalistic study factors which affect LOS of two groups of patients were investigated, focusing on the impact on LOS of medical comorbidity severe enough to require referral. Methods Active medical comorbidity was quantified using referral as the criterion. The study sample consisted of 200 inpatients with the diagnosis of schizophrenia and 228 inpatients suffering from bipolar diso...

  16. SOME IMPORTANT FACTORS AFFECTING EVOLUTION OF ACTIVITY BASED COSTING (ABC) SYSTEM IN EGYPTIAN MANUFACTURING FIRMS

    OpenAIRE

    Karim MAMDOUH ABBAS

    2014-01-01

    The present investigation aims to determine the factors affecting evolution of Activity Based Costing (ABC) system in Egyptian case. The study used the survey method to describe and analyze these factors in some Egyptian firms. The population of the study is Egyptian manufacturing firms. Accordingly, the number of received questionnaires was 392 (23 Egyptian manufacturing firms) in the first half of 2013. Finally, the study stated some influencing factors for evolution this system (ABC) in Eg...

  17. ATF4 mediation of NF1 functions in osteoblast reveals a nutritional basis for congenital skeletal dysplasiae

    OpenAIRE

    Elefteriou, Florent; Benson, M. Douglas; Sowa, Hideaki; Starbuck, Michael; Liu, Xiuyun; Ron, David; Parada, Luis F.; Karsenty, Gerard

    2006-01-01

    The transcription factor ATF4 enhances bone formation by favoring amino acid import and collagen synthesis in osteoblasts, a function requiring its phosphorylation by RSK2, the kinase inactivated in Coffin-Lowry Syndrome. Here, we show that in contrast, RSK2 activity, ATF4-dependent collagen synthesis, and bone formation are increased in mice lacking neurofibromin in osteoblasts (Nf1ob−/− mice). Independently of RSK2, ATF4 phosphorylation by PKA is enhanced in Nf1ob−/− mice, thereby increasin...

  18. The Degradation Interface of Magnesium Based Alloys in Direct Contact with Human Primary Osteoblast Cells

    OpenAIRE

    Ahmad Agha, Nezha; Willumeit-Römer, Regine; Laipple, Daniel; Luthringer, Bérengère; Feyerabend, Frank

    2016-01-01

    Magnesium alloys have been identified as a new generation material of orthopaedic implants. In vitro setups mimicking physiological conditions are promising for material / degradation analysis prior to in vivo studies however the direct influence of cell on the degradation mechanism has never been investigated. For the first time, the direct, active, influence of human primary osteoblasts on magnesium-based materials (pure magnesium, Mg-2Ag and Mg-10Gd alloys) is studied for up to 14 days. Se...

  19. Pyk2 Regulates Megakaryocyte-Induced Increases in Osteoblast Number and Bone Formation

    OpenAIRE

    Cheng, Ying-Hua; Hooker, R. Adam; NGUYEN, Khanh; Gerard-O’Riley, Rita; Waning, David L.; Chitteti, Brahmananda R; Meijome, Tomas E.; Chua, Hui Lin; Plett, Artur P.; Orschell, Christie M.; Srour, Edward F.; Mayo, Lindsey D.; Pavalko, Fredrick M; Bruzzaniti, Angela; Kacena, Melissa A.

    2013-01-01

    Pre-clinical and clinical evidence from megakaryocyte (MK) related diseases suggest that MKs play a significant role in maintaining bone homeostasis. Findings from our laboratories reveal that MKs significantly increase osteoblast (OB) number through direct MK-OB contact and the activation of integrins. We therefore examined the role of Pyk2, a tyrosine kinase known to be regulated downstream of integrins, in the MK-mediated enhancement of OBs. When OBs were co-cultured with MKs, total Pyk2 l...

  20. The Rho-GEF Kalirin regulates bone mass and the function of osteoblasts and osteoclasts

    OpenAIRE

    Huang, Su; Pierre P. Eleniste; Wayakanon, Kornchanok; Mandela, Prashant; Eipper, Betty A.; Mains, Richard E.; Allen, Matthew R; Bruzzaniti, Angela

    2013-01-01

    Bone homeostasis is maintained by the balance between bone resorption by osteoclasts and bone formation by osteoblasts. Dysregulation in the activity of the bone cells can lead to osteoporosis, a disease characterized by low bone mass and an increase in bone fragility and risk of fracture. Kalirin is a novel GTP-exchange factor protein that has been shown to play a role in cytoskeletal remodeling and dendritic spine formation in neurons. We examined Kalirin expression in skeletal tissue and f...

  1. Parathyroid Hormone Stimulates Phosphatidylethanolamine Hydrolysis by Phospholipase D in Osteoblastic Cells

    OpenAIRE

    Singh, Amareshwar T.K.; Frohman, Michael A.; Stern, Paula H.

    2005-01-01

    Parathyroid hormone (PTH) and phorbol-12,13-dibutyrate (PDBu) stimulate phospholipase D (PLD) activity and phosphatidylcholine (PC) hydrolysis in UMR-106 osteoblastic cells [1]. The current studies were designed to determine whether ethanolamine-containing phospholipids, and specifically phosphatidylethanolamine (PE), could also be substrates. In cells labeled with 14C-ethanolamine PTH and PDBu treatment decreased 14C-phosphatidylethanolamine. In cells co-labeled with 3H-choline and 14C-ethan...

  2. Mechanisms involved in regulation of osteoclastic differentiation by mechanical stress-loaded osteoblasts

    International Nuclear Information System (INIS)

    Highlights: → Effect of compressive force on osteoblasts were examined. → Compressive force induced OPG expression and suppressed osteoclastogenesis. → This enhancement of OPG is dependent on Wnt/Ca2+ signal pathway. -- Abstract: Mechanical stress is known to be important for regulation of bone turnover, though the detailed mechanisms are not fully understood. In the present study, we examined the effect of mechanical stress on osteoblasts using a novel compression model. Mouse osteoblastic MC3T3-E1 cells were embedded in three-dimensional (3D) gels and cultured with continuous compressive force (0-10.0 g/cm2) for 48 h, and the conditioned medium were collected. RAW264.7 cells were then incubated with the conditioned medium for various times in the presence of receptor activator of nuclear factor-κB ligand (RANKL). Conditioned medium was found to inhibit the differentiation of RAW264.7 cells into osteoclasts induced by RANKL via down-regulation of the expression of tumor necrosis factor receptor-associated factor 6 (TRAF6), phosphorylation of IκBα, and nuclear translocation of p50 and p65. Interestingly, the conditioned medium also had a high level of binding activity to RANKL and blocked the binding of RANK to RANKL. Furthermore, the binding activity of conditioned medium to RANKL was reduced when the 3D gel was supplemented with KN-93, an inhibitor of non-canonical Wnt/Ca2+ pathway. In addition, expression level of osteoprotegerin (OPG) mRNA was increased in time- and force-dependent manners, and remarkably suppressed by KN-93. These results indicate that osteoblastic cells subjected to mechanical stress produce OPG, which binds to RANKL. Furthermore, this binding activity strongly inhibited osteoclastogenesis through suppression of TRAF6 and the nuclear factor-kappa B (NF-κB) signaling pathway, suggesting that enhancement of OPG expression induced by mechanical stress is dependent on non-canonical Wnt/Ca2+ pathway.

  3. The affect of industrial activities on zinc in alluvial Egyptian soil determined using neutron activation analysis

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Thirty-two surface (0-20 cm) soil samples were collected from different locations in Egypt representing non-polluted,moderately and highly polluted soils.The aim of this study was to evaluate total Zn content in alluvial soils of Nile Delta in Egypt by using the delayed neutron activation analysis technique (DNAA),in the irradiation facilities of the first Egyptian research reactor (ET-RR-1).The gamma-ray spectra were recorded with a hyper pure germanium detection system.The well resolved gamma-ray peak at 1116.0 kev was efficiently used for 65Zn content determination.Zn content in non-polluted soil samples ranged between 74.1 and 103.8 ppm with an average of 98.5 + 5.1 ppm.Zn content in moderately polluted soils ranged between 136.0 and 232.5 ppm with an average of 180.1 + 32.6 ppm.The highest Zn levels ranging from 240.0 and 733.0 ppm with an average of 410.3 + 54.4 ppm,were observed in soil samples collected from,either highly polluted agricultural soils exposed to prolonged irrigation with industrial wastewater or surface soil samples from industrial sites.

  4. Osteoblast response to the surface of amino acid-functionalized hydroxyapatite.

    Science.gov (United States)

    Lee, Wing-Hin; Loo, Ching-Yee; Chrzanowski, Wojciech; Rohanizadeh, Ramin

    2015-06-01

    Interactions between proteins and the surface of biomaterials are crucial for the biological function and success of materials implanted in the human body. In this study, hydroxyapatite (HA) with negative and positive surface charges were fabricated by functionalizing the HA surface with acidic or basic amino acids. The influence of HA surface charge on protein adsorption and cell activities was studied. The crystallinity, morphology, and surface charge of amino acid-functionalized HA (AA-HA) particles and the stability of amino acids on the HA surface were determined. Both AA-HA and unmodified HA were studied for their capacity to adsorb proteins present in biological medium. The results showed that the presence of glutamic acid; Glu (acidic amino acids) and arginine; Arg (basic amino acids) on the HA surface resulted in higher protein adsorption owing to stronger electrostatic attraction between the HA particles and the proteins in medium. Functionalizing HA with Glu and Arg significantly promoted osteoblast adhesion on the surface of treated HA. No significant differences in cell proliferation between negatively and positively charged HA was observed. Significantly higher alkaline phosphatase (ALP) activity of osteoblasts on both charged surfaces was seen as compared to the unmodified HA. The study demonstrated that immobilization of amino acids (Glu and Arg) on the surface of HA promoted osteoblast proliferation and ALP activity. PMID:25346517

  5. Calcineurin/NFAT signaling in osteoblasts regulates bone mass.

    Science.gov (United States)

    Winslow, Monte M; Pan, Minggui; Starbuck, Michael; Gallo, Elena M; Deng, Lei; Karsenty, Gerard; Crabtree, Gerald R

    2006-06-01

    Development and repair of the vertebrate skeleton requires the precise coordination of bone-forming osteoblasts and bone-resorbing osteoclasts. In diseases such as osteoporosis, bone resorption dominates over bone formation, suggesting a failure to harmonize osteoclast and osteoblast function. Here, we show that mice expressing a constitutively nuclear NFATc1 variant (NFATc1(nuc)) in osteoblasts develop high bone mass. NFATc1(nuc) mice have massive osteoblast overgrowth, enhanced osteoblast proliferation, and coordinated changes in the expression of Wnt signaling components. In contrast, viable NFATc1-deficient mice have defects in skull bone formation in addition to impaired osteoclast development. NFATc1(nuc) mice have increased osteoclastogenesis despite normal levels of RANKL and OPG, indicating that an additional NFAT-regulated mechanism influences osteoclastogenesis in vivo. Calcineurin/NFATc signaling in osteoblasts controls the expression of chemoattractants that attract monocytic osteoclast precursors, thereby coupling bone formation and bone resorption. Our results indicate that NFATc1 regulates bone mass by functioning in both osteoblasts and osteoclasts. PMID:16740479

  6. Characteristics of the activity-affect association in inactive people: an ambulatory assessment study in daily life.

    Directory of Open Access Journals (Sweden)

    BirteVon Haaren

    2013-04-01

    Full Text Available Acute and regular exercise as well as physical activity is related to wellbeing and positive affect. Recent studies have shown that even daily, unstructured physical activities increase positive affect. However, the attempt to achieve adherence to physical activity or exercise in inactive people through public health interventions has often been unsuccessful. Most studies analyzing the activity-affect association in daily life, did not report participants´ habitual activity behavior. Thus, samples included active and inactive people, but they did not necessarily exhibit the same affective reactions to physical activity in daily life. Therefore the present study investigated whether the association between physical activity and subsequent affective state in daily life can also be observed in inactive individuals. We conducted a pilot study with 29 inactive university students (mean age 21.3 yrs ± 1.7 using the method of ambulatory assessment. Affect was assessed via electronic diary and physical activity was measured with accelerometers. Participants had to rate affect every two hours on a six item bipolar scale reflecting the three basic mood dimensions energetic arousal, valence and calmness. We calculated activity intensity level (mean Metabolic Equivalent (MET value and the amount of time spent in light activity over the last 15 minutes before every diary prompt and conducted within-subject correlations. We did not find significant associations between activity intensity and the three mood dimensions. Due to the high variability in within-subject correlations we conclude that not all inactive people show the same affective reactions to physical activity in daily life. Analyzing the physical activity-affect association of inactive people was difficult due to little variance and distribution of the assessed variables. Interactive assessment and randomized controlled trials might help solving these problems. Future studies should examine

  7. Exposure to GSM 900 MHz electromagnetic fields affects cerebral cytochrome c oxidase activity

    International Nuclear Information System (INIS)

    The world-wide and rapidly growing use of mobile phones has raised serious concerns about the biological and health-related effects of radio frequency (RF) radiation, particularly concerns about the effects of RFs upon the nervous system. The goal of this study was conducted to measure cytochrome oxidase (CO) levels using histochemical methods in order to evaluate regional brain metabolic activity in rat brain after exposure to a GSM 900 MHz signal for 45 min/day at a brain-averaged specific absorption rate (SAR) of 1.5 W/Kg or for 15 min/day at a SAR of 6 W/Kg over seven days. Compared to the sham and control cage groups, rats exposed to a GSM signal at 6 W/Kg showed decreased CO activity in some areas of the prefrontal and frontal cortex (infralimbic cortex, prelimbic cortex, primary motor cortex, secondary motor cortex, anterior cingulate cortex areas 1 and 2 (Cg1 and Cg2)), the septum (dorsal and ventral parts of the lateral septal nucleus), the hippocampus (dorsal field CA1, CA2 and CA3 of the hippocampus and dental gyrus) and the posterior cortex (retrosplenial agranular cortex, primary and secondary visual cortex, perirhinal cortex and lateral entorhinal cortex). However, the exposure to GSM at 1.5 W/Kg did not affect brain activity. Our results indicate that 6 W/Kg GSM 900 MHz microwaves may affect brain metabolism and neuronal activity in rats

  8. Leptin promotes osteoblast differentiation and mineralization of primary cultures of vascular smooth muscle cells by inhibiting glycogen synthase kinase (GSK)-3{beta}

    Energy Technology Data Exchange (ETDEWEB)

    Zeadin, Melec G.; Butcher, Martin K.; Shaughnessy, Stephen G. [Department of Medicine, McMaster University, Hamilton, ON (Canada); Thrombosis and Atherosclerosis Research Institute, Hamilton, ON (Canada); Werstuck, Geoff H., E-mail: Geoff.Werstuck@taari.ca [Department of Medicine, McMaster University, Hamilton, ON (Canada); Thrombosis and Atherosclerosis Research Institute, Hamilton, ON (Canada)

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer Leptin promotes osteoblast differentiation of primary smooth muscle cells. Black-Right-Pointing-Pointer Leptin regulates the expression of genes involved in osteoblast differentiation. Black-Right-Pointing-Pointer Constitutively active GSK-3{beta} attenuates leptin-induced osteoblast differentiation. Black-Right-Pointing-Pointer This suggests that leptin signals through GSK-3{beta} to promote osteoblast differentiation. -- Abstract: In this study, we begin to investigate the underlying mechanism of leptin-induced vascular calcification. We found that treatment of cultured bovine aortic smooth muscle cells (BASMCs) with leptin (0.5-4 {mu}g/ml) induced osteoblast differentiation in a dose-dependent manner. Furthermore, we found that leptin significantly increased the mRNA expression of osteopontin and bone sialoprotein, while down-regulating matrix gla protein (MGP) expression in BASMCs. Key factors implicated in osteoblast differentiation, including members of the Wnt signaling pathway, were examined. Exposure to leptin enhanced phosphorylation of GSK-3{beta} on serine-9 thereby inhibiting activity and promoting the nuclear accumulation of {beta}-catenin. Transfection of BASMCs with an adenovirus that expressed constitutively active GSK-3{beta} (Ad-GSK-3{beta} S9A) resulted in a >2-fold increase in GSK-3{beta} activity and a significant decrease in leptin-induced alkaline phosphatase (ALP) activity. In addition, qRT-PCR analysis showed that GSK-3{beta} activation resulted in a significant decrease in the expression of osteopontin and bone sialoprotein, but a marked increase in MGP mRNA expression. When taken together, our results suggest a mechanism by which leptin promotes osteoblast differentiation and vascular calcification in vivo.

  9. Leptin promotes osteoblast differentiation and mineralization of primary cultures of vascular smooth muscle cells by inhibiting glycogen synthase kinase (GSK)-3β

    International Nuclear Information System (INIS)

    Highlights: ► Leptin promotes osteoblast differentiation of primary smooth muscle cells. ► Leptin regulates the expression of genes involved in osteoblast differentiation. ► Constitutively active GSK-3β attenuates leptin-induced osteoblast differentiation. ► This suggests that leptin signals through GSK-3β to promote osteoblast differentiation. -- Abstract: In this study, we begin to investigate the underlying mechanism of leptin-induced vascular calcification. We found that treatment of cultured bovine aortic smooth muscle cells (BASMCs) with leptin (0.5–4 μg/ml) induced osteoblast differentiation in a dose-dependent manner. Furthermore, we found that leptin significantly increased the mRNA expression of osteopontin and bone sialoprotein, while down-regulating matrix gla protein (MGP) expression in BASMCs. Key factors implicated in osteoblast differentiation, including members of the Wnt signaling pathway, were examined. Exposure to leptin enhanced phosphorylation of GSK-3β on serine-9 thereby inhibiting activity and promoting the nuclear accumulation of β-catenin. Transfection of BASMCs with an adenovirus that expressed constitutively active GSK-3β (Ad-GSK-3β S9A) resulted in a >2-fold increase in GSK-3β activity and a significant decrease in leptin-induced alkaline phosphatase (ALP) activity. In addition, qRT-PCR analysis showed that GSK-3β activation resulted in a significant decrease in the expression of osteopontin and bone sialoprotein, but a marked increase in MGP mRNA expression. When taken together, our results suggest a mechanism by which leptin promotes osteoblast differentiation and vascular calcification in vivo.

  10. Glucocorticoids affect the metabolism of bone marrow stromal cells and lead to osteonecrosis of the femoral head: a review

    Institute of Scientific and Technical Information of China (English)

    TAN Gang; KANG Peng-de; PEI Fu-xing

    2012-01-01

    Objective To review the recent developments in the mechanisms of glucocorticoids induced osteonecrosis of femoral head (ONFH) and introduce a new theory of ONFH.Data sources Both Chinese- and English-language literatures were searched using MEDLINE (1997-2011),Pubmed (1997-2011 ) and the Index of Chinese-language Literature (1997-2011 ).Study selection Data from published articles about mechanisms of glucocorticoids induced ONFH in recent domestic and foreign literature were selected.Data extraction Data were mainly extracted from 61 articles which are listed in the reference section of this review.Results Glucocorticoids are steroid hormones secreted by the adrenal cortex that play a pivotal role in the regulation of a variety of developmental,metabolic and immune functions.However,high dose of exogenous glucocorticoids usage is the most common non-traumatic cause of ON FH.Glucocorticoids can affect the metabolisms of osteoblasts,osteoclasts,bone marrow stromal cells and adipocytes which decrease osteoblasts formation but increase adipocytes formation and cause ONFH finally.Conclusions Glucocorticoids affect the differentiation of mesenchymal stem cells,through activating or inhibiting the related transcript regulators of osteogenesis and adipogenesis.At last,the size and volume of mesenchymal stem cells derived adipocytes will increase amazingly,but the osteoblasts will be decreased obviously.In the meantime,the activity of the osteoclasts will be activated.So,these mechanisms work together and lead to ONFH.

  11. Patients With High Bone Mass Phenotype Exhibit Enhanced Osteoblast Differentiation and Inhibition of Adipogenesis of Human Mesenchymal Stem Cells

    DEFF Research Database (Denmark)

    Qiu, Weimin; Andersen, Tom; Bollerslev, Jens;

    2007-01-01

    ectopic mineralized bone when implanted subcutaneously with hydroxyapatite/tricalcium phosphate in SCID/NOD mice. Conclusions: LRP5 mutations and the level of Wnt signaling determine differentiation fate of hMSCs into osteoblasts or adipocytes. Activation of Wnt signaling can thus provide a novel approach...

  12. Effect of metformin on proliferation and related genes expression of human osteoblast MG63 under high glucose

    Institute of Scientific and Technical Information of China (English)

    曹小俊

    2013-01-01

    Objective To study the effect of metformin on proliferation and related genes expression of human osteoblast.Methods The proliferation of MG63 cells under high glucose intervened with metformin was measured by CCK-8 assay. The activity of intracellular alkaline phosphatase

  13. Osteoblast recruitment routes in human cancellous bone remodeling

    DEFF Research Database (Denmark)

    Kristensen, Helene B; Levin Andersen, Thomas; Marcussen, Niels;

    2014-01-01

    It is commonly proposed that bone forming osteoblasts recruited during bone remodeling originate from bone marrow perivascular cells, bone remodeling compartment canopy cells, or bone lining cells. However, an assessment of osteoblast recruitment during adult human cancellous bone remodeling is...... lacking. We addressed this question by quantifying cell densities, cell proliferation, osteoblast differentiation markers, and capillaries in human iliac crest biopsy specimens. We found that recruitment occurs on both reversal and bone-forming surfaces, as shown by the cell density and osterix levels on...

  14. EBF2 regulates osteoblast-dependent differentiation of osteoclasts

    DEFF Research Database (Denmark)

    Kieslinger, Matthias; Folberth, Stephanie; Dobreva, Gergana; Dorn, Tatjana; Croci, Laura; Erben, Reinhold; Consalez, G Giacomo; Grosschedl, Rudolf

    2005-01-01

    Communication between bone-depositing osteoblasts and bone-resorbing osteoclasts is required for bone development and homeostasis. Here, we identify EBF2, a member of the early B cell factor (EBF) family of transcription factors that is expressed in osteoblast progenitors, as a regulator of osteo...... in the Opg promoter and transactivates the Opg promoter in synergy with the Wnt-responsive LEF1/TCF:beta-catenin pathway. Taken together, these data identify EBF2 as a regulator of RANK-RANKL signaling and osteoblast-dependent differentiation of osteoclasts....

  15. Osteoblast Adhesion of Breast Cancer Cells with Scanning Acoustic Microscopy

    Science.gov (United States)

    Miyasaka, C.; Mercer, R. R.; Mastro, A. M.

    Conditioned medium was collected from a bone-metastatic breast cancer cell line, MDA-MB-231, and cultured with an immature osteoblast cell line, MC3T3-E1. Under these conditions the osteoblasts acquired a changed morphology and appeared to adhere in a different way to the substrate and to each other. To characterize cellular adhesion, MC3T3-E1 osteoblasts were cultured with or without MDA-MB-231 conditioned medium for two days. With mechanical scanning acoustic reflection microscopy, we were able to detect a change in the adhesive condition of the interface between the cell and the substrate, but not with optical microscopy

  16. Cannabinoid Modulation of Frontolimbic Activation and Connectivity During Volitional Regulation of Negative Affect.

    Science.gov (United States)

    Gorka, Stephanie M; Phan, K Luan; Lyons, Maryssa; Mori, Shoko; Angstadt, Mike; Rabinak, Christine A

    2016-06-01

    Behavioral and brain research indicates that administration of Δ(9)-tetrahydrocannabinol (THC) alters threat perception and enhances the suppression of conditioned fear responses via modulation of the frontolimbic circuit. No prior studies, however, have examined whether THC also affects volitional forms of emotion processing such as cognitive reappraisal. The aim of the current study was therefore to examine the effects of THC on frontolimbic activation and functional connectivity during cognitive reappraisal in a sample of healthy adults. The study was a randomized, double-blind, placebo-controlled, between-subject design and all participants ingested either an oral dose of synthetic THC (n=41) or placebo (n=37) before completion of an emotion regulation task during functional magnetic resonance imaging (fMRI). Functional connectivity was assessed using generalized psychophysiological interaction (gPPI) analyses. Results indicated that although there were no group differences in self-reported attenuation of negative affect during cognitive reappraisal, relative to placebo, THC increased amygdala activation and reduced amygdala and dorsolateral prefrontal cortex (dlPFC) functional coupling during cognitive reappraisal of emotionally negative pictures. This suggests that in addition to automatic emotional processes, THC affects frontolimbic functioning during cognitive reappraisal. PMID:26647971

  17. Ways of experiencing participation and factors affecting the current activity level after non-reconstructed ACL injury

    OpenAIRE

    Österberg, Annika; Kvist, Joanna; Abrandt Dahlgren, Madeleine

    2011-01-01

    Current assessment methods after an ACL injury do not consider how the individuals themselves experience their participation in activities or what factors they think have affected their current activity level.

  18. Biocompatibility evaluation in vitro. Part Ⅲ: Cytotoxicity expression of human and animal osteoblasts on the biomaterials

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Exclusion and imflammation in the clinic are observed for various reasons including material chemical composition, physical properties as well as macro- and micro-structure of the implants, surface condition of the implants, and also patient dependent factors. Cytotoxicity expression of cells is a central issue in current biocompatibility to screen the potential implant materials and drugs. This study was aimed at investigation reaction between the potential implant materials and surround tissue. Cytotoxicity of human and mt osteoblast in the material extracts was determinated by testing standards such as GHS assay, MTI assay, alkaline phosphatase activity assay, LDH assay, and Lowry assay. Research results demonstrated that compared with the control condition polystyrene culture plate both human and rat osteoblast cells have normal phenotypie expression in hydroxyapatite extract, and this expression was statistically restricted in hydroxyapatite-spinel extract. However, this restrict, e.g. cytotoxicity could be partially eliminated by immersion treatment of the materials in culture medium.

  19. Ca uptake and its influence by growth hormone in osteoblasts of fetal rat calvaria

    International Nuclear Information System (INIS)

    Uptake and release of Ca2+ are important functions of osteoblasts. In the paper we studied the uptake of calcium and influence by Growth Hormone in osteoblasts of fetal rat calvaria by liquid scintillation spectrometry of 45Ca2+. In short-term cultures of the bone derived cells, the uptake of 45Ca2+ increased steadily. The activity of 45Ca2+ in the cells of 15 minute cultures was 2∼3 times of that in the 0 minute cultures. It continued to increase in the cells of 30 minute cultures. Exposure of the bone cells to GH at 55.3 ng/ml increased the uptake of 45Ca2+ by 2.3 times in the 30 minute cultures and 1.5 times in the 60 minute cultures than those of the control

  20. Dysregulated Gene Expression in the Primary Osteoblasts and Osteocytes Isolated from Hypophosphatemic Hyp Mice

    Science.gov (United States)

    Miyagawa, Kazuaki; Yamazaki, Miwa; Kawai, Masanobu; Nishino, Jin; Koshimizu, Takao; Ohata, Yasuhisa; Tachikawa, Kanako; Mikuni-Takagaki, Yuko; Kogo, Mikihiko; Ozono, Keiichi; Michigami, Toshimi

    2014-01-01

    Osteocytes express multiple genes involved in mineral metabolism including PHEX, FGF23, DMP1 and FAM20C. In Hyp mice, a murine model for X-linked hypophosphatemia (XLH), Phex deficiency results in the overproduction of FGF23 in osteocytes, which leads to hypophosphatemia and impaired vitamin D metabolism. In this study, to further clarify the abnormality in osteocytes of Hyp mice, we obtained detailed gene expression profiles in osteoblasts and osteocytes isolated from the long bones of 20-week-old Hyp mice and wild-type (WT) control mice. The expression of Fgf23, Dmp1, and Fam20c was higher in osteocytic cells than in osteoblastic cells in both genotypes, and was up-regulated in Hyp cells. Interestingly, the up-regulation of these genes in Hyp bones began before birth. On the other hand, the expression of Slc20a1 encoding the sodium/phosphate (Na+/Pi) co-transporter Pit1 was increased in osteoblasts and osteocytes from adult Hyp mice, but not in Hyp fetal bones. The direct effects of extracellular Pi and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on isolated osteoblastic and osteocytic cells were also investigated. Twenty-four-hour treatment with 10−8 M 1,25(OH)2D3 increased the expression of Fgf23 in WT osteoblastic cells but not in osteocytic cells. Dmp1 expression in osteocytic cells was increased due to the 24-hour treatment with 10 mM Pi and was suppressed by 10−8 M 1,25(OH)2D3 in WT osteocytic cells. We also found the up-regulation of the genes for FGF1, FGF2, their receptors, and Egr-1 which is a target of FGF signaling, in Hyp osteocytic cells, suggesting the activation of FGF/FGFR signaling. These results implicate the complex gene dysregulation in osteoblasts and osteocytes of Hyp mice, which might contribute to the pathogenesis. PMID:24710520

  1. Dysregulated gene expression in the primary osteoblasts and osteocytes isolated from hypophosphatemic Hyp mice.

    Directory of Open Access Journals (Sweden)

    Kazuaki Miyagawa

    Full Text Available Osteocytes express multiple genes involved in mineral metabolism including PHEX, FGF23, DMP1 and FAM20C. In Hyp mice, a murine model for X-linked hypophosphatemia (XLH, Phex deficiency results in the overproduction of FGF23 in osteocytes, which leads to hypophosphatemia and impaired vitamin D metabolism. In this study, to further clarify the abnormality in osteocytes of Hyp mice, we obtained detailed gene expression profiles in osteoblasts and osteocytes isolated from the long bones of 20-week-old Hyp mice and wild-type (WT control mice. The expression of Fgf23, Dmp1, and Fam20c was higher in osteocytic cells than in osteoblastic cells in both genotypes, and was up-regulated in Hyp cells. Interestingly, the up-regulation of these genes in Hyp bones began before birth. On the other hand, the expression of Slc20a1 encoding the sodium/phosphate (Na+/Pi co-transporter Pit1 was increased in osteoblasts and osteocytes from adult Hyp mice, but not in Hyp fetal bones. The direct effects of extracellular Pi and 1,25-dihydroxyvitamin D3 [1,25(OH2D3] on isolated osteoblastic and osteocytic cells were also investigated. Twenty-four-hour treatment with 10-8 M 1,25(OH2D3 increased the expression of Fgf23 in WT osteoblastic cells but not in osteocytic cells. Dmp1 expression in osteocytic cells was increased due to the 24-hour treatment with 10 mM Pi and was suppressed by 10-8 M 1,25(OH2D3 in WT osteocytic cells. We also found the up-regulation of the genes for FGF1, FGF2, their receptors, and Egr-1 which is a target of FGF signaling, in Hyp osteocytic cells, suggesting the activation of FGF/FGFR signaling. These results implicate the complex gene dysregulation in osteoblasts and osteocytes of Hyp mice, which might contribute to the pathogenesis.

  2. Receptors and effects of gut hormones in three osteoblastic cell lines

    Directory of Open Access Journals (Sweden)

    Wilson Peter JM

    2011-07-01

    obestatin. ALP showed higher levels in Saos-2 after GIP, GHR and OB and in TE-85 after GHR. P1NP showed higher levels after GIP and OB in Saos-2. Decreased levels of P1NP were observed in TE-85 and MG-63 after GLP-1, GLP-2 and OB. MG-63 showed opposite responses in osteocalcin levels after GLP-2. Conclusions These results suggest that osteoblast activity modulation varies according to different development stage under different nutrition related-peptides.

  3. Biofilm activity and sludge characteristics affected by exogenous N-acyl homoserine lactones in biofilm reactors.

    Science.gov (United States)

    Hu, Huizhi; He, Junguo; Liu, Jian; Yu, Huarong; Zhang, Jie

    2016-07-01

    This study verified the effect of N-acyl homoserine lactone (AHL) concentrations on mature biofilm systems. Three concentrations of an AHL mixture were used in the batch test. Introducing of 5nM AHLs significantly increased biofilm activity and increased sludge characteristics, which resulted in better pollutant removal performance, whereas exogenous 50nM and 500nM AHLs limited pollutant removal, especially COD and nitrogen removal. To further identify how exogenous signal molecular affects biofilm system nitrogen removal, analyzing of nitrifying bacteria through real-time polymerase chain reaction (RT-PCR) revealed that these additional signal molecules affect nitrifying to total bacteria ratio. In addition, the running state of the system was stable during 15days of operation without an AHL dose, which suggests that the changes in the system due to AHL are irreversible. PMID:27030953

  4. In vitro vitamin K(2) and 1α,25-dihydroxyvitamin D(3) combination enhances osteoblasts anabolism of diabetic mice.

    Science.gov (United States)

    Poon, Christina C W; Li, Rachel W S; Seto, Sai Wang; Kong, Siu Kai; Ho, Ho Pui; Hoi, Maggie P M; Lee, Simon M Y; Ngai, Sai Ming; Chan, Shun Wan; Leung, George P H; Kwan, Yiu Wa

    2015-11-15

    In this study, we evaluated the anabolic effect and the underlying cellular mechanisms involved of vitamin K2 (10 nM) and 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) (10 nM), alone and in combination, on primary osteoblasts harvested from the iliac crests of C57BL/KsJ lean (+/+) and obese/diabetic (db/db) mice. A lower alkaline phosphatase (ALP) activity plus a reduced expression of bone anabolic markers and bone formation transcription factors (osteocalcin, Runx2, Dlx5, ATF4 and OSX) were consistently detected in osteoblasts of db/db mice compared to lean mice. A significantly higher calcium deposits formation in osteoblasts was observed in lean mice when compared to db/db mice. Co-administration of vitamin K2 (10 nM) and 1,25(OH)2D3 (10 nM) caused an enhancement of calcium deposits in osteoblasts in both strains of mice. Vitamins K2 and 1,25(OH)2D3 co-administration time-dependently (7, 14 and 21 days) increased the levels of bone anabolic markers and bone formation transcription factors, with a greater magnitude of increase observed in osteoblasts of db/db mice. Combined vitamins K2 plus 1,25(OH)2D3 treatment significantly enhanced migration and the re-appearance of surface microvilli and ruffles of osteoblasts of db/db mice. Thus, our results illustrate that vitamins K2 plus D3 combination could be a novel therapeutic strategy in treating diabetes-associated osteoporosis. PMID:26452518

  5. Vitamin D receptor overexpression in osteoblasts and osteocytes prevents bone loss during vitamin D-deficiency.

    Science.gov (United States)

    Lam, Nga N; Triliana, Rahma; Sawyer, Rebecca K; Atkins, Gerald J; Morris, Howard A; O'Loughlin, Peter D; Anderson, Paul H

    2014-10-01

    There are several lines of evidence that demonstrate the ability of 1,25-dihydroxyvitamin D (1,25(OH)2D3), acting via the vitamin D receptor (VDR) to mediate negative or positive effects in bone. Transgenic over-expression of VDR in osteoblasts and osteocytes in a mouse model (OSVDR) has been previously shown to inhibit processes of bone resorption and enhance bone formation, under conditions of adequate calcium intake. While these findings suggest that vitamin D signalling in osteoblasts and osteocytes promotes bone mineral accrual, the vitamin D requirement for this action is not well understood. In this study, 4 week old female OSVDR and wild-type (WT) mice were fed either a vitamin D-replete (1000IU/kg diet, D+) or vitamin D-deficient (D-) diet for 4 months to observe changes to bone mineral homeostasis. Tibial bone mineral volume was analysed by micro-CT and changes to bone cell activities were measured using standard dynamic histomorphometric techniques. While vitamin D-deplete WT mice demonstrated a reduction in periosteal bone accrual and overall bone mineral volume, OSVDR mice, however, displayed increased cortical and cancellous bone volume in mice which remained higher during vitamin D-depletion due to a reduced osteoclast number and increased bone formation rate. These data suggest that increased VDR-mediated activity in osteoblast and osteocytes prevents bone loss due to vitamin D-deficiency. This article is part of a Special Issue entitled '16th Vitamin D Workshop'. PMID:24434283

  6. Estrogen and phenol red free medium for osteoblast culture: study of the mineralization ability.

    Science.gov (United States)

    de Faria, A N; Zancanela, D C; Ramos, A P; Torqueti, M R; Ciancaglini, P

    2016-08-01

    To design an estrogen and phenol red free medium for cell culture and check its effectiveness and safety on osteoblast growth it is necessary to maintain the estrogen receptors free for tests. For this purpose, we tested some modifications of the traditional culture media: estrogen depleted fetal bovine serum; estrogen charcoal stripped fetal bovine serum and phenol red free α-MEM. The aim of this work is to examine the effects of its depletion in the proliferation, differentiation, and toxicity of mesenchymal stromal cells differentiated into osteoblasts to obtain an effective interference free culture medium for in vitro studies, focused on non-previously studied estrogen receptors. We performed viability tests using the following techniques: MTT, alkaline phosphatase specific activity, formation of mineralized matrix by Alizarin technique and analysis of SEM/EDX of mineralized nodules. The results showed that the culture media with estrogen free α-MEM + phenol red free α-MEM did not impact viability, alkaline phosphatase activity and mineralization of the osteoblasts culture compared to control. In addition, its nodules possess Ca/P ratio similar to hydroxyapatite nodules on the 14th and 21st day. In conclusion, the modified culture medium with phenol red free α-MEM with estrogen depleted fetal bovine serum can be safely used in experiments where the estrogen receptors need to be free. PMID:25634598

  7. Low-intensity pulsed ultrasound regulates proliferation and differentiation of osteoblasts through osteocytes

    Energy Technology Data Exchange (ETDEWEB)

    Li, Lei, E-mail: geraldleelei@163.com [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu (China); Yang, Zheng [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu (China); Zhang, Hai [Department of Restorative Dentistry, School of Dentistry, University of Washington, Seattle, WA (United States); Chen, Wenchuan [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu (China); Chen, Mengshi [Department of Biomechanics, Sichuan University, Chengdu (China); Zhu, Zhimin, E-mail: hxzhimin@163.com [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu (China)

    2012-02-10

    Highlights: Black-Right-Pointing-Pointer CM from LIPUS-stimulated osteocytes inhibits proliferation of osteoblasts. Black-Right-Pointing-Pointer CM from LIPUS-stimulated osteocytes enhances differentiation of osteoblasts. Black-Right-Pointing-Pointer LIPUS stimulates MLO-Y4 cells to secrete PGE{sub 2} and NO. -- Abstract: Low-intensity pulsed ultrasound (LIPUS) has been used as a safe and effective modality to enhance fracture healing. As the most abundant cells in bone, osteocytes orchestrate biological activities of effector cells via direct cell-to-cell contacts and by soluble factors. In this study, we have used the osteocytic MLO-Y4 cells to study the effects of conditioned medium from LIPUS-stimulated MLO-Y4 cells on proliferation and differentiation of osteoblastic MC3T3-E1 cells. Conditioned media from LIPUS-stimulated MLO-Y4 cells (LIPUS-Osteocyte-CM) were collected and added on MC3T3-E1 cell cultures. MC3T3-E1 cells cultured in LIPUS-Osteocyte-CM demonstrated a significant inhibition of proliferation and an increased alkaline phosphatase activity. The results of PGE{sub 2} and NO assay showed that LIPUS could enhance PGE{sub 2} and NO secretion from MLO-Y4 cells at all time points within 24 h after LIPUS stimulation. We conclude that LIPUS regulates proliferation and differentiation of osteoblasts through osteocytes in vitro. Increased secretion of PGE{sub 2} from osteocytes may play a role in this effect.

  8. Environmental noise levels affect the activity budget of the Florida manatee

    Science.gov (United States)

    Miksis-Olds, Jennifer L.; Donaghay, Percy L.; Miller, James H.; Tyack, Peter L.

    2005-09-01

    Manatees inhabit coastal bays, lagoons, and estuaries because they are dependent on the aquatic vegetation that grows in shallow waters. Food requirements force manatees to occupy the same areas in which human activities are the greatest. Noise produced from human activities has the potential to affect these animals by eliciting responses ranging from mild behavioral changes to extreme aversion. This study quantifies the behavioral responses of manatees to both changing levels of ambient noise and transient noise sources. Results indicate that elevated environmental noise levels do affect the overall activity budget of this species. The proportion of time manatees spend feeding, milling, and traveling in critical habitats changed as a function of noise level. More time was spent in the directed, goal-oriented behaviors of feeding and traveling, while less time was spent milling when noise levels were highest. The animals also responded to the transient noise of approaching vessels with changes in behavioral state and movements out of the geographical area. This suggests that manatees detect and respond to changes in environmental noise levels. Whether these changes legally constitute harassment and produce biologically significant effects need to be addressed with hypothesis-driven experiments and long-term monitoring. [For Animal Bioacoustics Best Student Paper Award.

  9. Soil acidity as affecting micronutrients concentration, nitrato reductase enzyme activity and yield in upland rice plants

    Directory of Open Access Journals (Sweden)

    Edemar Moro

    2013-12-01

    Full Text Available The lowest grain yield of rice under no-tillage system (NTS in relation to the conventional system may be due to the predominance nitrate in the soil and the low nitrate reductase activity. Another reason may be caused by micronutrient deficiency because of superficially soil acidity corrections. Therefore, the objective of this study was to evaluate the changes caused by soil pH in the N forms in the soil, micronutrients concentration in rice plants, nitrate reductase activity, yield of rice and its components. The experiment was performed in a greenhouse conditions. The experimental design was a completely randomized in a factorial three (levels of soil acidity x five (micronutrients sources with four replications. The addition of micronutrients does not affect levels of nitrate and ammonium in the soil; soil acidity significantly affects levels of nitrate and ammonium in the soil, concentration of micronutrients in rice plants and crop yield and its components; medium soil acidity (pH 5.5 result in medium to high levels of Cu and Fe, medium level of Zn and Mn, high nitrate reductase activity, resulting in higher dry matter, tillers, panicles, spikelets, weight of 100 grains and hence grain yield.

  10. Affective and physiological responses to the suffering of others: compassion and vagal activity.

    Science.gov (United States)

    Stellar, Jennifer E; Cohen, Adam; Oveis, Christopher; Keltner, Dacher

    2015-04-01

    Compassion is an affective response to another's suffering and a catalyst of prosocial behavior. In the present studies, we explore the peripheral physiological changes associated with the experience of compassion. Guided by long-standing theoretical claims, we propose that compassion is associated with activation in the parasympathetic autonomic nervous system through the vagus nerve. Across 4 studies, participants witnessed others suffer while we recorded physiological measures, including heart rate, respiration, skin conductance, and a measure of vagal activity called respiratory sinus arrhythmia (RSA). Participants exhibited greater RSA during the compassion induction compared with a neutral control (Study 1), another positive emotion (Study 2), and a prosocial emotion lacking appraisals of another person's suffering (Study 3). Greater RSA during the experience of compassion compared with the neutral or control emotion was often accompanied by lower heart rate and respiration but no difference in skin conductance. In Study 4, increases in RSA during compassion positively predicted an established composite of compassion-related words, continuous self-reports of compassion, and nonverbal displays of compassion. Compassion, a core affective component of empathy and prosociality, is associated with heightened parasympathetic activity. PMID:25621856

  11. Recql4 haploinsufficiency in mice leads to defects in osteoblast progenitors: Implications for low bone mass phenotype

    International Nuclear Information System (INIS)

    The cellular and molecular mechanisms that underlie skeletal abnormalities in defective Recql4-related syndromes are poorly understood. Our objective in this study was to explore the function of Recql4 in osteoblast biology both in vitro and in vivo. Immunohistochemistry on adult mouse bone showed Recql4 protein localization in active osteoblasts around growth plate, but not in fully differentiated osteocytes. Consistent with this finding, Recql4 gene expression was high in proliferating mouse osteoblastic MC3T3.E1 cells and decreased as cells progressively lost their proliferation activity during differentiation. Recql4 overexpression in osteoblastic cells exhibited higher proliferation activity, while its depletion impeded cell growth. In addition, bone marrow stromal cells from male Recql4+/- mice had fewer progenitor cells, including osteoprogenitors, indicated by reduced total fibroblast colony forming units (CFU-f) and alkaline phosphatase-positive CFU-f colonies concomitant with reduced bone mass. These findings provide evidence that Recql4 functions as a regulatory protein during osteoprogenitor proliferation, a critical cellular event during skeleton development

  12. Magnetic nanocomposite scaffolds combined with static magnetic field in the stimulation of osteoblastic differentiation and bone formation.

    Science.gov (United States)

    Yun, Hyung-Mun; Ahn, Su-Jin; Park, Kyung-Ran; Kim, Mi-Joo; Kim, Jung-Ju; Jin, Guang-Zhen; Kim, Hae-Won; Kim, Eun-Cheol

    2016-04-01

    Magnetism has recently been implicated to play significant roles in the regulation of cell responses. Allowing cells to experience a magnetic field applied externally or scaffolding them in a material with intrinsic magnetic properties has been a possible way of utilizing magnetism. Here we aim to investigate the combined effects of the external static magnetic field (SMF) with magnetic nanocomposite scaffold made of polycaprolactone/magnetic nanoparticles on the osteoblastic functions and bone formation. The SMF synergized with the magnetic scaffolds in the osteoblastic differentiation of primary mouse calvarium osteoblasts, including the expression of bone-associated genes (Runx2 and Osterix) and alkaline phosphatase activity. The synergism was demonstrated in the activation of integrin signaling pathways, such as focal adhesion kinase, paxillin, RhoA, mitogen-activated protein kinase, and nuclear factor-kappaB, as well as in the up-regulation of bone morphogenetic protein-2 and phosphorylation of Smad1/5/8. Furthermore, the SMF/magnetic scaffold-stimulated osteoblasts promoted the angiogenic responses of endothelial cells, including the expression of vascular endothelial growth factor and angiogenin-1 genes and the formation of capillary tubes. When the magnetic scaffolds were implanted in mouse calvarium defects, the application of SMF significantly enhanced the new bone formation at 6 weeks, as revealed by the histological and micro-computed tomographic analyses. Current findings suggest that the combinatory application of external (SMF) and internal (scaffold) magnetism can be a promising tool to regenerative engineering of bone. PMID:26854394

  13. Do government brochures affect physical activity cognition? A pilot study of Canada's physical activity guide to healthy active living.

    Science.gov (United States)

    Kliman, Aviva M; Rhodes, Ryan

    2008-08-01

    Health Canada has published national physical activity (PA) guidelines, which are included in their 26-page Physical Activity Guide to Healthy Active Living (CPAG). To date, the use of CPAG as a motivational instrument for PA promotion has not been evaluated. The purpose of this study was to determine whether reading CPAG 1) increased motivational antecedents to engage in regular PA, and 2) increased regular PA intention and behaviour over 1 month. Participants included 130 randomly sampled Canadian adults (18 years or older) who were randomly mailed pack ages consisting of either 1) a questionnaire and a copy of CPAG, or 2) a questionnaire. Questionnaire items pertained to participants' sociodemographics, previous PA behaviours (Godin Leisure-Time Questionnaire) and PA motivation (theory of planned behaviour). Participants were then sent a follow-up questionnaire pertaining to their PA behaviours throughout the previous month. Results revealed significant interactions between the guide condition and previous activity status on instrumental behavioural beliefs about strength activities and subjective norms about endurance activities (p behavioural control) and outcomes (intention, behaviour) seem unaffected. PMID:18825580