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Sample records for affects osteoblast behaviour

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

    SciTech Connect

    Schindeler, Aaron . E-mail: AaronS@chw.edu.au; Little, David G.

    2005-12-16

    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 [{sup 14}C]-labeled ZA confirmed that the bisphosphonate bound in a concentration-dependent manner over the 1-50 {mu}M 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.

  2. Osteoblasts growth behaviour on bio-based calcium carbonate aragonite nanocrystal.

    PubMed

    Shafiu Kamba, Abdullahi; Zakaria, Zuki Abu Bakar

    2014-01-01

    Calcium carbonate (CaCO3) nanocrystals derived from cockle shells emerge to present a good concert in bone tissue engineering because of their potential to mimic the composition, structure, and properties of native bone. The aim of this study was to evaluate the biological response of CaCO3 nanocrystals on hFOB 1.19 and MC3T3 E-1 osteoblast cells in vitro. Cell viability and proliferation were assessed by MTT and BrdU assays, and LDH was measured to determine the effect of CaCO3 nanocrystals on cell membrane integrity. Cellular morphology was examined by SEM and fluorescence microscopy. The results showed that CaCO3 nanocrystals had no toxic effects to some extent. Cell proliferation, alkaline phosphatase activity, and protein synthesis were enhanced by the nanocrystals when compared to the control. Cellular interactions were improved, as indicated by SEM and fluorescent microscopy. The production of VEGF and TGF-1 was also affected by the CaCO3 nanocrystals. Therefore, bio-based CaCO3 nanocrystals were shown to stimulate osteoblast differentiation and improve the osteointegration process. PMID:24734228

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

    PubMed

    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

  4. Osteoblast-specific expression of Fra-2/AP-1 controls adiponectin and osteocalcin expression and affects metabolism.

    PubMed

    Bozec, Aline; Bakiri, Latifa; Jimenez, Maria; Rosen, Evan D; Catalá-Lehnen, Philip; Schinke, Thorsten; Schett, Georg; Amling, Michael; Wagner, Erwin F

    2013-12-01

    Recent studies have established that the skeleton functions as an endocrine organ affecting metabolism through the osteoblast-derived hormone osteocalcin (Ocn). However, it is not fully understood how many transcription factors expressed in osteoblasts regulate the endocrine function. Here, we show that mice with osteoblast-specific deletion of Fra-2 (Fosl2) have low bone mass but increased body weight. In contrast, transgenic expression of Fra-2 in osteoblasts leads to increased bone mass and decreased body weight accompanied by reduced serum glucose and insulin levels, improved glucose tolerance and insulin sensitivity. In addition, mice lacking Fra-2 have reduced levels of circulating Ocn, but high adiponectin (Adipoq), whereas Fra-2 transgenic mice exhibit high Ocn and low Adipoq levels. Moreover, we found that Adipoq was expressed in osteoblasts and that this expression was transcriptionally repressed by Fra-2. These results demonstrate that Fra-2 expression in osteoblasts represents a novel paradigm for a transcription factor controlling the endocrine function of the skeleton. PMID:24046454

  5. Bioactive glass coatings affect the behavior of osteoblast-like cells

    PubMed Central

    Foppiano, Silvia; Marshall, Sally J.; Marshall, Grayson W.; Saiz, Eduardo; Tomsia, Antoni P.

    2007-01-01

    Functionally graded coatings (FGCs) of bioactive glass on titanium alloy (Ti6Al4V) were fabricated by the enameling technique. These innovative coatings may be an alternative to plasma-sprayed, hydroxyapatite-coated implants. Previously we determined that a preconditioning treatment in simulated body fluid (SBF) helped to stabilize FGCs (Foppiano, S., et al., Acta Biomater, 2006; 2(2):133-42). The primary goal of this work was to assess the in vitro cytocompatibility of preconditioned FGCs with MC3T3-E1.4 mouse pre-osteoblastic cells. We evaluated cell adhesion, proliferation and mineralization on FGCs in comparison to uncoated Ti6Al4V and tissue culture polystyrene (TCPS). No difference in cell adhesion was identified, whereas proliferation was significantly different on all materials, being highest on FGCs followed by TCPS and Ti6Al4V. Qualitative and quantitative mineralization assays indicated that mineralization occurred on all materials. The amount of inorganic phosphate released by the mineralizing layers was significantly different, being highest on TCPS, followed by FGC and uncoated Ti6Al4V. The secondary objective of this work was to assess the ability of the FGCs to affect gene expression, indirectly, by means of their dissolution products, which was assessed by real-time reverse-transcription polymerase chain reaction. The FGC dissolution products induced a 2-fold increase in the expression of Runx-2, and a 20% decrease in the expression of collagen type 1 with respect to TCPS extract. These genes are regulators of osteoblast differentiation and mineralization, respectively. The findings of this study indicate that preconditioned FGCs are cytocompatible and suggest that future work may allow composition changes to induce preferred gene expression. PMID:17466608

  6. Symbiont infection affects aphid defensive behaviours.

    PubMed

    Dion, Emilie; Polin, Sarah Erika; Simon, Jean-Christophe; Outreman, Yannick

    2011-10-23

    Aphids harbour both an obligate bacterial symbiont, Buchnera aphidicola, and a wide range of facultative ones. Facultative symbionts can modify morphological, developmental and physiological host traits that favour their spread within aphid populations. We experimentally investigated the idea that symbionts may also modify aphid behavioural traits to enhance their transmission. Aphids exhibit many behavioural defences against enemies. Despite their benefits, these behaviours have some associated costs leading to reduction in aphid reproduction. Some aphid individuals harbour a facultative symbiont Hamiltonella defensa that provides protection against parasitoids. By analysing aphid behaviours in the presence of parasitoids, we showed that aphids infected with H. defensa exhibited reduced aggressiveness and escape reactions compared with uninfected aphids. The aphid and the symbiont have both benefited from these behavioural changes: both partners reduced the fitness decrements associated with the behavioural defences. Such symbiont-induced changes of behavioural defences may have consequences for coevolutionary processes between host organisms and their enemies. PMID:21490007

  7. Mobbing Behaviour: Victims and the Affected

    ERIC Educational Resources Information Center

    Erturk, Abbas

    2013-01-01

    The purpose of this research was to identify the level of mobbing behaviour faced by teachers and managers working in primary schools, their responses to such behaviour and the difference in these responses according to the gender variable. The sample of the research consists of a total of 1,316 teachers and managers including 691 men and 625…

  8. Innovative macroporous granules of nanostructured-hydroxyapatite agglomerates: bioactivity and osteoblast-like cell behaviour.

    PubMed

    Laranjeira, M S; Fernandes, M H; Monteiro, F J

    2010-12-01

    To modulate the biological response of implantable granules, two types of bioactive porous granules composed of nanostructured-hydroxyapatite (HA) agglomerates and microstructured-HA, respectively, were prepared using a polyurethane sponge impregnation and burnout method. The resulting granules presented a highly porous structure with interconnected porosity. Both types of granules were characterized using Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), and mercury intrusion porosimetry. Results showed that nanostructed-HA granules presented higher surface area and porosity than microstructured-HA granules. In vitro testing using MG63 human osteoblast-like cells showed that on both types of surfaces cells were able to adhere, proliferate, and migrate through the macropores, and a higher growth rate was achieved on nanostructured-HA granules than on microstructured-HA granules (76 and 40%, respectively). In addition, these cells maintained similar expression levels of osteoblastic-associated markers namely collagen type I, alkaline phosphatase, bone morphogenetic protein-2, macrophage colony-stimulating factor, and osteoprotegerin. These innovative nanostructured-HA granules may be considered as promising bioceramic alternative matrixes for bone regeneration and drug release application. PMID:20845490

  9. Cobalt and chromium exposure affects osteoblast function and impairs the mineralization of prosthesis surfaces in vitro.

    PubMed

    Shah, Karan M; Wilkinson, Jeremy Mark; Gartland, Alison

    2015-11-01

    Cobalt (Co) and chromium (Cr) ions and nanoparticles equivalent to those released through tribo-corrosion of prosthetic metal-on-metal (MOM) bearings and taper junctions are detrimental to osteoblast activity and function in vitro when examined as individual species. Here we examined the effects of Co(2+):Cr(3+) and Co(2+):Cr(6+) combinations on osteoblast-like SaOS-2 cellular activity, alkaline phosphatase (ALP) activity and mineralization to better reflect clinical exposure conditions in vivo. We also assessed the effect of Co(2+):Cr(3+) combinations and Co:Cr nanoparticles on SaOS-2 cell osteogenic responses on grit-blasted, plasma-sprayed titanium-coated, and hydroxyapatite-coated prosthesis surfaces. Cellular activity and ALP activity were reduced to a greater extent with combination treatments compared to individual ions. Co(2+) and Cr(3+) interacted additively and synergistically to reduce cellular activity and ALP activity, respectively, while the Co(2+) with Cr(6+) combination was dominated by the effect of Cr(6+) alone. Mineralization by osteoblasts was greater on hydroxyapatite-coated surfaces compared to grit-blasted and plasma-sprayed titanium-coated surfaces. Treatments with Co(2+):Cr(3+) ions and Co:Cr nanoparticles reduced the percentage mineralization on all surfaces, with hydroxyapatite-coated surfaces having the least reduction. In conclusion, our data suggests that previous studies investigating individual metal ions underestimate their potential clinical effects on osteoblast activity. Furthermore, the data suggests that hydroxyapatite-coated surfaces may modulate osteoblast responses to metal debris. PMID:25929464

  10. Web-based Factors Affecting Online Purchasing Behaviour

    NASA Astrophysics Data System (ADS)

    Ariff, Mohd Shoki Md; Sze Yan, Ng; Zakuan, Norhayati; Zaidi Bahari, Ahamad; Jusoh, Ahmad

    2013-06-01

    The growing use of internet and online purchasing among young consumers in Malaysia provides a huge prospect in e-commerce market, specifically for B2C segment. In this market, if E-marketers know the web-based factors affecting online buyers' behaviour, and the effect of these factors on behaviour of online consumers, then they can develop their marketing strategies to convert potential customers into active one, while retaining existing online customers. Review of previous studies related to the online purchasing behaviour in B2C market has point out that the conceptualization and empirical validation of the online purchasing behaviour of Information and Communication Technology (ICT) literate users, or ICT professional, in Malaysia has not been clearly addressed. This paper focuses on (i) web-based factors which online buyers (ICT professional) keep in mind while shopping online; and (ii) the effect of web-based factors on online purchasing behaviour. Based on the extensive literature review, a conceptual framework of 24 items of five factors was constructed to determine web-based factors affecting online purchasing behaviour of ICT professional. Analysis of data was performed based on the 310 questionnaires, which were collected using a stratified random sampling method, from ICT undergraduate students in a public university in Malaysia. The Exploratory factor analysis performed showed that five factors affecting online purchase behaviour are Information Quality, Fulfilment/Reliability/Customer Service, Website Design, Quick and Details, and Privacy/Security. The result of Multiple Regression Analysis indicated that Information Quality, Quick and Details, and Privacy/Security affect positively online purchase behaviour. The results provide a usable model for measuring web-based factors affecting buyers' online purchase behaviour in B2C market, as well as for online shopping companies to focus on the factors that will increase customers' online purchase.

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

    SciTech Connect

    Ehnert, Sabrina; Freude, Thomas; Ihle, Christoph; Mayer, Larissa; Braun, Bianca; Graeser, Jessica; Flesch, Ingo; 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.

  12. Episodic disorders of behaviour and affect after acquired brain injury.

    PubMed

    Eames, Peter Eames; Wood, Rodger Ll

    2003-01-01

    Psychological disorders that follow traumatic brain injury are possibly more complex and diverse than those associated with other forms of "brain damage". These may include organic aggressive, or organic affective syndromes that are episodic in nature and therefore require a more specific diagnosis, a different classification, and a different approach to treatment. Consequently, it is necessary for clinicians to learn to distinguish between "primary" psychiatric illnesses and those disorders of behavioural control and mood that stem specifically from brain injury. There is relatively little in the clinical literature that explains the relationship between variable states of behaviour, mood or temperament, and clinical disorders that may have long-term implications for patient management. This concept paper therefore addresses abnormalities of mood and behaviour that are episodic in character and are not recognisably included in the DSM and ICD classifications of psychological or psychiatric disorders. PMID:21854336

  13. Slight Changes in the Mechanical Stimulation Affects Osteoblast- and Osteoclast-Like Cells in Co-Culture

    PubMed Central

    Kadow-Romacker, Anke; Duda, Georg N.; Bormann, Nicole; Schmidmaier, Gerhard; Wildemann, Britt

    2013-01-01

    Summary Background Osteoblast- and osteoclast-like cells are responsible for coordinated bone maintenance, illustrated by a balanced formation and resorption. Both parameters appear to be influenced by mechanical constrains acting on each of these cell types individually. We hypothesized that the interactions between both cell types are also influenced by mechanical stimulation. Methods Co-cultures of osteoblast- and osteoclast-like cells were stimulated with 1,100 µstrain, 0.1 or 0.3 Hz for 1–5 min/day over 5 days. Two different setups depending on the differentiation of the osteoclast-like cells were used: i) differentiation assay for the fusion of pre-osteoclasts to osteoclasts, ii) resorption assay to determine the activity level of osteoclast-like cells. Results In the differentiation assay (co-culture of osteoblasts with unfused osteoclast precursor cells) the mechanical stimulation resulted in a significant decrease of collagen-1 and osteocalcin produced by osteoblast-like cells. Significantly more TRAP-iso5b was measured after stimulation for 3 min with 0.1 Hz, indicating enhanced osteoclastogenesis. In the resorption assay (co-culture of osteoblasts with fused osteoclasts) the stimulation for 3 min with 0.3 Hz significantly increased the resorption activity of osteoclasts measured by the pit formation and the collagen resorption. The same mechanical stimulation resulted in an increased collagen-1 production by the osteoblast-like cells. The ratio of RANKL/OPG was not different between the groups. Conclusion These findings demonstrate that already small changes in duration or frequency of mechanical stimulation had significant consequences for the behavior of osteoblast- and osteoclast-like cells in co-culture, which partially depend on the differentiation status of the osteoclast-like cells. PMID:24474895

  14. Behavioural phenotype affects social interactions in an animal network

    PubMed Central

    Pike, Thomas W; Samanta, Madhumita; Lindström, Jan; Royle, Nick J

    2008-01-01

    Animal social networks can be extremely complex and are characterized by highly non-random interactions between group members. However, very little is known about the underlying factors affecting interaction preferences, and hence network structure. One possibility is that behavioural differences between individuals, such as how bold or shy they are, can affect the frequency and distribution of their interactions within a network. We tested this using individually marked three-spined sticklebacks (Gasterosteus aculeatus), and found that bold individuals had fewer overall interactions than shy fish, but tended to distribute their interactions more evenly across all group members. Shy fish, on the other hand, tended to associate preferentially with a small number of other group members, leading to a highly skewed distribution of interactions. This was mediated by the reduced tendency of shy fish to move to a new location within the tank when they were interacting with another individual; bold fish showed no such tendency and were equally likely to move irrespective of whether they were interacting or not. The results show that animal social network structure can be affected by the behavioural composition of group members and have important implications for understanding the spread of information and disease in social groups. PMID:18647713

  15. Large Gradient High Magnetic Fields Affect Osteoblast Ultrastructure and Function by Disrupting Collagen I or Fibronectin/αβ1 Integrin

    PubMed Central

    Qian, Ai-Rong; Gao, Xiang; Zhang, Wei; Li, Jing-Bao; Wang, Yang; Di, Sheng-Meng; Hu, Li-Fang; Shang, Peng

    2013-01-01

    The superconducting magnet generates a field and field gradient product that can levitate diamagnetic materials. In this study a specially designed superconducting magnet with a large gradient high magnetic field (LG-HMF), which can provide three apparent gravity levels (μ-g, 1-g, and 2-g), was used to simulate a space-like gravity environment. The effects of LG-HMF on the ultrastructure and function of osteoblast-like cells (MG-63 and MC3T3-E1) and the underlying mechanism were investigated by transmission electromicroscopy (TEM), MTT, and cell western (ICW) assays. Under LG-HMF significant morphologic changes in osteoblast-like cells occurred, including expansion of endoplasmic reticulum and mitochondria, an increased number of lysosomes, distorted microvilli, and aggregates of actin filaments. Compared to controls, cell viability and alkaline phosphatase (ALP) secretion were significantly increased, and collagen I (col I), fibronectin (FN), vinculin, integrin α3, αv, and β1 expression were changed under LG-HMF conditions. In conclusion, LG-HMF affects osteoblast ultrastructure, cell viability, and ALP secretion, and the changes caused by LG-HMF may be related to disrupting col I or FN/αβ1 integrin. PMID:23382804

  16. Can the structure of an explosive caldera affect eruptive behaviour?

    NASA Astrophysics Data System (ADS)

    Willcox, C. P.; Branney, M.; Carrasco-Nuñez, G.; Barford, D.

    2010-12-01

    Explosive caldera volcanoes cause catastrophic events at the Earth’s surface, yet we know little about how their internal structures evolve with time, and whether this can affect both differentiation and eruptive behaviour. Distinguishing how structural evolution impacts upon eruption behaviour and periodicity is challenging because the resolution of eruption frequencies can be difficult at ancient exhumed calderas, whereas at young volcanoes, most of the caldera floor faults and associated conduits are hidden. Some exhumed calderas reveal caldera floor faults and conduits; some of these apparently underwent a single collapse event that was piecemeal, i.e. fragmentation into several, variously subsided fault-blocks (e.g. Scafell caldera, UK). In contrast, the present study tests whether some caldera volcanoes may become more intensely fractured with time as a result of successive distinct caldera-collapse eruptions (“multi-cyclic calderas”). It has been proposed that this scenario could lead to an increase in eruption frequency, with smaller eruptions over time. Magma leakage through the increasingly fractured volcano might also lead to less evolved compositions with time due to shorter residence times. We have returned to the volcano where this hypothesis was formulated, the ~ 20 km diameter, hydrothermally active Los Humeros caldera in eastern central México. We aim to see how well the structural evolution of this modern caldera can be reconstructed, and whether changes in structure affected the styles and periodicity of large explosive eruptions. How a caldera evolves structurally could have important implications for predicting future catastrophic eruptions. Detailed structural mapping (e.g. of fault scarps, vent positions, and tilted strata), documentation of draping and cross-cutting field relations, together with logging, optical and SEM petrography, XRF major and trace element geochemistry and new 40Ar-39Ar and radiocarbon dating of the pyroclastic

  17. Effect of hydroxyapatite-based biomaterials on human osteoblast phenotype.

    PubMed

    Trombelli, L; Penolazzi, L; Torreggiani, E; Farina, R; Lambertini, E; Vecchiatini, R; Piva, R

    2010-03-01

    The present study evaluated human primary osteoblasts and two different osteoblast-like cell lines behaviour when cultured in presence of different hydroxyapatite-based (HA) biomaterials (SINTlife-FIN-CERAMICA S.p.a., Faenza, Italy; Bio-Oss, Geistlich Biomaterials, Woulhusen, Switzerland; Biostite-GABA Vebas, San Giuliano Milanese, MI, Italy), focusing attention on the effect of HA/Biostite in terms of modulation of osteoblastic differentiation. Analysis were about adhesion, proliferation and mineralization activity. Runt-related transcription factor 2 (Runx2), Estrogen Receptor alpha (ERalfa) expression and alkaline phosphatase activity (ALP) were measured as osteoblastic differentiation markers. Determination of viable cells was done with MTT colorimetric assay. Scanning electron microscopy (SEM) analysis was performed on biomaterial-treated cells. All hydroxyapatite-based biomaterials didn't affect cells morphology and viability, whereas only presence of HA/Biostite improved cells adhesion, growth and differentiation. Adhesion and spreading of the primary cells on HA/Biostite were the same showed by two different osteoblast-like cell lines. These results have important implications for both tissue-engineered bone grafts and enhancement of HA implants performance, to develop new teeth's supporting structure therapies and replacement. PMID:20357737

  18. Personality affects defensive behaviour of Porcellio scaber (Isopoda, Oniscidea)

    PubMed Central

    Tuf, Ivan Hadrián; Drábková, Lucie; Šipoš, Jan

    2015-01-01

    Abstract We evaluated individual behavioural patterns of isopods expressed as tonic immobility following some intrusive treatments. Common rough woodlice, Porcellio scaber, were kept individually in plastic boxes and tested for tonic immobility repeatedly. Reactivity, sensitivity (number of stimuli needed to respond), and endurance of tonic immobility (TI) according three types of treatments (touch, squeeze, drop) were evaluated. Touch was the weakest treatment and it was necessary to repeat it a number of times to obtain a response; while squeeze and drop induced TI more frequently. Nevertheless, duration of the response persisted for a longer time with the touch treatment. Within each set of the three treatment, the strongest response was the third one, regardless of treatment type. Duration of reaction was affected by the size of the woodlouse, the smallest individuals feigning death for the shortest time. Despite body size, we found a significant individual pattern of endurance of TI among tested woodlice, which was stable across treatments as well as across time (5 repetitions during a 3 week period). Porcellio scaber is one of the first species of terrestrial isopods with documented personality traits. PMID:26261447

  19. Affective and Behavioural Variables: Reforms as Experiments to Produce a Civil Society

    ERIC Educational Resources Information Center

    Fitz-Gibbon, Carol T.

    2006-01-01

    Affective and behavioural indicators of the effects of schooling, and of other interventions, might be more important than cognitive indicators, particularly in the long run and considering the urgent need for civil societies. Examples are provided of the statistical properties of affective and behavioural indicators, and of their current use in…

  20. Targeted overexpression of the two colony-stimulating factor-1 isoforms in osteoblasts differentially affects bone loss in ovariectomized mice

    PubMed Central

    Yao, Gang-Qing; Wu, Jian-Jun; Ovadia, Shira; Troiano, Nancy; Sun, Ben Hua; Insogna, Karl

    2009-01-01

    Colony-stimulating factor-1 (CSF1) is one of two cytokines required for normal osteoclastogenesis. There are two major isoforms of CSF1, the cell-surface or membrane-bound isoform (mCSF1) and soluble CSF1 (sCSF1). Whether these isoforms serve nonredundant functions in bone is unclear. To explore this question, we generated transgenic mice expressing human sCSF1, human mCSF1, or both (s/mCSF1) in osteoblasts using the 2.3-kb rat αI-collagen promoter. Bone density determined by peripheral quantitative computed tomography was significantly reduced in mCSF1, sCSF1, and s/mCSF1 transgenic mice compared with wild-type animals. When analyzed by sex, sCSF1, and s/mCSF1, female animals but not mCSF1 female mice were found to have greater bone loss than their male littermates (−20 vs. −9.2%; P < 0.05 for sCSF1 and −21.6 vs. −11.2% for s/mCSF1; P < 0.01). By breeding CSF1 isoform-selective transgenic mice to an op/op background, mice were generated in which a single CSF1 isoform was the only source of the cytokine (sCSF1op/op and mCSF1op/op). Unlike osteoblast-targeted overexpression of mCSF1, selective transgenic expression of sCSF1 did not completely correct the op/op phenotype in 5-mo-old animals. Interestingly, compared with sham-ovariectomized mice of the same genotype, ovariectomy in sCSF1op/op mice led to a greater loss of spinal bone mineral density (22.1%) than was seen in either mCSF1op/op mice (12.9%) or in wild-type animals (10.9%). Our findings support the conclusion that sCSF1 and mCSF1 serve nonredundant functions in bone and that sCSF1 may play a role in mediating estrogen-deficiency bone loss. PMID:19141689

  1. Dicer ablation in osteoblasts by Runx2 driven cre-loxP recombination affects bone integrity, but not glucocorticoid-induced suppression of bone formation.

    PubMed

    Liu, Peng; Baumgart, Mario; Groth, Marco; Wittmann, Jürgen; Jäck, Hans-Martin; Platzer, Matthias; Tuckermann, Jan P; Baschant, Ulrike

    2016-01-01

    Glucocorticoid-induced osteoporosis (GIO) is one of the major side effects of long-term glucocorticoid (GC) therapy mediated mainly via the suppression of bone formation and osteoblast differentiation independently of GC receptor (GR) dimerization. Since microRNAs play a critical role in osteoblast differentiation processes, we investigated the role of Dicer dependent microRNAs in the GC-induced suppression of osteoblast differentiation. MicroRNA sequencing of dexamethasone-treated wild-type and GR dimer-deficient mesenchymal stromal cells revealed GC-controlled miRNA expression in a GR dimer-dependent and GR dimer-independent manner. To determine the functional relevance of mature miRNAs in GC-induced osteoblast suppression, mice with an osteoblast-specific deletion of Dicer (Dicer(Runx2Cre)) were exposed to glucocorticoids. In vitro generated Dicer-deficient osteoblasts were treated with dexamethasone and analyzed for proliferation, differentiation and mineralization capacity. In vivo, abrogation of Dicer-dependent miRNA biogenesis in osteoblasts led to growth retardation and impaired bone formation. However, subjecting these mice to GIO showed that bone formation was similar reduced in Dicer(Runx2Cre) mice and littermate control mice upon GC treatment. In line, differentiation of Dicer deficient osteoblasts was suppressed to the same extent as wild type cells by GC treatment. Therefore, Dicer-dependent small RNA biogenesis in osteoblasts plays only a minor role in the pathogenesis of GC-induced inhibition of bone formation. PMID:27554624

  2. Dicer ablation in osteoblasts by Runx2 driven cre-loxP recombination affects bone integrity, but not glucocorticoid-induced suppression of bone formation

    PubMed Central

    Liu, Peng; Baumgart, Mario; Groth, Marco; Wittmann, Jürgen; Jäck, Hans-Martin; Platzer, Matthias; Tuckermann, Jan P.; Baschant, Ulrike

    2016-01-01

    Glucocorticoid-induced osteoporosis (GIO) is one of the major side effects of long-term glucocorticoid (GC) therapy mediated mainly via the suppression of bone formation and osteoblast differentiation independently of GC receptor (GR) dimerization. Since microRNAs play a critical role in osteoblast differentiation processes, we investigated the role of Dicer dependent microRNAs in the GC-induced suppression of osteoblast differentiation. MicroRNA sequencing of dexamethasone-treated wild-type and GR dimer-deficient mesenchymal stromal cells revealed GC-controlled miRNA expression in a GR dimer-dependent and GR dimer-independent manner. To determine the functional relevance of mature miRNAs in GC-induced osteoblast suppression, mice with an osteoblast-specific deletion of Dicer (DicerRunx2Cre) were exposed to glucocorticoids. In vitro generated Dicer-deficient osteoblasts were treated with dexamethasone and analyzed for proliferation, differentiation and mineralization capacity. In vivo, abrogation of Dicer-dependent miRNA biogenesis in osteoblasts led to growth retardation and impaired bone formation. However, subjecting these mice to GIO showed that bone formation was similar reduced in DicerRunx2Cre mice and littermate control mice upon GC treatment. In line, differentiation of Dicer deficient osteoblasts was suppressed to the same extent as wild type cells by GC treatment. Therefore, Dicer-dependent small RNA biogenesis in osteoblasts plays only a minor role in the pathogenesis of GC-induced inhibition of bone formation. PMID:27554624

  3. Factors Affecting Mothers' Healthcare-Seeking Behaviour for Childhood Illnesses in a Rural Nigerian Setting

    ERIC Educational Resources Information Center

    Abdulraheem, I. S.; Parakoyi, D. B.

    2009-01-01

    Appropriate healthcare-seeking behaviour could prevent a significant number of child deaths and complications due to ill health. Improving mothers' care-seeking behaviour could also contribute in reducing a large number of child morbidity and mortality in developing countries. This article aims to determine factors affecting healthcare-seeking…

  4. Penile spines affect copulatory behaviour in a primate (Callithrix jacchus).

    PubMed

    Dixson, A F

    1991-03-01

    Androgen-dependent, keratinized "spines" occur on the glans penis in many rodents, primates and other mammals. Since penile spines overlie dermal tactile receptors, they may play a role in copulatory behaviour. An experiment was conducted to test this hypothesis. Sixteen sexually experienced adult male marmosets were paired with ovariectomized females before, and after, removal of penile spines (using thioglycollate cream applied to the glans under anaesthesia) or a sham operation. Spine removal resulted in an increased duration of preintromission pelvic thrusting (mean +/- s.e.m. from 6.87 +/- 1.09 to 14.94 +/- 3.32 s, p = 0.05) and of intromitted thrusting (from 1.73 +/- 0.11 to 2.0 +/- 0.11 s, p less than 0.05). Three males exhibited partial intromissions during some postspinectomy tests, an effect which had not been observed prior to the operation. Sham operations had no behavioural effects. Results indicate that penile spines play a significant (but not indispensible) role in sensory feedback during copulation in this primate species. PMID:2062934

  5. Rising CO2 concentrations affect settlement behaviour of larval damselfishes

    NASA Astrophysics Data System (ADS)

    Devine, B. M.; Munday, P. L.; Jones, G. P.

    2012-03-01

    Reef fish larvae actively select preferred benthic habitat, relying on olfactory, visual and acoustic cues to discriminate between microhabitats at settlement. Recent studies show exposure to elevated carbon dioxide (CO2) impairs olfactory cue recognition in larval reef fishes. However, whether this alters the behaviour of settling fish or disrupts habitat selection is unknown. Here, the effect of elevated CO2 on larval behaviour and habitat selection at settlement was tested in three species of damselfishes (family Pomacentridae) that differ in their pattern of habitat use: Pomacentrus amboinensis (a habitat generalist), Pomacentrus chrysurus (a rubble specialist) and Pomacentrus moluccensis (a live coral specialist). Settlement-stage larvae were exposed to current-day CO2 levels or CO2 concentrations that could occur by 2100 (700 and 850 ppm) based on IPCC emission scenarios. First, pair-wise choice tests were performed using a two-channel flume chamber to test olfactory discrimination between hard coral, soft coral and coral rubble habitats. The habitat selected by settling fish was then compared among treatments using a multi-choice settlement experiment conducted overnight. Finally, settlement timing between treatments was compared across two lunar cycles for one of the species, P. chrysurus. Exposure to elevated CO2 disrupted the ability of larvae to discriminate between habitat odours in olfactory trials. However, this had no effect on the habitats selected at settlement when all sensory cues were available. The timing of settlement was dramatically altered by CO2 exposure, with control fish exhibiting peak settlement around the new moon, whereas fish exposed to 850 ppm CO2 displaying highest settlement rates around the full moon. These results suggest larvae can rely on other sensory information, such as visual cues, to compensate for impaired olfactory ability when selecting settlement habitat at small spatial scales. However, rising CO2 could cause larvae

  6. Large-scale climatic anomalies affect marine predator foraging behaviour and demography

    NASA Astrophysics Data System (ADS)

    Bost, Charles A.; Cotté, Cedric; Terray, Pascal; Barbraud, Christophe; Bon, Cécile; Delord, Karine; Gimenez, Olivier; Handrich, Yves; Naito, Yasuhiko; Guinet, Christophe; Weimerskirch, Henri

    2015-10-01

    Determining the links between the behavioural and population responses of wild species to environmental variations is critical for understanding the impact of climate variability on ecosystems. Using long-term data sets, we show how large-scale climatic anomalies in the Southern Hemisphere affect the foraging behaviour and population dynamics of a key marine predator, the king penguin. When large-scale subtropical dipole events occur simultaneously in both subtropical Southern Indian and Atlantic Oceans, they generate tropical anomalies that shift the foraging zone southward. Consequently the distances that penguins foraged from the colony and their feeding depths increased and the population size decreased. This represents an example of a robust and fast impact of large-scale climatic anomalies affecting a marine predator through changes in its at-sea behaviour and demography, despite lack of information on prey availability. Our results highlight a possible behavioural mechanism through which climate variability may affect population processes.

  7. Large-scale climatic anomalies affect marine predator foraging behaviour and demography

    PubMed Central

    Bost, Charles A.; Cotté, Cedric; Terray, Pascal; Barbraud, Christophe; Bon, Cécile; Delord, Karine; Gimenez, Olivier; Handrich, Yves; Naito, Yasuhiko; Guinet, Christophe; Weimerskirch, Henri

    2015-01-01

    Determining the links between the behavioural and population responses of wild species to environmental variations is critical for understanding the impact of climate variability on ecosystems. Using long-term data sets, we show how large-scale climatic anomalies in the Southern Hemisphere affect the foraging behaviour and population dynamics of a key marine predator, the king penguin. When large-scale subtropical dipole events occur simultaneously in both subtropical Southern Indian and Atlantic Oceans, they generate tropical anomalies that shift the foraging zone southward. Consequently the distances that penguins foraged from the colony and their feeding depths increased and the population size decreased. This represents an example of a robust and fast impact of large-scale climatic anomalies affecting a marine predator through changes in its at-sea behaviour and demography, despite lack of information on prey availability. Our results highlight a possible behavioural mechanism through which climate variability may affect population processes. PMID:26506134

  8. Large-scale climatic anomalies affect marine predator foraging behaviour and demography.

    PubMed

    Bost, Charles A; Cotté, Cedric; Terray, Pascal; Barbraud, Christophe; Bon, Cécile; Delord, Karine; Gimenez, Olivier; Handrich, Yves; Naito, Yasuhiko; Guinet, Christophe; Weimerskirch, Henri

    2015-01-01

    Determining the links between the behavioural and population responses of wild species to environmental variations is critical for understanding the impact of climate variability on ecosystems. Using long-term data sets, we show how large-scale climatic anomalies in the Southern Hemisphere affect the foraging behaviour and population dynamics of a key marine predator, the king penguin. When large-scale subtropical dipole events occur simultaneously in both subtropical Southern Indian and Atlantic Oceans, they generate tropical anomalies that shift the foraging zone southward. Consequently the distances that penguins foraged from the colony and their feeding depths increased and the population size decreased. This represents an example of a robust and fast impact of large-scale climatic anomalies affecting a marine predator through changes in its at-sea behaviour and demography, despite lack of information on prey availability. Our results highlight a possible behavioural mechanism through which climate variability may affect population processes. PMID:26506134

  9. FGF-2 induces the proliferation of human periodontal ligament cells and modulates their osteoblastic phenotype by affecting Runx2 expression in the presence and absence of osteogenic inducers

    PubMed Central

    AN, SHAOFENG; HUANG, XIANGYA; GAO, YAN; LING, JUNQI; HUANG, YIHUA; XIAO, YIN

    2015-01-01

    The exact phenotype of human periodontal ligament cells (hPDLCs) remains a controversial area. Basic fibroblast growth factor (FGF-2) exhibits various functions and its effect on hPDLCs is also controversial. Therefore, the present study examined the effect of FGF-2 on the growth and osteoblastic phenotype of hPDLCs with or without osteogenic inducers (dexamethasone and β-glycerophosphate). FGF-2 was added to defined growth culture medium and osteogenic inductive culture medium. Cell proliferation, osteogenic differentiation and mineralization were measured. The selected differentiation markers, Runx2, collagen type I, α1 (Col1a1), osteocalcin (OCN) and epidermal growth factor receptor (EGFR), were investigated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Runx2 and OCN protein expression was measured by western blotting. FGF-2 significantly increased the proliferation of hPDLCs, but did not affect alkaline phosphatase activity. RT-qPCR analysis revealed enhanced mRNA expression of Runx2, OCN and EGFR, but suppressed Col1a1 gene expression in the absence of osteogenic inducers, whereas all these gene levels had no clear trend in their presence. The Runx2 protein expression was clearly increased, but the OCN protein level showed no evident trend. The mineralization assay demonstrated that FGF-2 inhibited mineralized matrix deposition with osteogenic inducers. These results suggested that FGF-2 induces the growth of immature hPDLCs, which is a competitive inhibitor of epithelial downgrowth, and suppresses their differentiation into mineralized tissue by affecting Runx2 expression. Therefore, this may lead to the acceleration of periodontal regeneration. PMID:26133673

  10. Mother-Child Affect and Emotion Socialization Processes across the Late Preschool Period: Predictions of Emerging Behaviour Problems

    ERIC Educational Resources Information Center

    Newland, Rebecca P.; Crnic, Keith A.

    2011-01-01

    The current study examined concurrent and longitudinal relations between maternal negative affective behaviour and child negative emotional expression in preschool age children with (n=96) or without (n=126) an early developmental risk, as well as the predictions of later behaviour problems. Maternal negative affective behaviour, child…

  11. How will climate change affect vine behaviour in different soils?

    NASA Astrophysics Data System (ADS)

    Leibar, Urtzi; Aizpurua, Ana; Morales, Fermin; Pascual, Inmaculada; Unamunzaga, Olatz

    2014-05-01

    and water-deficit had a clear influence on the grape phenological development and composition, whilst soil affected root configuration and anthocyanins concentration. Effects of climate change and water availability on different soil conditions should be considered to take full advantage or mitigate the consequences of the future climate conditions.

  12. Teacher Interpersonal Behaviour and Secondary Students' Cognitive, Affective and Moral Outcomes in Hong Kong

    ERIC Educational Resources Information Center

    Sivan, Atara; Chan, Dennis W. K.

    2013-01-01

    This study validated the Chinese version of the Questionnaire on Teacher Interaction (QTI) in the Hong Kong context as well as examined the relationship between students' perceptions of interpersonal teacher behaviour and their cognitive, affective and moral learning outcomes. Data were collected with the QTI and four other measures of student…

  13. Comorbid LD and ADHD in Childhood: Socioemotional and Behavioural Adjustment and Parents' Positive and Negative Affect

    ERIC Educational Resources Information Center

    Al-Yagon, Michal

    2009-01-01

    The present study examined how vulnerability and protective factors at the individual level (child's disabilities; patterns of attachment), and at the family level (fathers'/mothers' affect), help explain differences in socioemotional and behavioural adjustment among children aged 8-12 years with comorbid learning disability (LD) and attention…

  14. Larval traits carry over to affect post-settlement behaviour in a common coral reef fish.

    PubMed

    Dingeldein, Andrea L; White, J Wilson

    2016-07-01

    Most reef fishes begin life as planktonic larvae before settling to the reef, metamorphosing and entering the benthic adult population. Different selective forces determine survival in the planktonic and benthic life stages, but traits established in the larval stage may carry over to affect post-settlement performance. We tested the hypothesis that larval traits affect two key post-settlement fish behaviours: social group-joining and foraging. Certain larval traits of reef fishes are permanently recorded in the rings in their otoliths. In the bluehead wrasse (Thalassoma bifasciatum), prior work has shown that key larval traits recorded in otoliths (growth rate, energetic condition at settlement) carry over to affect post-settlement survival on the reef, with higher-larval-condition fish experiencing less post-settlement mortality. We hypothesized that this selective mortality is mediated by carry-over effects on post-settlement antipredator behaviours. We predicted that better-condition fish would forage less and be more likely to join groups, both behaviours that would reduce predation risk. We collected 550 recently settled bluehead wrasse (Thalassoma bifasciatum) from three reef sites off St. Croix (USVI) and performed two analyses. First, we compared each settler's larval traits to the size of its social group to determine whether larval traits influenced group-joining behaviour. Secondly, we observed foraging behaviour in a subset of grouped and solitary fish (n = 14) for 1-4 days post-settlement. We then collected the fish and tested whether larval traits influenced the proportion of time spent foraging. Body length at settlement, but not condition, affected group-joining behaviour; smaller fish were more likely to remain solitary or in smaller groups. However, both greater length and better condition were associated with greater proportions of time spent foraging over four consecutive days post-settlement. Larval traits carry over to affect post

  15. Chronic low dose tumor necrosis factor-α (TNF) suppresses early bone accrual in young mice by inhibiting osteoblasts without affecting osteoclasts.

    PubMed

    Gilbert, L C; Chen, H; Lu, X; Nanes, M S

    2013-09-01

    The inflammatory cytokine tumor necrosis factor-α (TNF-α) is known to cause bone resorption and inhibit bone formation in arthritis and aging but less is known about TNF effects in the young growing skeleton. While investigating the mechanism of bone loss in TNF transgenic mice, we identified an early TNF-sensitive period marked by suppression of osteoblasts and bone accrual as the sole mechanism of TNF action, without an effect on osteoclasts or bone resorption. TgTNF mice express low concentrations of hTNFα (≤5 pg/ml). Osteoblasts cultured from TgTNF mice express reduced levels of RUNX2, Osx, alkaline phosphatase, bone sialoprotein, and osteocalcin and have delayed formation of mineralized nodules. Early accrual of bone in TgTNF mice is suppressed until 6 weeks of age, after which the rate of bone accrual normalizes without catch up. Histomorphometry revealed that TgTNF mice fail to generate a transient surge in osteoblast number that is seen in wild type (WT) mice at 4 weeks. Osteoclasts, TRAP staining, erosive surfaces, serum CTx, and OPG/RANKL expression did not differ between young TgTNF and WT mice. Canonical Wnts and signaling through β-catenin were reduced in TgTNF mice at 4 weeks and partially recovered by 12 weeks, associated with reduced cytoplasm to nuclear transfer of β-catenin and Wnt regulated genes. TgTNF mice were crossed with BatGal Wnt reporter mice. Active Wnt signaling in tibial trabecular lining cells was reduced in TgTNF mice at 4 weeks compared to control littermates. Our results demonstrate that a low dose inflammatory stimulus is sufficient to inhibit the early surge in osteoblasts and optimal bone formation of young mice independent of changes in osteoclasts. TNF inhibition of the Wnt pathway contributes to the suppression of osteoblasts. PMID:23756233

  16. Detection of genetic variants affecting cattle behaviour and their impact on milk production: a genome-wide association study.

    PubMed

    Friedrich, Juliane; Brand, Bodo; Ponsuksili, Siriluck; Graunke, Katharina L; Langbein, Jan; Knaust, Jacqueline; Kühn, Christa; Schwerin, Manfred

    2016-02-01

    Behaviour traits of cattle have been reported to affect important production traits, such as meat quality and milk performance as well as reproduction and health. Genetic predisposition is, together with environmental stimuli, undoubtedly involved in the development of behaviour phenotypes. Underlying molecular mechanisms affecting behaviour in general and behaviour and productions traits in particular still have to be studied in detail. Therefore, we performed a genome-wide association study in an F2 Charolais × German Holstein cross-breed population to identify genetic variants that affect behaviour-related traits assessed in an open-field and novel-object test and analysed their putative impact on milk performance. Of 37,201 tested single nucleotide polymorphism (SNPs), four showed a genome-wide and 37 a chromosome-wide significant association with behaviour traits assessed in both tests. Nine of the SNPs that were associated with behaviour traits likewise showed a nominal significant association with milk performance traits. On chromosomes 14 and 29, six SNPs were identified to be associated with exploratory behaviour and inactivity during the novel-object test as well as with milk yield traits. Least squares means for behaviour and milk performance traits for these SNPs revealed that genotypes associated with higher inactivity and less exploratory behaviour promote higher milk yields. Whether these results are due to molecular mechanisms simultaneously affecting behaviour and milk performance or due to a behaviour predisposition, which causes indirect effects on milk performance by influencing individual reactivity, needs further investigation. PMID:26515756

  17. Protection behaviour: a phenomenon affecting organ and tissue donation in the 21st century?

    PubMed

    Kent, B C

    2004-03-01

    UK statistics show that whilst waiting lists for transplantation surgery continue to increase, donor numbers are static. This paper describes the hermeneutic phase of a mixed method study and puts forward the concept of protection behaviour as one explanation for nurses' reticence to discuss post-mortem donation wishes with patients' relatives. The desire to protect appears to influence attitudes, confidence levels and perceived ability to become involved in donor identification and donation discussion, consequently affecting the availability of transplantable organs and tissue. By understanding more fully why protective behaviours are employed, it increases the likelihood of a solution being found. PMID:14967184

  18. Under a neighbour's influence: public information affects stress hormones and behaviour of a songbird

    PubMed Central

    Cornelius, Jamie M.; Breuner, Creagh W.; Hahn, Thomas P.

    2010-01-01

    Socially acquired information improves the accuracy and efficiency of environmental assessments and can increase fitness. Public information may be especially useful during unpredictable food conditions, or for species that depend on resources made less predictable by human disturbance. However, the physiological mechanisms by which direct foraging assessments and public information are integrated to affect behaviour remain largely unknown. We tested for potential effects of public information on the behavioural and hormonal response to food reduction by manipulating the social environment of captive red crossbills (Loxia curvirostra). Red crossbills are irruptive migrants that are considered sensitive to changes in food availability and use public information in decision making. Here, we show that public information can attenuate or intensify the release of glucocorticoids (i.e. stress hormones) during food shortage in red crossbills. The observed modulation of corticosterone may therefore be a physiological mechanism linking public information, direct environmental assessments and behavioural change. This mechanism would not only allow for public information to affect individual behaviour, but might also facilitate group decision making by bringing group members into more similar physiological states. The results further suggest that stressors affecting entire populations may be magnified in individual physiology through social interactions. PMID:20356895

  19. Autonomous motivation is associated with the maintenance stage of behaviour change in people with affective disorders.

    PubMed

    Vancampfort, Davy; Moens, Herman; Madou, Tomas; De Backer, Tanja; Vallons, Veerle; Bruyninx, Peter; Vanheuverzwijn, Sarah; Mota, Cindy Teixeira; Soundy, Andy; Probst, Michel

    2016-06-30

    The present study examined whether in people with affective disorders motives for adopting and maintaining physical activity recommendations (as formulated by the self-determination theory) differed across the stages of behaviour change (identified by the transtheoretical model). A total of 165 (105♀) persons (45.6±14.2years) with affective disorders [major depressive disorder (n=96) or bipolar disorder (n=69)] completed the Behavioural Regulation in Exercise Questionnaire-2 and the Patient-centred Assessment and Counselling for Exercise questionnaire. Discriminant and multivariate analyses demonstrated that persons with affective disorders at the early stages of change have less autonomous and more controlled physical activity motives than those at the later stages. Our results suggest that autonomous motivation may have an important role to play in the maintenance of health recommendations in persons with affective disorders. Longitudinal and intervention studies should be designed in people with affective disorders to identify the causal pathways between motives for maintaining health recommendations, effective changes in health behaviour and physical and mental health outcomes. PMID:27131627

  20. Telomerized presenescent osteoblasts prevent bone mass loss in vivo.

    PubMed

    Yudoh, K; Nishioka, K

    2004-06-01

    Previously, we showed that human osteoblasts expressing the human telomerase reverse transcriptase (hTERT) gene exhibited specific survival advantages--the result of breaching the replicative senescence barrier and maintaining the phenotypic and functional properties of primary osteoblasts in vitro over the total replicative capacity of primary osteoblasts. We postulated that rejuvenated osteoblasts may have a potential to correct bone loss or osteopenia in age-related osteoporotic diseases. In the present study, we studied whether telomerized presenescent osteoblasts prevent bone mass loss in vivo. After obtaining the informed consent from a patient with osteoarthritis who underwent the arthroplastic knee surgery, osteoblastic cells were isolated from donor bone sample. We transfected the gene encoding hTERT into human osteoblastic cells. Human bone fragments from a donor were incubated with human hTERT-transfected presenescent (in vitro aged) osteoblasts or mock-transfected presenescent osteoblasts in culture medium containing Matrigel. We subcutaneously implanted human bone fragments with telomerized presenescent osteoblasts or primary presenescent osteoblasts as three-dimensional Matrigel xenografts in severe combined immunodeficiency (SCID) mice (each group: six mice) and analyzed the grafts at 6 weeks after implantation. We also determined whether telomerized osteoblasts affect the bone-forming capacity in vivo, using a well-established mouse transplantation model in which ceramic hydroxyapatite/tricalcium phosphate particles are used as carrier vehicle. Telomerized presenescent osteoblasts were rejuvenated, and maintained the functional properties of young osteoblasts in vitro. Bone mineral content (BMC) and bone mineral density (BMD) were measured by ash weight and dual-energy X-ray absorptiometry, respectively. Whereas BMC and BMD of human bone fragments, which were inoculated with aged osteoblasts in SCID mice, decreased with time, telomerized

  1. Comparison of elicitation methods for moral and affective beliefs in the theory of planned behaviour.

    PubMed

    Dean, M; Arvola, A; Vassallo, M; Lähteenmäki, L; Raats, M M; Saba, A; Shepherd, R

    2006-09-01

    Although the theory of planned behaviour (TPB) has been applied successfully in the area of food choice, it has been criticized for its pure utilitarian approach to the factors determining behaviour. Despite the increase in predictive power of the model with added components such as affective attitude and moral and ethical concerns, in most studies the elicitation process still only addresses people's utilitarian beliefs about the behaviour with little attention paid to other aspects. This study compares the traditional method of elicitation of advantages and disadvantages with two other methods (word association and open-ended) in the elicitations of beliefs, attitudes and moral concerns in relation to the consumption of organic foods. Results show the traditional method to be best for eliciting cognitive beliefs, open-ended emotion task for eliciting emotional beliefs and open-ended beliefs task best for moral concerns. The advantages and disadvantages of each method are discussed. PMID:16782230

  2. Osteoblast-derived VEGF regulates osteoblast differentiation and bone formation during bone repair

    PubMed Central

    Hu, Kai; Olsen, Bjorn R.

    2016-01-01

    Osteoblast-derived VEGF is important for bone development and postnatal bone homeostasis. Previous studies have demonstrated that VEGF affects bone repair and regeneration; however, the cellular mechanisms by which it works are not fully understood. In this study, we investigated the functions of osteoblast-derived VEGF in healing of a bone defect. The results indicate that osteoblast-derived VEGF plays critical roles at several stages in the repair process. Using transgenic mice with osteoblast-specific deletion of Vegfa, we demonstrated that VEGF promoted macrophage recruitment and angiogenic responses in the inflammation phase, and optimal levels of VEGF were required for coupling of angiogenesis and osteogenesis in areas where repair occurs by intramembranous ossification. VEGF likely functions as a paracrine factor in this process because deletion of Vegfr2 in osteoblastic lineage cells enhanced osteoblastic maturation and mineralization. Furthermore, osteoblast- and hypertrophic chondrocyte–derived VEGF stimulated recruitment of blood vessels and osteoclasts and promoted cartilage resorption at the repair site during the periosteal endochondral ossification stage. Finally, osteoblast-derived VEGF stimulated osteoclast formation in the final remodeling phase of the repair process. These findings provide a basis for clinical strategies to improve bone regeneration and treat defects in bone healing. PMID:26731472

  3. Enriching early adult environment affects the copulation behaviour of a tephritid fly.

    PubMed

    Díaz-Fleischer, Francisco; Arredondo, José; Aluja, Martín

    2009-07-01

    Early adult experiences in enriched environments favours animal brain and behavioural development ultimately resulting in an increased fitness. However, measuring the effect of environmental enrichment in animal behaviour in nature is often a complicated task, considering the complexity of the natural environment. We expanded previous studies to evaluate how early experience in an enriched environment affects copulation behaviour when animals are confronted with a complex semi-natural environment. Anastrepha ludens flies are an ideal model system for studying these effects because their natural habitats differ significantly from the cage environments in which these flies are reared for biological control purposes. For example, in the field, males form leks of up to six individuals. Each male defends a territory represented by a tree leaf whereas in rearing cages, territories are completely reduced because of the high population density. In a series of three experiments, we observed that male density represented the most influential stimulus for A. ludens male copulation success. Males that experienced lower densities in early adulthood obtained the highest proportion of copulations. By contrast, female copulation behaviour was not altered by female density. However, exposure to natural or artificial leaves in cages in which flies were kept until tested influenced female copulation behaviour. Females that were exposed to enriched environments exhibited a shorter latency to mate and shorter copulation durations with males than females reared in poor environments. We discuss the influence of early experience on male copulation success and female-mating choosiness. PMID:19525439

  4. Effects of Hypogravity on Osteoblast Differentiation

    NASA Technical Reports Server (NTRS)

    Globus, Ruth; Doty, Steven

    1997-01-01

    Weightbearing is essential for normal skeletal function. Without weightbearing, the rate of bone formation by osteoblasts decreases in the growing rat. Defective formation may account for the decrease in the maturation, strength and mass of bone that is caused by spaceflight. These skeletal defects may be mediated by a combination of physiologic changes triggered by spaceflight, including skeletal unloading, fluid shifts, and stress-induced endocrine factors. The fundamental question of whether the defects in osteoblast function due to weightlessness are mediated by localized skeletal unloading or by systemic physiologic adaptations such as fluid shifts has not been answered. Furthermore, bone-forming activity of osteoblasts during unloading may be affected by paracrine signals from vascular, monocytic, and neural cells that also reside in skeletal tissue. Therefore we proposed to examine whether exposure of cultured rat osteoblasts to spaceflight inhibits cellular differentiation and impairs mineralization when isolated from the influence of both systemic factors and other skeletal cells.

  5. Sepsis-Induced Osteoblast Ablation Causes Immunodeficiency.

    PubMed

    Terashima, Asuka; Okamoto, Kazuo; Nakashima, Tomoki; Akira, Shizuo; Ikuta, Koichi; Takayanagi, Hiroshi

    2016-06-21

    Sepsis is a host inflammatory response to severe infection associated with high mortality that is caused by lymphopenia-associated immunodeficiency. However, it is unknown how lymphopenia persists after the accelerated lymphocyte apoptosis subsides. Here we show that sepsis rapidly ablated osteoblasts, which reduced the number of common lymphoid progenitors (CLPs). Osteoblast ablation or inducible deletion of interleukin-7 (IL-7) in osteoblasts recapitulated the lymphopenic phenotype together with a lower CLP number without affecting hematopoietic stem cells (HSCs). Pharmacological activation of osteoblasts improved sepsis-induced lymphopenia. This study demonstrates a reciprocal interaction between the immune and bone systems, in which acute inflammation induces a defect in bone cells resulting in lymphopenia-associated immunodeficiency, indicating that bone cells comprise a therapeutic target in certain life-threatening immune reactions. PMID:27317262

  6. Aerobic Glycolysis in Osteoblasts

    PubMed Central

    Esen, Emel; Long, Fanxin

    2014-01-01

    Osteoblasts, the chief bone-making cells in the body, are a focus of osteoporosis research. Although teriparatite, a synthetic fragment of the human parathyroid hormone (PTH), has been an effective bone anabolic drug, there remains a clinical need for additional therapeutics that safely stimulates osteoblast number and function. Work in the past several decades has provided unprecedented clarity about the roles of growth factors and transcription factors in regulating osteoblast differentiation and activity, but whether these factors may regulate cellular metabolism to influence cell fate and function has been largely unexplored. The past few years have witnessed a resurgence of interest in the cellular metabolism of osteoblasts, with the hope that elucidation of their metabolic profile may open new avenues for developing bone anabolic agents. Here we review the current understanding about glucose metabolism in osteoblasts. PMID:25200872

  7. Associations of negative affect and eating behaviour in obese women with and without binge eating disorder.

    PubMed

    Schulz, S; Laessle, R G

    2010-12-01

    The present study was planned to investigate differences in psychopathological features, eating behaviour and eating habits between obese women with and without BED. It also aimed to identify specific relationships between affective symptoms and eating behaviour in obese women with BED. Eighty-four obese women were studied (40 with BED, 44 non-BED). Psychiatric comorbidities were assessed with the structured diagnostic interview for DSM-IV (SCID). Depressive symptoms were measured with the Beck Depression Inventory (BDI) and anxiety with the state-trait anxiety inventory (STAI). Eating habits (emotional and restrained eating) were assessed by the Dutch eating behaviour questionnaire (DEBQ). Food diaries were used for assessing naturalistic eating behaviour (food intake) and mood before and after food intake. BED subjects exhibited higher levels of comorbidity (in particular mood disorders, anxiety disorders and substance-related disorders), higher depressive symptoms, trait anxiety, external and emotional eating scores than non-BED subjects. Regression analyses revealed that anxiety and emotional eating were significant predictors for BED status. In the BED group, depressive symptoms were significantly related to emotional eating and food intake and negatively related to restraint. Anxiety was significantly related to emotional eating. In general, food intake significantly enhanced mood. Mood was worse on the days with self-reported binge eating episodes than on nonbinge days. These results are discussed with regard to aetiological models for BED and for BED being a distinct diagnostic category separate from obesity. PMID:21406953

  8. Inflorescence architecture affects pollinator behaviour and mating success in Spiranthes sinensis (Orchidaceae).

    PubMed

    Iwata, Tatsunori; Nagasaki, Osamu; Ishii, Hiroshi S; Ushimaru, Atushi

    2012-01-01

    • Despite the wide inflorescence diversity among angiosperms, the effects of inflorescence architecture (three-dimensional flower arrangement) on pollinator behaviour and mating success have not been sufficiently studied in natural plant populations. • Here, we investigated how inflorescence architecture affected inter- and intra-plant pollinator movements and consequent mating success in a field population of Spiranthes sinensis var. amoena (S. sinensis). In this species, the flowers are helically arranged around the stem, and the degree of twisting varies greatly among individuals. The large variation in inflorescence architecture in S. sinensis results from variation in a single structural parameter, the helical angle (the angular distance between neighbour-flower directions). • The numbers of visits per inflorescence and successive probes per visit by leaf-cutting bees decreased with helical angle, indicating that individual flowers of tightly twisted inflorescences received less visitations. As expected from pollinator behaviour, pollinia removal and fruit set of individual flowers decreased with helical angle. Meanwhile, geitonogamy decreased in tightly twisted inflorescences. • Our novel findings demonstrate that natural variation in inflorescence architecture significantly affects pollinator behaviour and reproductive success, suggesting that inflorescence architecture can evolve under pollinator-mediated natural selection in plant populations. We also discuss how diverse inflorescence architectures may have been maintained in S. sinensis populations. PMID:21919912

  9. Thermoregulatory behaviour affects prevalence of chytrid fungal infection in a wild population of Panamanian golden frogs.

    PubMed

    Richards-Zawacki, Corinne L

    2010-02-22

    Predicting how climate change will affect disease dynamics requires an understanding of how the environment affects host-pathogen interactions. For amphibians, global declines and extinctions have been linked to a pathogenic chytrid fungus, Batrachochytrium dendrobatidis. Using a combination of body temperature measurements and disease assays conducted before and after the arrival of B. dendrobatidis, this study tested the hypothesis that body temperature affects the prevalence of infection in a wild population of Panamanian golden frogs (Atelopus zeteki). The timing of first detection of the fungus was consistent with that of a wave of epidemic infections spreading south and eastward through Central America. During the epidemic, many golden frogs modified their thermoregulatory behaviour, raising body temperatures above their normal set point. Odds of infection decreased with increasing body temperature, demonstrating that even slight environmental or behavioural changes have the potential to affect an individual's vulnerability to infection. The thermal dependency of the relationship between B. dendrobatidis and its amphibian hosts demonstrates how the progression of an epidemic can be influenced by complex interactions between host and pathogen phenotypes and the environments in which they are found. PMID:19864287

  10. The roles of the amygdala in the affective regulation of body, brain, and behaviour

    NASA Astrophysics Data System (ADS)

    Mirolli, Marco; Mannella, Francesco; Baldassarre, Gianluca

    2010-09-01

    Despite the great amount of knowledge produced by the neuroscientific literature on affective phenomena, current models tackling non-cognitive aspects of behaviour are often bio-inspired but rarely bio-constrained. This paper presents a theoretical account of affective systems centred on the amygdala (Amg). This account aims to furnish a general framework and specific pathways to implement models that are more closely related to biological evidence. The Amg, which receives input from brain areas encoding internal states, innately relevant stimuli, and innately neutral stimuli, plays a fundamental role in the motivational and emotional processes of organisms. This role is based on the fact that Amg implements the two associative processes at the core of Pavlovian learning (conditioned stimulus (CS)-unconditioned stimulus (US) and CS-unconditioned response (UR) associations), and that it has the capacity of modulating these associations on the basis of internal states. These functionalities allow the Amg to play an important role in the regulation of the three fundamental classes of affective responses (namely, the regulation of body states, the regulation of brain states via neuromodulators, and the triggering of a number of basic behaviours fundamental for adaptation) and in the regulation of three high-level cognitive processes (namely, the affective labelling of memories, the production of goal-directed behaviours, and the performance of planning and complex decision-making). Our analysis is conducted within a methodological approach that stresses the importance of understanding the brain within an evolutionary/adaptive framework and with the aim of isolating general principles that can potentially account for the wider possible empirical evidence in a coherent fashion.

  11. Keeper-Animal Interactions: Differences between the Behaviour of Zoo Animals Affect Stockmanship.

    PubMed

    Ward, Samantha J; Melfi, Vicky

    2015-01-01

    Stockmanship is a term used to describe the management of animals with a good stockperson someone who does this in a in a safe, effective, and low-stress manner for both the stock-keeper and animals involved. Although impacts of unfamiliar zoo visitors on animal behaviour have been extensively studied, the impact of stockmanship i.e familiar zoo keepers is a new area of research; which could reveal significant ramifications for zoo animal behaviour and welfare. It is likely that different relationships are formed dependant on the unique keeper-animal dyad (human-animal interaction, HAI). The aims of this study were to (1) investigate if unique keeper-animal dyads were formed in zoos, (2) determine whether keepers differed in their interactions towards animals regarding their attitude, animal knowledge and experience and (3) explore what factors affect keeper-animal dyads and ultimately influence animal behaviour and welfare. Eight black rhinoceros (Diceros bicornis), eleven Chapman's zebra (Equus burchellii), and twelve Sulawesi crested black macaques (Macaca nigra) were studied in 6 zoos across the UK and USA. Subtle cues and commands directed by keepers towards animals were identified. The animals latency to respond and the respective behavioural response (cue-response) was recorded per keeper-animal dyad (n = 93). A questionnaire was constructed following a five-point Likert Scale design to record keeper demographic information and assess the job satisfaction of keepers, their attitude towards the animals and their perceived relationship with them. There was a significant difference in the animals' latency to appropriately respond after cues and commands from different keepers, indicating unique keeper-animal dyads were formed. Stockmanship style was also different between keepers; two main components contributed equally towards this: "attitude towards the animals" and "knowledge and experience of the animals". In this novel study, data demonstrated unique dyads

  12. Keeper-Animal Interactions: Differences between the Behaviour of Zoo Animals Affect Stockmanship

    PubMed Central

    Ward, Samantha J.; Melfi, Vicky

    2015-01-01

    Stockmanship is a term used to describe the management of animals with a good stockperson someone who does this in a in a safe, effective, and low-stress manner for both the stock-keeper and animals involved. Although impacts of unfamiliar zoo visitors on animal behaviour have been extensively studied, the impact of stockmanship i.e familiar zoo keepers is a new area of research; which could reveal significant ramifications for zoo animal behaviour and welfare. It is likely that different relationships are formed dependant on the unique keeper-animal dyad (human-animal interaction, HAI). The aims of this study were to (1) investigate if unique keeper-animal dyads were formed in zoos, (2) determine whether keepers differed in their interactions towards animals regarding their attitude, animal knowledge and experience and (3) explore what factors affect keeper-animal dyads and ultimately influence animal behaviour and welfare. Eight black rhinoceros (Diceros bicornis), eleven Chapman’s zebra (Equus burchellii), and twelve Sulawesi crested black macaques (Macaca nigra) were studied in 6 zoos across the UK and USA. Subtle cues and commands directed by keepers towards animals were identified. The animals latency to respond and the respective behavioural response (cue-response) was recorded per keeper-animal dyad (n = 93). A questionnaire was constructed following a five-point Likert Scale design to record keeper demographic information and assess the job satisfaction of keepers, their attitude towards the animals and their perceived relationship with them. There was a significant difference in the animals’ latency to appropriately respond after cues and commands from different keepers, indicating unique keeper-animal dyads were formed. Stockmanship style was also different between keepers; two main components contributed equally towards this: “attitude towards the animals” and “knowledge and experience of the animals”. In this novel study, data demonstrated

  13. Impaired oculo-motor behaviour affects both reading and scene perception in neglect patients.

    PubMed

    Primativo, Silvia; Arduino, Lisa S; Daini, Roberta; De Luca, Maria; Toneatto, Carlo; Martelli, Marialuisa

    2015-04-01

    Unilateral spatial neglect (USN) is a common neuropsychological disorder following a right-sided brain lesion. Although USN is mostly characterized by symptoms involving the left hemispace, other symptoms are not left lateralized. Recently, it was shown that patients with neglect dyslexia, a reading disturbance that affects about 40% of USN patients, manifest a non-lateralized impairment of eye movement behaviour in association with their reading deficit when they read aloud and perform non-verbal saccadic tasks (Primativo et al., 2013). In the present paper, we aimed to demonstrate that the eye movement impairment shown by some USN patients reflects a more general oculo-motor disorder that is not confined to orthographic material, the horizontal axis or constrained saccadic tasks. We conjectured that inaccurate oculo-motor behaviour in USN patients indicates the presence of a reading deficit. With this aim we evaluated 20 patients, i.e., 10 right-sided brain-damaged patients without neglect and 10 patients affected by USN. On the basis of the patients' eye movement patterns during a scene exploration task, we found that 4 out of the 10 USN patients presented an abnormal oculo-motor pattern. These same four patients (but not the others) also failed in performing 5 different saccadic tasks and produced neglect dyslexia reading errors in both single words and texts. First, we show that a large proportion of USN patients have inaccurate eye movement behaviour in non-reading tasks. Second, we demonstrate that this exploratory deficit is predictive of the reading impairment. Thus, we conclude that the eye movement deficit prevents reading and impairs the performance on many other perceptual tests, including scene exploration. The large percentage of patients with impaired eye-movement pattern suggests that particular attention should be paid to eye movement behaviour during the diagnostic phase in order to program the best rehabilitation strategy for each patient. PMID

  14. Do Bells Affect Behaviour and Heart Rate Variability in Grazing Dairy Cows?

    PubMed Central

    Johns, Julia; Patt, Antonia; Hillmann, Edna

    2015-01-01

    In alpine regions cows are often equipped with bells. The present study investigated the impact of wearing a bell on behaviour and heart rate variability in dairy cows. Nineteen non-lactating Brown-Swiss cows with bell experience were assigned to three different treatments. For 3 days each, cows were equipped with no bell (control), with a bell with inactivated clapper (silent bell) or with a functional bell (functional bell). The bells weighed 5.5 kg and had frequencies between 532 Hz and 2.8 kHz and amplitudes between 90 and 113 dB at a distance of 20 cm. Data were collected on either the first and third or on all 3 days of each treatment. Whereas duration of rumination was reduced with a functional bell and a silent bell compared with no bell, feeding duration was reduced with a silent bell and was intermediate with a functional bell. Head movements were reduced when wearing a silent bell compared with no bell and tended to be reduced when wearing a functional compared to no bell. With a functional bell, lying duration was reduced by almost 4 hours on the third day of treatment compared with the first day with a functional bell and compared with no bell or a silent bell. All additional behavioural measures are consistent with the hypothesis of a restriction in the behaviour of the cows wearing bells, although this pattern did not reach significance. There was no treatment effect on heart rate variability, suggesting that the bells did not affect vago-sympathetic balance. An effect of experimental day was found for only 1 out of 10 behavioural parameters, as shown by a decrease in lying with a functional bell on day 3. The results indicate behavioural changes in the cows wearing a bell over 3 days, without indication of habituation to the bell. Altogether, the behavioural changes suggest that the behaviour of the cows was disturbed by wearing a bell. If long-lasting, these effects may have implications for animal welfare. PMID:26110277

  15. The geomagnetic environment in which sea turtle eggs incubate affects subsequent magnetic navigation behaviour of hatchlings

    PubMed Central

    Fuxjager, Matthew J.; Davidoff, Kyla R.; Mangiamele, Lisa A.; Lohmann, Kenneth J.

    2014-01-01

    Loggerhead sea turtle hatchlings (Caretta caretta) use regional magnetic fields as open-ocean navigational markers during trans-oceanic migrations. Little is known, however, about the ontogeny of this behaviour. As a first step towards investigating whether the magnetic environment in which hatchlings develop affects subsequent magnetic orientation behaviour, eggs deposited by nesting female loggerheads were permitted to develop in situ either in the natural ambient magnetic field or in a magnetic field distorted by magnets placed around the nest. In orientation experiments, hatchlings that developed in the normal ambient field oriented approximately south when exposed to a field that exists near the northern coast of Portugal, a direction consistent with their migratory route in the northeastern Atlantic. By contrast, hatchlings that developed in a distorted magnetic field had orientation indistinguishable from random when tested in the same north Portugal field. No differences existed between the two groups in orientation assays involving responses to orbital movements of waves or sea-finding, neither of which involves magnetic field perception. These findings, to our knowledge, demonstrate for the first time that the magnetic environment present during early development can influence the magnetic orientation behaviour of a neonatal migratory animal. PMID:25100699

  16. The impact of maternal control on children's anxious cognitions, behaviours and affect: an experimental study.

    PubMed

    Thirlwall, Kerstin; Creswell, Cathy

    2010-10-01

    Controlling parenting is associated with child anxiety however the direction of effects remains unclear. The present study implemented a Latin-square experimental design to assess the impact of parental control on children's anxious affect, cognitions and behaviour. A non-clinical sample of 24 mothers of children aged 4-5 years were trained to engage in (a) controlling and (b) autonomy-granting behaviours in interaction with their child during the preparation of a speech. When mothers engaged in controlling parenting behaviours, children made more negative predictions about their performance prior to delivering their speech and reported feeling less happy about the task, and this was moderated by child trait anxiety. In addition, children with higher trait anxiety displayed a significant increase in observed child anxiety in the controlling condition. The pattern of results was maintained when differences in mothers' levels of negativity and habitual levels of control were accounted for. These findings are consistent with theories that suggest that controlling parenting is a risk factor in the development of childhood anxiety. PMID:20594546

  17. Factors affecting farmers' behaviour in pesticide use: Insights from a field study in northern China.

    PubMed

    Fan, Liangxin; Niu, Haipeng; Yang, Xiaomei; Qin, Wei; Bento, Célia P M; Ritsema, Coen J; Geissen, Violette

    2015-12-15

    Quantitative understanding of farmers' behaviour in pesticide use is critical to enhance sustainability of chemical pest control and protect farmers' health and the environment. However, reports on the levels of knowledge and awareness of farmers and the practices of pesticide use are often insufficient. Here, we conducted a comprehensive analysis on the effects of knowledge and awareness of farmers as well as the influence of the associated stakeholders (i.e. pesticide retailers and the government) on farmers' behaviour in pesticide use by using a detailed survey of 307 agricultural households (79 grain farms, 65 fruit farms, 53 vegetable farms and 110 mixed-crop farms) in the Wei River basin in northern China. Eight protective behaviours (PBs) were exhibited by farmers. Careful and safe storage of pesticides, changing clothes or showering after applying pesticides, and reading instructions of the container labels before application were the most frequent PBs. Vegetable and fruit farmers had higher levels of education and knowledge than grain farmers, but the former were less willing to reduce pesticide use because of fear of low profits and lack of trust in the government and pesticide retailers. The PBs of farmers were strongly affected by the perception of the consequences of their behaviour (standardised path coefficient, SPC=0.42) and the level of farmers' knowledge (SPC=0.33). Pesticide retailers and the government had a moderate and weak influence, respectively, on farmers' PBs, suggesting a large gap of trust among farmers, pesticide retailers, and the government. Training and supervising retailers, educating farmers, and improving information transparency across farmers, pesticide retailers and the staff of the Agricultural Extension and Technology Service are recommended for bridging the gap of trust between farmers and the associated stakeholders as well as for promoting the use of PBs among farmers. PMID:26282770

  18. Myotonic dystrophy CTG expansion affects synaptic vesicle proteins, neurotransmission and mouse behaviour

    PubMed Central

    Hernández-Hernández, Oscar; Guiraud-Dogan, Céline; Sicot, Géraldine; Huguet, Aline; Luilier, Sabrina; Steidl, Esther; Saenger, Stefanie; Marciniak, Elodie; Obriot, Hélène; Chevarin, Caroline; Nicole, Annie; Revillod, Lucile; Charizanis, Konstantinos; Lee, Kuang-Yung; Suzuki, Yasuhiro; Kimura, Takashi; Matsuura, Tohru; Cisneros, Bulmaro; Swanson, Maurice S.; Trovero, Fabrice; Buisson, Bruno; Bizot, Jean-Charles; Hamon, Michel; Humez, Sandrine; Bassez, Guillaume; Metzger, Friedrich; Buée, Luc; Munnich, Arnold; Sergeant, Nicolas; Gourdon, Geneviève

    2013-01-01

    Myotonic dystrophy type 1 is a complex multisystemic inherited disorder, which displays multiple debilitating neurological manifestations. Despite recent progress in the understanding of the molecular pathogenesis of myotonic dystrophy type 1 in skeletal muscle and heart, the pathways affected in the central nervous system are largely unknown. To address this question, we studied the only transgenic mouse line expressing CTG trinucleotide repeats in the central nervous system. These mice recreate molecular features of RNA toxicity, such as RNA foci accumulation and missplicing. They exhibit relevant behavioural and cognitive phenotypes, deficits in short-term synaptic plasticity, as well as changes in neurochemical levels. In the search for disease intermediates affected by disease mutation, a global proteomics approach revealed RAB3A upregulation and synapsin I hyperphosphorylation in the central nervous system of transgenic mice, transfected cells and post-mortem brains of patients with myotonic dystrophy type 1. These protein defects were associated with electrophysiological and behavioural deficits in mice and altered spontaneous neurosecretion in cell culture. Taking advantage of a relevant transgenic mouse of a complex human disease, we found a novel connection between physiological phenotypes and synaptic protein dysregulation, indicative of synaptic dysfunction in myotonic dystrophy type 1 brain pathology. PMID:23404338

  19. From Affective Experience to Motivated Action: Tracking Reward-Seeking and Punishment-Avoidant Behaviour in Real-Life

    PubMed Central

    Wichers, Marieke; Kasanova, Zuzana; Bakker, Jindra; Thiery, Evert; Derom, Catherine; Jacobs, Nele; van Os, Jim

    2015-01-01

    Many of the decisions and actions in everyday life result from implicit learning processes. Important to psychopathology are, for example, implicit reward-seeking and punishment-avoidant learning processes. It is known that when specific actions get associated with a rewarding experience, such as positive emotions, that this will increase the likelihood that an organism will engage in similar actions in the future. Similarly, when actions get associated with punishing experiences, such as negative emotions, this may reduce the likelihood that the organism will engage in similar actions in the future. This study examines whether we can observe these implicit processes prospectively in the flow of daily life. If such processes take place then we expect that current behaviour can be predicted by how similar behaviour was experienced (in terms of positive and negative affect) at previous measurement moments. This was examined in a sample of 621 female individuals that had participated in an Experience Sampling data collection. Measures of affect and behaviour were collected at 10 semi-random moments of the day for 5 consecutive days. It was examined whether affective experience that was paired with certain behaviours (physical activity and social context) at previous measurements modified the likelihood to show similar behaviours at next measurement moments. Analyses were performed both at the level of observations (a time scale with units of ± 90 min) and at day level (a time scale with units of 24 h). As expected, we found that affect indeed moderated the extent to which previous behaviour predicted similar behaviour later in time, at both beep- and day-level. This study showed that it is feasible to track reward-seeking and punishment-avoidant behaviour prospectively in humans in the flow of daily life. This opens up a new toolbox to examine processes determining goal-oriented behaviour in relation to psychopathology in humans. PMID:26087323

  20. Attitudinal Factors Affecting Viral Advertising Pass-On Behaviour of Online Consumers in Food Industry

    NASA Astrophysics Data System (ADS)

    Mohd Salleh, Nurhidayah; Ariff, Mohd Shoki Md; Zakuan, Norhayati; Sulaiman, Zuraidah; Zameri Mat Saman, Muhamad

    2016-05-01

    The increase number of active users of social media, especially Facebook, stimulates viral advertising behaviour among them, thus attracting e-marketers to focus on viral advertising in promoting their products. In global market, use of Facebook platform indicated that food services/restaurant of food industry is ranked number 11 with 18.8% users’ response rate within the platform. This development calls for e-marketers in Malaysia to use Facebook as their viral advertising channel. Attitudinal factors affecting the viral advertising pass-on behaviour (VAPB) especially among members of social media is of interest to many researchers. The typical attitudinal factors used were attitude toward social media (ATSM), attitude toward advertising in social media (AASM) and attitude toward advertising in general (AAIG). Attitude toward advertised brand (ATAB) is important in fast food industry because users of social media tend to share their experience about tastes and features of the food. However, ATAB is less emphasized in the conceptual model between attitudinal factors and VAPB. These four factors of consumer attitude served as independent variables in the conceptual model of this study and their effect on viral advertising pass-on behaviour among members of Domino's Pizza Malaysia Facebook page was examined. Online survey using a set of questionnaire which was sent to the members of this group via private message was employed. A total of 254 sets of usable questionnaires were collected from the respondents. All the attitudinal factors, except for AASM, were found to have positive and significant effect on VAPB. AAIG exerted the strongest effect on VAPB. Therefore, e-marketers should emphasize on developing a favourable attitude toward advertising in general among members of a social media to get them involve in viral advertising. In addition, instilling a favourable attitude towards advertised brand is also vital as it influences the members to viral the brand

  1. Routine handling methods affect behaviour of three-spined sticklebacks in a novel test of anxiety

    PubMed Central

    Thompson, Ralph R.J.; Paul, Elizabeth S.; Radford, Andrew N.; Purser, Julia; Mendl, Michael

    2016-01-01

    Fish are increasingly popular subjects in behavioural and neurobiological research. It is therefore important that they are housed and handled appropriately to ensure good welfare and reliable scientific findings, and that species-appropriate behavioural tests (e.g. of cognitive/affective states) are developed. Routine handling of captive animals may cause physiological stress responses that lead to anxiety-like states (e.g. increased perception of danger). In fish, these may be particularly pronounced when handling during tank-to-tank transfer involves removal from water into air. Here we develop and use a new combined scototaxis (preference for dark over light areas) and novel-tank-diving test, alongside conventional open-field and novel-object tests, to measure the effects of transferring three-spined sticklebacks (Gasterosteus aculeatus) between tanks using a box or net (in and out of water respectively). Preference tests for dark over light areas confirmed the presence of scototaxis in this species. Open-field and novel-object tests failed to detect any significant differences between net and box-handled fish. However, the combined diving and scototaxis detected consistent differences between the treatments. Net-handled fish spent less time on the dark side of the tank, less time in the bottom third, and kept a greater distance from the ‘safe’ bottom dark area than box-handled fish. Possible explanations for this reduction in anxiety-like behaviour in net-handled fish are discussed. The combined diving and scototaxis test may be a sensitive and taxon-appropriate method for measuring anxiety-like states in fish. PMID:26965568

  2. Routine handling methods affect behaviour of three-spined sticklebacks in a novel test of anxiety.

    PubMed

    Thompson, Ralph R J; Paul, Elizabeth S; Radford, Andrew N; Purser, Julia; Mendl, Michael

    2016-06-01

    Fish are increasingly popular subjects in behavioural and neurobiological research. It is therefore important that they are housed and handled appropriately to ensure good welfare and reliable scientific findings, and that species-appropriate behavioural tests (e.g. of cognitive/affective states) are developed. Routine handling of captive animals may cause physiological stress responses that lead to anxiety-like states (e.g. increased perception of danger). In fish, these may be particularly pronounced when handling during tank-to-tank transfer involves removal from water into air. Here we develop and use a new combined scototaxis (preference for dark over light areas) and novel-tank-diving test, alongside conventional open-field and novel-object tests, to measure the effects of transferring three-spined sticklebacks (Gasterosteus aculeatus) between tanks using a box or net (in and out of water respectively). Preference tests for dark over light areas confirmed the presence of scototaxis in this species. Open-field and novel-object tests failed to detect any significant differences between net and box-handled fish. However, the combined diving and scototaxis detected consistent differences between the treatments. Net-handled fish spent less time on the dark side of the tank, less time in the bottom third, and kept a greater distance from the 'safe' bottom dark area than box-handled fish. Possible explanations for this reduction in anxiety-like behaviour in net-handled fish are discussed. The combined diving and scototaxis test may be a sensitive and taxon-appropriate method for measuring anxiety-like states in fish. PMID:26965568

  3. Flexible responses to visual and olfactory stimuli by foraging Manduca sexta: larval nutrition affects adult behaviour

    PubMed Central

    Goyret, Joaquín; Kelber, Almut; Pfaff, Michael; Raguso, Robert A.

    2009-01-01

    Here, we show that the consequences of deficient micronutrient (β-carotene) intake during larval stages of Manduca sexta are carried across metamorphosis, affecting adult behaviour. Our manipulation of larval diet allowed us to examine how developmental plasticity impacts the interplay between visual and olfactory inputs on adult foraging behaviour. Larvae of M. sexta were reared on natural (Nicotiana tabacum) and artificial laboratory diets containing different concentrations of β-carotene (standard diet, low β-carotene, high β-carotene and cornmeal). This vitamin-A precursor has been shown to be crucial for photoreception sensitivity in the retina of M. sexta. After completing development, post-metamorphosis, starved adults were presented with artificial feeders that could be either scented or unscented. Regardless of their larval diet, adult moths fed with relatively high probabilities on scented feeders. When feeders were unscented, moths reared on tobacco were more responsive than moths reared on β-carotene-deficient artificial diets. Strikingly, moths reared on artificial diets supplemented with increasing amounts of β-carotene (low β and high β) showed increasing probabilities of response to scentless feeders. We discuss these results in relationship to the use of complex, multi-modal sensory information by foraging animals. PMID:19419987

  4. Ghrelin stimulates milk intake by affecting adult type feeding behaviour in postnatal rats.

    PubMed

    Piao, H; Hosoda, H; Kangawa, K; Murata, T; Narita, K; Higuchi, T

    2008-03-01

    The influence of ghrelin on feeding behaviour during infancy is unknown. To determine whether ghrelin influences milk intake in rat pups, newborn rats received a single i.p. injection of either rat ghrelin (100 microg/kg) or rabbit anti-ghrelin immunoglobulin G (100 microg/kg) every 5 days from postpartum day 5 to day 30 (P5-P30). Milk intake was then assessed by body weight gain following a 2-h suckling period. Ghrelin significantly increased weight gain relative to vehicle-injected controls in P20, P25 and P30 pups, but not in younger animals. Similarly, after 8 h of milk restriction, anti-ghrelin injections significantly decreased weight gain in P25 and P30, but not in younger pups. Interestingly, however, ghrelin did increase independent feeding in P10 and P15 pups using a paradigm in which pups consumed milk from a milk-soaked paper towel. We therefore conclude that ghrelin stimulates milk intake at an early postnatal stage, primarily by affecting adult-type feeding behaviour. PMID:18194428

  5. Brain size affects the behavioural response to predators in female guppies (Poecilia reticulata)

    PubMed Central

    van der Bijl, Wouter; Thyselius, Malin; Kotrschal, Alexander; Kolm, Niclas

    2015-01-01

    Large brains are thought to result from selection for cognitive benefits, but how enhanced cognition leads to increased fitness remains poorly understood. One explanation is that increased cognitive ability results in improved monitoring and assessment of predator threats. Here, we use male and female guppies (Poecilia reticulata), artificially selected for large and small brain size, to provide an experimental evaluation of this hypothesis. We examined their behavioural response as singletons, pairs or shoals of four towards a model predator. Large-brained females, but not males, spent less time performing predator inspections, an inherently risky behaviour. Video analysis revealed that large-brained females were further away from the model predator when in pairs but that they habituated quickly towards the model when in shoals of four. Males stayed further away from the predator model than females but again we found no brain size effect in males. We conclude that differences in brain size affect the female predator response. Large-brained females might be able to assess risk better or need less sensory information to reach an accurate conclusion. Our results provide experimental support for the general idea that predation pressure is likely to be important for the evolution of brain size in prey species. PMID:26203003

  6. Brain size affects the behavioural response to predators in female guppies (Poecilia reticulata).

    PubMed

    van der Bijl, Wouter; Thyselius, Malin; Kotrschal, Alexander; Kolm, Niclas

    2015-08-01

    Large brains are thought to result from selection for cognitive benefits, but how enhanced cognition leads to increased fitness remains poorly understood. One explanation is that increased cognitive ability results in improved monitoring and assessment of predator threats. Here, we use male and female guppies (Poecilia reticulata), artificially selected for large and small brain size, to provide an experimental evaluation of this hypothesis. We examined their behavioural response as singletons, pairs or shoals of four towards a model predator. Large-brained females, but not males, spent less time performing predator inspections, an inherently risky behaviour. Video analysis revealed that large-brained females were further away from the model predator when in pairs but that they habituated quickly towards the model when in shoals of four. Males stayed further away from the predator model than females but again we found no brain size effect in males. We conclude that differences in brain size affect the female predator response. Large-brained females might be able to assess risk better or need less sensory information to reach an accurate conclusion. Our results provide experimental support for the general idea that predation pressure is likely to be important for the evolution of brain size in prey species. PMID:26203003

  7. Breathing hypoxic gas affects the physiology as well as the diving behaviour of tufted ducks.

    PubMed

    Halsey, Lewis G; Butler, Patrick J; Woakes, Anthony J

    2005-01-01

    We measured the effects of exposure to hypoxia (15% and 11% oxygen) and hypercapnia (up to 4.5% carbon dioxide) on rates of respiratory gas exchange both between and during dives in tufted ducks, Aythya fuligula, to investigate to what extent these may explain changes in diving behaviour. As found in previous studies, the ducks decreased dive duration (t(d)) and increased surface duration when diving from a hypoxic or hypercapnic gas mix. In the hypercapnic conditions, oxygen consumption during the dive cycle was not affected. Oxygen uptake between dives was reduced by only 17% when breathing a hypoxic gas mix of 11% oxygen. However, estimates of the rate of oxygen metabolism during the foraging periods of dives decreased nearly threefold in 11% oxygen. Given that tufted ducks normally dive well within their aerobic dive limits and that they significantly reduced their t(d) during hypoxia, it is not at all clear why they make this physiological adjustment. PMID:15778946

  8. Feeding behaviour of an intertidal snail: Does past environmental stress affect predator choices and prey vulnerability?

    NASA Astrophysics Data System (ADS)

    Gestoso, Ignacio; Arenas, Francisco; Olabarria, Celia

    2015-03-01

    Predation is one of the most important factors in determining structure and dynamics of communities on intertidal rocky shores. Such regulatory role may be of special relevance in novel communities resulting from biological invasions. Non-indigenous species frequently escape natural predators that limit their distribution and abundance in the native range. However, biological interactions also can limit the establishment and spread of non-native populations. There is a growing concern that climate change might affect predator-prey interactions exacerbating the ecological impacts of non-indigenous species. However, mechanisms underlying such interactions are poorly understood in marine ecosystems. Here, we explored if past environmental stress, i.e., increasing temperature and decreasing pH, could affect the vulnerability of two mussel prey, the native Mytilus galloprovincialis and the non-indigenous Xenostrobus securis, to predation by the native dogwhelk Nucella lapillus. In addition, we evaluated the consequences on the feeding behaviour of N. lapillus. First, we exposed monospecific assemblages of each mussel species to combined experimental conditions of increasing temperature and decreasing pH in mesocosms for 3 weeks. Then assemblages were placed on a rocky shore and were enclosed in cages with dogwhelks where they remained for 3 weeks. Despite the lack of preference, consumption was much greater on the native than on the invasive mussels, which barely were consumed by dogwhelks. However, this trend was diverted when temperature increased. Thus, under a coastal warming scenario shifts in dogwhelks feeding behaviour may help to contain invader's populations, especially in estuarine areas where these predators are abundant.

  9. Food chain transport of nanoparticles affects behaviour and fat metabolism in fish.

    PubMed

    Cedervall, Tommy; Hansson, Lars-Anders; Lard, Mercy; Frohm, Birgitta; Linse, Sara

    2012-01-01

    Nano-sized (10(-9)-10(-7) m) particles offer many technical and biomedical advances over the bulk material. The use of nanoparticles in cosmetics, detergents, food and other commercial products is rapidly increasing despite little knowledge of their effect on organism metabolism. We show here that commercially manufactured polystyrene nanoparticles, transported through an aquatic food chain from algae, through zooplankton to fish, affect lipid metabolism and behaviour of the top consumer. At least three independent metabolic parameters differed between control and test fish: the weight loss, the triglycerides∶cholesterol ratio in blood serum, and the distribution of cholesterol between muscle and liver. Moreover, we demonstrate that nanoparticles bind to apolipoprotein A-I in fish serum in-vitro, thereby restraining them from properly utilising their fat reserves if absorbed through ingestion. In addition to the metabolic effects, we show that consumption of nanoparticle-containing zooplankton affects the feeding behaviour of the fish. The time it took the fish to consume 95% of the food presented to them was more than doubled for nanoparticle-exposed compared to control fish. Since many nano-sized products will, through the sewage system, end up in freshwater and marine habitats, our study provides a potential bioassay for testing new nano-sized material before manufacturing. In conclusion, our study shows that from knowledge of the molecular composition of the protein corona around nanoparticles it is possible to make a testable molecular hypothesis and bioassay of the potential biological risks of a defined nanoparticle at the organism and ecosystem level. PMID:22384193

  10. How do different data logger sizes and attachment positions affect the diving behaviour of little penguins?

    NASA Astrophysics Data System (ADS)

    Ropert-Coudert, Yan; Knott, Nathan; Chiaradia, André; Kato, Akiko

    2007-02-01

    It is crucial in any bio-logging study to establish the potential effect that attachment of loggers may have on the animal. This ensures that the behaviour monitored by the loggers has a biological relevance, as well as for ethical reasons. Evaluation of the effects of externally attached loggers shows that they increase the drag of swimming animals and increase their energy expenditure. Nevertheless, little research has been done on the effects of size or position of such loggers. In this study, we tested whether the size (i.e. large: 4.9% versus small: 3.4% of the bird's frontal area) or the place of attachment (middle versus lower back) affected the diving behaviour of male and female little penguins ( Eudyptula minor). The positioning of the data logger on the middle or lower section of little penguins' back had little, if no effect, on the diving variables measured in this study. Size of the loggers, however, had strong effects. Birds with large loggers made shorter dives and reached shallower depths than those with small loggers. In addition, birds with large loggers made more dives probably to compensate for the extra cost of carrying a large logger. The measured variables also differed between the sexes, with males diving deeper and longer than females. Logger size had a sex-specific effect on the trip duration and descent speed, with males equipped with large loggers staying longer at sea than those with small loggers, and females with large loggers descending faster than those with small loggers. From our results, it appears that effects of logger position do not exist or are very small in comparison with the effects of logger size. The results of the current study indicate that the effects of size of loggers be evaluated more commonly in bio-logging research into the diving activity of free-ranging birds.

  11. Tubulin perturbation leads to unexpected cell wall modifications and affects stomatal behaviour in Populus

    PubMed Central

    Swamy, Prashant S.; Hu, Hao; Pattathil, Sivakumar; Maloney, Victoria J.; Xiao, Hui; Xue, Liang-Jiao; Chung, Jeng-Der; Johnson, Virgil E.; Zhu, Yingying; Peter, Gary F.; Hahn, Michael G.; Mansfield, Shawn D.; Harding, Scott A.; Tsai, Chung-Jui

    2015-01-01

    Cortical microtubules are integral to plant morphogenesis, cell wall synthesis, and stomatal behaviour, presumably by governing cellulose microfibril orientation. Genetic manipulation of tubulins often leads to abnormal plant development, making it difficult to probe additional roles of cortical microtubules in cell wall biogenesis. Here, it is shown that expressing post-translational C-terminal modification mimics of α-tubulin altered cell wall characteristics and guard cell dynamics in transgenic Populus tremula x alba that otherwise appear normal. 35S promoter-driven transgene expression was high in leaves but unusually low in xylem, suggesting high levels of tubulin transgene expression were not tolerated in wood-forming tissues during regeneration of transformants. Cellulose, hemicellulose, and lignin contents were unaffected in transgenic wood, but expression of cell wall-modifying enzymes, and extractability of lignin-bound pectin and xylan polysaccharides were increased in developing xylem. The results suggest that pectin and xylan polysaccharides deposited early during cell wall biogenesis are more sensitive to subtle tubulin perturbation than cellulose and matrix polysaccharides deposited later. Tubulin perturbation also affected guard cell behaviour, delaying drought-induced stomatal closure as well as light-induced stomatal opening in leaves. Pectins have been shown to confer cell wall flexibility critical for reversible stomatal movement, and results presented here are consistent with microtubule involvement in this process. Taken together, the data show the value of growth-compatible tubulin perturbations for discerning microtubule functions, and add to the growing body of evidence for microtubule involvement in non-cellulosic polysaccharide assembly during cell wall biogenesis. PMID:26246616

  12. Tubulin perturbation leads to unexpected cell wall modifications and affects stomatal behaviour in Populus.

    PubMed

    Swamy, Prashant S; Hu, Hao; Pattathil, Sivakumar; Maloney, Victoria J; Xiao, Hui; Xue, Liang-Jiao; Chung, Jeng-Der; Johnson, Virgil E; Zhu, Yingying; Peter, Gary F; Hahn, Michael G; Mansfield, Shawn D; Harding, Scott A; Tsai, Chung-Jui

    2015-10-01

    Cortical microtubules are integral to plant morphogenesis, cell wall synthesis, and stomatal behaviour, presumably by governing cellulose microfibril orientation. Genetic manipulation of tubulins often leads to abnormal plant development, making it difficult to probe additional roles of cortical microtubules in cell wall biogenesis. Here, it is shown that expressing post-translational C-terminal modification mimics of α-tubulin altered cell wall characteristics and guard cell dynamics in transgenic Populus tremula x alba that otherwise appear normal. 35S promoter-driven transgene expression was high in leaves but unusually low in xylem, suggesting high levels of tubulin transgene expression were not tolerated in wood-forming tissues during regeneration of transformants. Cellulose, hemicellulose, and lignin contents were unaffected in transgenic wood, but expression of cell wall-modifying enzymes, and extractability of lignin-bound pectin and xylan polysaccharides were increased in developing xylem. The results suggest that pectin and xylan polysaccharides deposited early during cell wall biogenesis are more sensitive to subtle tubulin perturbation than cellulose and matrix polysaccharides deposited later. Tubulin perturbation also affected guard cell behaviour, delaying drought-induced stomatal closure as well as light-induced stomatal opening in leaves. Pectins have been shown to confer cell wall flexibility critical for reversible stomatal movement, and results presented here are consistent with microtubule involvement in this process. Taken together, the data show the value of growth-compatible tubulin perturbations for discerning microtubule functions, and add to the growing body of evidence for microtubule involvement in non-cellulosic polysaccharide assembly during cell wall biogenesis. PMID:26246616

  13. Genotype at the PMEL17 locus affects social and explorative behaviour in chickens.

    PubMed

    Karlsson, A-C; Kerje, S; Andersson, L; Jensen, P

    2010-04-01

    1. We studied behaviour and brain gene expression in homozygous PMEL17 genotypes, using chickens originating from an advanced White Leghorn x red junglefowl intercross. The behavioural studies consisted of three social and one explorative behaviour test. There were significant differences between the genotypes in both social and explorative behaviour. 2. Gene expression studies showed no PMEL17 expression in brain, so the genotype differences must depend on extra-neural gene expression or expression during embryonic development. However, linkage or spurious family effects (genetic drift) can not be excluded. 3. The study strongly suggests a correlated effect between plumage colour and behaviour, and we conclude that PMEL17 may have a pleiotropic effect on social and explorative behaviour in chickens. PMID:20461577

  14. Heterozygosity for Nuclear Factor One X Affects Hippocampal-Dependent Behaviour in Mice

    PubMed Central

    Harris, Lachlan; Dixon, Chantelle; Cato, Kathleen; Heng, Yee Hsieh Evelyn; Kurniawan, Nyoman D.; Ullmann, Jeremy F. P.; Janke, Andrew L.; Gronostajski, Richard M.; Richards, Linda J.; Burne, Thomas H. J.; Piper, Michael

    2013-01-01

    Identification of the genes that regulate the development and subsequent functioning of the hippocampus is pivotal to understanding the role of this cortical structure in learning and memory. One group of genes that has been shown to be critical for the early development of the hippocampus is the Nuclear factor one (Nfi) family, which encodes four site-specific transcription factors, NFIA, NFIB, NFIC and NFIX. In mice lacking Nfia, Nfib or Nfix, aspects of early hippocampal development, including neurogenesis within the dentate gyrus, are delayed. However, due to the perinatal lethality of these mice, it is not clear whether this hippocampal phenotype persists to adulthood and affects hippocampal-dependent behaviour. To address this we examined the hippocampal phenotype of mice heterozygous for Nfix (Nfix+/−), which survive to adulthood. We found that Nfix+/− mice had reduced expression of NFIX throughout the brain, including the hippocampus, and that early hippocampal development in these mice was disrupted, producing a phenotype intermediate to that of wild-type mice and Nfix−/− mice. The abnormal hippocampal morphology of Nfix+/− mice persisted to adulthood, and these mice displayed a specific performance deficit in the Morris water maze learning and memory task. These findings demonstrate that the level of Nfix expression during development and within the adult is essential for the function of the hippocampus during learning and memory. PMID:23776487

  15. Heterozygosity for nuclear factor one x affects hippocampal-dependent behaviour in mice.

    PubMed

    Harris, Lachlan; Dixon, Chantelle; Cato, Kathleen; Heng, Yee Hsieh Evelyn; Kurniawan, Nyoman D; Ullmann, Jeremy F P; Janke, Andrew L; Gronostajski, Richard M; Richards, Linda J; Burne, Thomas H J; Piper, Michael

    2013-01-01

    Identification of the genes that regulate the development and subsequent functioning of the hippocampus is pivotal to understanding the role of this cortical structure in learning and memory. One group of genes that has been shown to be critical for the early development of the hippocampus is the Nuclear factor one (Nfi) family, which encodes four site-specific transcription factors, NFIA, NFIB, NFIC and NFIX. In mice lacking Nfia, Nfib or Nfix, aspects of early hippocampal development, including neurogenesis within the dentate gyrus, are delayed. However, due to the perinatal lethality of these mice, it is not clear whether this hippocampal phenotype persists to adulthood and affects hippocampal-dependent behaviour. To address this we examined the hippocampal phenotype of mice heterozygous for Nfix (Nfix (+/-)), which survive to adulthood. We found that Nfix (+/-) mice had reduced expression of NFIX throughout the brain, including the hippocampus, and that early hippocampal development in these mice was disrupted, producing a phenotype intermediate to that of wild-type mice and Nfix(-/-) mice. The abnormal hippocampal morphology of Nfix (+/-) mice persisted to adulthood, and these mice displayed a specific performance deficit in the Morris water maze learning and memory task. These findings demonstrate that the level of Nfix expression during development and within the adult is essential for the function of the hippocampus during learning and memory. PMID:23776487

  16. Fibronectin is a survival factor for differentiated osteoblasts

    NASA Technical Reports Server (NTRS)

    Globus, R. K.; Doty, S. B.; Lull, J. C.; Holmuhamedov, E.; Humphries, M. J.; Damsky, C. H.

    1998-01-01

    The skeletal extracellular matrix produced by osteoblasts contains the glycoprotein fibronectin, which regulates the adhesion, differentiation and function of various adherent cells. Interactions with fibronectin are required for osteoblast differentiation in vitro, since fibronectin antagonists added to cultures of immature fetal calvarial osteoblasts inhibit their progressive differentiation. To determine if fibronectin plays a unique role in fully differentiated osteoblasts, cultures that had already formed mineralized nodules in vitro were treated with fibronectin antagonists. Fibronectin antibodies caused >95% of the cells in the mature cultures to display characteristic features of apoptosis (nuclear condensation, apoptotic body formation, DNA laddering) within 24 hours. Cells appeared to acquire sensitivity to fibronectin antibody-induced apoptosis as a consequence of differentiation, since antibodies failed to kill immature cells and the first cells killed were those associated with mature nodules. Intact plasma fibronectin, as well as fragments corresponding to the amino-terminal, cell-binding, and carboxy-terminal domains of fibronectin, independently induced apoptosis of mature (day-13), but not immature (day-4), osteoblasts. Finally, transforming growth factor-beta1 partially protected cells from the apoptotic effects of fibronectin antagonists. Thus, in the course of maturation cultured osteoblasts switch from depending on fibronectin for differentiation to depending on fibronectin for survival. These data suggest that fibronectin, together with transforming growth factor-beta1, may affect bone formation, in part by regulating the survival of osteoblasts.

  17. Effect of magnesium ion on human osteoblast activity

    PubMed Central

    He, L.Y.; Zhang, X.M.; Liu, B.; Tian, Y.; Ma, W.H.

    2016-01-01

    Magnesium, a promising biodegradable metal, has been reported in several studies to increase bone formation. Although there is some information regarding the concentrations of magnesium ions that affect bone remodeling at a cellular level, little is known about the effect of magnesium ions on cell gap junctions. Therefore, this study aimed to systematically investigate the effects of different concentrations of magnesium on bone cells, and further evaluate its effect on gap junctions of osteoblasts. Cultures of normal human osteoblasts were treated with magnesium ions at concentrations of 1, 2 and 3 mM, for 24, 48 and 72 h. The effects of magnesium ions on viability and function of normal human osteoblasts and on gap junction intercellular communication (GJIC) in osteoblasts were investigated. Magnesium ions induced significant (P<0.05) increases in cell viability, alkaline phosphate activity and osteocalcin levels of human osteoblasts. These stimulatory actions were positively associated with the concentration of magnesium and the time of exposure. Furthermore, the GJIC of osteoblasts was significantly promoted by magnesium ions. In conclusion, this study demonstrated that magnesium ions induced the activity of osteoblasts by enhancing GJIC between cells, and influenced bone formation. These findings may contribute to a better understanding of the influence of magnesium on bone remodeling and to the advance of its application in clinical practice. PMID:27383121

  18. The osteoblastic niche in the context of multiple myeloma.

    PubMed

    Toscani, Denise; Bolzoni, Marina; Accardi, Fabrizio; Aversa, Franco; Giuliani, Nicola

    2015-01-01

    The osteoblastic niche has a critical role in the regulation of hemopoietic stem cell (HSC) quiescence and self-renewal and in the support of hematopoiesis. Several mechanisms are involved in the crosstalk between stem cells and osteoblasts, including soluble cytokines, adhesion molecules, and signal pathways such as the wingless-Int (Wnt), Notch, and parathyroid hormone pathways. According to the most recent evidence, there is an overlap between osteoblastic and perivascular niches that affects HSC function involving mesenchymal stromal and endothelial cells and a gradient of oxygen regulated by hypoxia inducible factor (HIF)-1α. Derived from plasma cells, multiple myeloma (MM) is a hematopoietic malignancy characterized by a peculiar dependency on the bone microenvironment. Quiescent MM cells may reside in the osteoblastic niche for protection from apoptotic stimuli; in turn, MM cells suppress osteoblast formation and function, leading to impairment of bone formation and the development of osteolytic lesions. Several recent studies have investigated the mechanisms involved in the relationship between osteoblasts and MM cells and identified potential therapeutic targets in the osteoblastic niche, including the HIF-1α, Runx2, and Wnt (both canonical and noncanonical) signaling pathways. PMID:25424768

  19. Interpersonal engagement mediates the relation between maternal affect and externalising behaviour in young children with type 1 diabetes.

    PubMed

    Chisholm, Vivienne; Gonzalez, Andrea; Atkinson, Leslie

    2014-01-01

    Mother-child interactions around a shared activity have been shown to play a key role in the development of young children's capacity to interact cooperatively with others. This evidence is particularly germane to type 1 diabetes (T1D) management in younger children where cooperation with parental treatment efforts is crucial for treatment success and where maternal distress and child behavioural problems are risk factors for treatment management, biomedical and psychological outcomes. In 49 4-to-8 year old children with T1D, we investigated whether the association between maternal affect and child problematic behaviour is mediated by mother-child interactions in the context of a T1D-relevant collaborative problem-solving activity. Mothers completed standardised measures of maternal and child psychological adjustment and interacted with their children in the problem-solving activity, analysed for quality of interpersonal engagement based on evaluations of maternal (sensitivity and cognitive stimulation) and dyadic (joint attention and warmth) behaviours. Mediation analyses confirmed the hypothesis that interpersonal engagement mediates the relation between maternal affective state and child behavioural problems. Specifically, more negative maternal affect is associated with lower levels of interpersonal engagement; these less engaged interactions in turn are associated with more behavioural problems in children. These findings are consistent with research involving typically developing children. The implications of our findings are twofold. First, in the context of psychological adjustment to T1D, maternal affect and mother-child interactions are 2 potential targets for interventions which promote cooperative interactions. Second, understanding and caring for children at biological risk requires attention to developmental psychology theory and method; in particular, research addressing parent-child cooperation carries both conceptual and clinical relevance. PMID

  20. Interpersonal Engagement Mediates the Relation between Maternal Affect and Externalising Behaviour in Young Children with Type 1 Diabetes

    PubMed Central

    Chisholm, Vivienne; Gonzalez, Andrea; Atkinson, Leslie

    2014-01-01

    Mother-child interactions around a shared activity have been shown to play a key role in the development of young children’s capacity to interact cooperatively with others. This evidence is particularly germane to type 1 diabetes (T1D) management in younger children where cooperation with parental treatment efforts is crucial for treatment success and where maternal distress and child behavioural problems are risk factors for treatment management, biomedical and psychological outcomes. In 49 4-to-8 year old children with T1D, we investigated whether the association between maternal affect and child problematic behaviour is mediated by mother-child interactions in the context of a T1D-relevant collaborative problem-solving activity. Mothers completed standardised measures of maternal and child psychological adjustment and interacted with their children in the problem-solving activity, analysed for quality of interpersonal engagement based on evaluations of maternal (sensitivity and cognitive stimulation) and dyadic (joint attention and warmth) behaviours. Mediation analyses confirmed the hypothesis that interpersonal engagement mediates the relation between maternal affective state and child behavioural problems. Specifically, more negative maternal affect is associated with lower levels of interpersonal engagement; these less engaged interactions in turn are associated with more behavioural problems in children. These findings are consistent with research involving typically developing children. The implications of our findings are twofold. First, in the context of psychological adjustment to T1D, maternal affect and mother-child interactions are 2 potential targets for interventions which promote cooperative interactions. Second, understanding and caring for children at biological risk requires attention to developmental psychology theory and method; in particular, research addressing parent-child cooperation carries both conceptual and clinical relevance. PMID

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

    PubMed

    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

  2. EFFECTS OF PAMIDRONATE ON HUMAN ALVEOLAR OSTEOBLASTS IN VITRO

    PubMed Central

    Marolt, Darja; Cozin, Matthew; Vunjak-Novakovic, Gordana; Cremers, Serge; Landesberg, Regina

    2011-01-01

    Purpose Administration of bisphosphonates has recently been associated with the development of osteonecrotic lesions of the jaw (ONJ). To elucidate the potential contributions of osteogenic cells to the development and regeneration of ONJ, we have isolated primary cells from human alveolar and long/iliac bones, and examined the effects of pamidronate on cell viability, proliferation, osteogenesis and wound healing. Materials and Methods Primary human osteoblasts and bone marrow stromal cells were isolated from alveolar and iliac/long bone and marrow tissue. Cellular proliferation, alkaline phosphatase activity, apoptosis (TUNEL, Caspase-3, and DAPI assays) and wound healing in an in vitro scratch assay were assessed after exposure to pamidronate at a range of clinically relevant doses. Results Primary alveolar osteoblasts proliferated at significantly higher rates than long/iliac bone osteoblasts in vitro. Upon exposure of alveolar osteoblasts and long/iliac bone marrow stromal cells to pamidronate for more than 72h, we have observed significantly decreased cell viability, proliferation, osteogenesis and in vitro wound healing at ≥6 × 10−5 M pamidronate, with the induction of apoptosis in ~20% of cell population. Conclusions The remodeling activity of alveolar bone, indicated by higher proliferation of alveolar osteoblasts, could be negatively affected by exposure to high concentrations of pamidronate over extended periods of time. The absence of anabolic effects of pamidronate on alveolar osteoblasts, and induction of apoptosis in osteogenic cells could negatively affect bone balance at this site, and contribute to osteonecrosis of the jaw. PMID:21856057

  3. Regulation of Osteoblast Survival by the Extracellular Matrix and Gravity

    NASA Technical Reports Server (NTRS)

    Globus. Ruth K.; Almeida, Eduardo A. C.; Searby, Nancy D.; Bowley, Susan M. (Technical Monitor)

    2000-01-01

    Spaceflight adversely affects the skeleton, posing a substantial risk to astronaut's health during long duration missions. The reduced bone mass observed in growing animals following spaceflight is due at least in part to inadequate bone formation by osteoblasts. Thus, it is of central importance to identify basic cellular mechanisms underlying normal bone formation. The fundamental ideas underlying our research are that interactions between extracellular matrix proteins, integrin adhesion receptors, cytoplasmic signaling and cytoskeletal proteins are key ingredients for the proper functioning of osteoblasts, and that gravity impacts these interactions. As an in vitro model system we used primary fetal rat calvarial cells which faithfully recapitulate osteoblast differentiation characteristically observed in vivo. We showed that specific integrin receptors ((alpha)3(beta)1), ((alpha)5(beta)1), ((alpha)8(betal)1) and extracellular matrix proteins (fibronectin, laminin) were needed for the differentiation of immature osteoblasts. In the course of maturation, cultured osteoblasts switched from depending on fibronectin and laminin for differentiation to depending on these proteins for their very survival. Furthermore, we found that manipulating the gravity vector using ground-based models resulted in activation of key intracellular survival signals generated by integrin/extracellular matrix interactions. We are currently testing the in vivo relevance of some of these observations using targeted transgenic technology. In conclusion, mechanical factors including gravity may participate in regulating survival via cellular interactions with the extracellular matrix. This leads us to speculate that microgravity adversely affects the survival of osteoblasts and contributes to spaceflight-induced osteoporosis.

  4. MEK5 suppresses osteoblastic differentiation

    SciTech Connect

    Kaneshiro, Shoichi; Otsuki, Dai; Yoshida, Kiyoshi; Yoshikawa, Hideki; Higuchi, Chikahisa

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  6. Medial prefrontal serotonin in the rat is involved in goal-directed behaviour when affect guides decision making

    PubMed Central

    La Fors, Sabrina S. B. M.; Meerkerk, Dorie T. J.; Joosten, Ruud N. J. M. A.; Uylings, Harry B. M.; Feenstra, Matthijs G. P.

    2007-01-01

    Rationale Across species, serotonin (5-HT) depletion in the prefrontal cortex (PFC) has been shown to cause impaired performance on tests of cognitive flexibility and the processing of affective information (e.g. information with an ‘emotional’ content). While recent work has explored the specific role of the orbital PFC herein, the role of the medial PFC remains unclear. Objectives The aim of our current experiments was to study the role of medial PFC 5-HT in both the processing of affective information and reversal learning across stimulus modalities. Materials and methods To this end, we selectively destroyed 5-HT terminals in the medial PFC of male Wistar rats by means of local infusion of the toxin 5,7-dihydroxytryptamine. Both control and lesioned animals were tested in two reversal learning paradigms with either spatial or odour cues and an affective switch from non-preferred to preferred food rewards. Results Our results indicate that a pellet switch during reversal learning impaired performance in control animals but not in lesioned animals, independent of the stimulus modality. Conclusion These results indicate that lesioned animals are not guided in their behaviour by the affective value of the reward like intact animals and thus that medial prefrontal 5-HT is needed for affective processing in goal-directed behaviour. PMID:17874235

  7. The Influence of Authoritative Teaching on Children's School Adjustment: Are Children with Behavioural Problems Differentially Affected?

    ERIC Educational Resources Information Center

    Baker, Jean A.; Clark, Teresa P.; Crowl, Alicia; Carlson, John S.

    2009-01-01

    Children with significant behaviour problems are at risk for poor classroom adjustment and school failure. Given this likelihood for a poor developmental trajectory, there is a need to better understand environmental influences within classrooms that help to effectively socialize children to those settings. The current study evaluated the effects…

  8. DOES FEEDBACK FROM A NEST AFFECT PERIPARTURIENT BEHAVIOUR, HORMONES AND HEART RATE IN GILTS?

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To determine the effects of feedback from a farrowing nest on sow welfare, as determined by behaviour, hormones and heart rate, 20 gilts housed in central-nest-farrowing-pens were permitted to build a nest of peat, straw and branches. Ten sows then had their nest removed (NR) 10h after the onset of ...

  9. mTORC1 Signaling Promotes Osteoblast Differentiation from Preosteoblasts

    PubMed Central

    Chen, Jianquan; Long, Fanxin

    2015-01-01

    Preosteoblasts are precursor cells that are committed to the osteoblast lineage. Differentiation of these cells to mature osteoblasts is regulated by the extracellular factors and environmental cues. Recent studies have implicated mTOR signaling in the regulation of osteoblast differentiation. However, mTOR exists in two distinct protein complexes (mTORC1 and mTORC2), and the specific role of mTORC1 in regulating the progression of preosteoblasts to mature osteoblastis still unclear. In this study, we first deleted Raptor, a unique and essential component of mTORC1, in primary calvarial cells. Deletion of Raptor resulted in loss of mTORC1 but an increase in mTORC2 signaling without overtly affecting autophagy. Under the osteogenic culture condition, Raptor-deficient cells exhibited a decrease in matrix synthesis and mineralization. qPCR analyses revealed that deletion of Raptor reduced the expression of late-stage markers for osteoblast differentiation (Bglap, Ibsp, and Col1a), while slightly increasing early osteoblast markers (Runx2, Sp7, and Alpl). Consistent with the findings in vitro, genetic ablation of Raptor in osterix-expressing cells led to osteopenia in mice. Together, our findings have identified a specific role for mTORC1 in the transition from preosteoblasts to mature osteoblasts. PMID:26090674

  10. The Relations of Parental Affect and Encouragement to Children's Moral Emotions and Behaviour.

    ERIC Educational Resources Information Center

    Spinrad, Tracy L.; Losoya, Sandra H.; Eisenburg, Nancy; Fabes, Richard A.; Shepard, Stephanie A.; Cumberland, Amanda; Guthrie, Ivanna K.; Murphy, Bridget C.

    1999-01-01

    Explores the role of observed parental affect and encouragement in children's empathy-related responding and moral behavior, specifically cheating on a puzzle activity. Finds that (1) parents' affect and encouragement positively related to children's sympathy (not empathy) and (2) boys' cheating on the puzzle correlated to parents' affect and…

  11. Liver irradiation causes distal bystander effects in the rat brain and affects animal behaviour

    PubMed Central

    Kovalchuk, Anna; Mychasiuk, Richelle; Muhammad, Arif; Hossain, Shakhawat; Ilnytskyy, Slava; Ghose, Abhijit; Kirkby, Charles; Ghasroddashti, Esmaeel; Kovalchuk, Olga; Kolb, Bryan

    2016-01-01

    Radiation therapy can not only produce effects on targeted organs, but can also influence shielded bystander organs, such as the brain in targeted liver irradiation. The brain is sensitive to radiation exposure, and irradiation causes significant neuro-cognitive deficits, including deficits in attention, concentration, memory, and executive and visuospatial functions. The mechanisms of their occurrence are not understood, although they may be related to the bystander effects. We analyzed the induction, mechanisms, and behavioural repercussions of bystander effects in the brain upon liver irradiation in a well-established rat model. Here, we show for the first time that bystander effects occur in the prefrontal cortex and hippocampus regions upon liver irradiation, where they manifest as altered gene expression and somewhat increased levels of γH2AX. We also report that bystander effects in the brain are associated with neuroanatomical and behavioural changes, and are more pronounced in females than in males. PMID:26678032

  12. Wind speed affects prey-catching behaviour in an orb web spider

    NASA Astrophysics Data System (ADS)

    Turner, Joe; Vollrath, Fritz; Hesselberg, Thomas

    2011-12-01

    Wind has previously been shown to influence the location and orientation of spider web sites and also the geometry and material composition of constructed orb webs. We now show that wind also influences components of prey-catching behaviour within the web. A small wind tunnel was used to generate different wind speeds. Araneus diadematus ran more slowly towards entangled Drosophila melanogaster in windy conditions, which took less time to escape the web. This indicates a lower capture probability and a diminished overall predation efficiency for spiders at higher wind speeds. We conclude that spiders' behaviour of taking down their webs as wind speed increases may therefore not be a response only to possible web damage.

  13. Group member prototypicality and intergroup negotiation: how one's standing in the group affects negotiation behaviour.

    PubMed

    Van Kleef, Gerben A; Steinel, Wolfgang; van Knippenberg, Daan; Hogg, Michael A; Svensson, Alicia

    2007-03-01

    How does a representative's position in the group influence behaviour in intergroup negotiation? Applying insights from the social identity approach (specifically self-categorization theory), the effects of group member prototypicality, accountability and group attractiveness on competitiveness in intergroup bargaining were examined. As representatives of their group, participants engaged in a computer-mediated negotiation with a simulated out-group opponent. In Experiment 1 (N=114), representatives with a peripheral status in the group sent more competitive and fewer cooperative messages to the opponent than did prototypical representatives, but only under accountability. Experiment 2 (N=110) replicated this finding, and showed that, under accountability, peripherals also made higher demands than did prototypicals, but only when group membership was perceived as attractive. Results are discussed in relation to impression management and strategic behaviour. PMID:17355722

  14. Inequity Aversion Negatively Affects Tolerance and Contact-Seeking Behaviours towards Partner and Experimenter.

    PubMed

    Brucks, Désirée; Essler, Jennifer L; Marshall-Pescini, Sarah; Range, Friederike

    2016-01-01

    Inequity aversion has been proposed to act as a limiting factor for cooperation, thus preventing subjects from disadvantageous cooperative interactions. While a recent study revealed that also dogs show some sensitivity to inequity, the underlying mechanisms of this behaviour are still unclear. The aim of the current study was threefold: 1) to replicate the study by Range et al. (2009, PNAS, 106, 340-345); 2) to investigate the emotional mechanisms involved in the inequity response by measuring the heart rate and 3) to explore the link between inequity aversion and cooperation in terms of behaviours shown towards the partner dog and towards the experimenter who caused the inequity. Dog tested in dyads were alternately asked to give their paw and were either equally or unequally rewarded by the experimenter. After each social test condition, we conducted food tolerance tests and free interaction tests in which the subjects' social behaviour towards the partner and the experimenter were observed. As in the previous study, subjects refused to continue giving their paw when only the partner was rewarded, but not when both dogs were rewarded with rewards of different quality. Although subjects did not react to this quality inequity during the test, we did find reduced durations of food sharing in the subsequent tolerance test, indicating that dogs perceived the inequity but were not able to react to it in the test context. Moreover, subjects avoided their partner and the experimenter more during the free interaction time following unequal compared to equal treatment. Despite the clear behavioural reactions to inequity, we could not detect any changes in heart rate. Results suggest that inequity aversion might in fact be mediated by simple emotional mechanisms: sharing a negative experience, like inequity, might reduce future cooperation by decreasing the likelihood of proximity being maintained between partners. PMID:27081852

  15. Inequity Aversion Negatively Affects Tolerance and Contact-Seeking Behaviours towards Partner and Experimenter

    PubMed Central

    Brucks, Désirée; Essler, Jennifer L.; Marshall-Pescini, Sarah; Range, Friederike

    2016-01-01

    Inequity aversion has been proposed to act as a limiting factor for cooperation, thus preventing subjects from disadvantageous cooperative interactions. While a recent study revealed that also dogs show some sensitivity to inequity, the underlying mechanisms of this behaviour are still unclear. The aim of the current study was threefold: 1) to replicate the study by Range et al. (2009, PNAS, 106, 340–345); 2) to investigate the emotional mechanisms involved in the inequity response by measuring the heart rate and 3) to explore the link between inequity aversion and cooperation in terms of behaviours shown towards the partner dog and towards the experimenter who caused the inequity. Dog tested in dyads were alternately asked to give their paw and were either equally or unequally rewarded by the experimenter. After each social test condition, we conducted food tolerance tests and free interaction tests in which the subjects’ social behaviour towards the partner and the experimenter were observed. As in the previous study, subjects refused to continue giving their paw when only the partner was rewarded, but not when both dogs were rewarded with rewards of different quality. Although subjects did not react to this quality inequity during the test, we did find reduced durations of food sharing in the subsequent tolerance test, indicating that dogs perceived the inequity but were not able to react to it in the test context. Moreover, subjects avoided their partner and the experimenter more during the free interaction time following unequal compared to equal treatment. Despite the clear behavioural reactions to inequity, we could not detect any changes in heart rate. Results suggest that inequity aversion might in fact be mediated by simple emotional mechanisms: sharing a negative experience, like inequity, might reduce future cooperation by decreasing the likelihood of proximity being maintained between partners. PMID:27081852

  16. From global change to a butterfly flapping: biophysics and behaviour affect tropical climate change impacts.

    PubMed

    Bonebrake, Timothy C; Boggs, Carol L; Stamberger, Jeannie A; Deutsch, Curtis A; Ehrlich, Paul R

    2014-10-22

    Difficulty in characterizing the relationship between climatic variability and climate change vulnerability arises when we consider the multiple scales at which this variation occurs, be it temporal (from minute to annual) or spatial (from centimetres to kilometres). We studied populations of a single widely distributed butterfly species, Chlosyne lacinia, to examine the physiological, morphological, thermoregulatory and biophysical underpinnings of adaptation to tropical and temperate climates. Microclimatic and morphological data along with a biophysical model documented the importance of solar radiation in predicting butterfly body temperature. We also integrated the biophysics with a physiologically based insect fitness model to quantify the influence of solar radiation, morphology and behaviour on warming impact projections. While warming is projected to have some detrimental impacts on tropical ectotherms, fitness impacts in this study are not as negative as models that assume body and air temperature equivalence would suggest. We additionally show that behavioural thermoregulation can diminish direct warming impacts, though indirect thermoregulatory consequences could further complicate predictions. With these results, at multiple spatial and temporal scales, we show the importance of biophysics and behaviour for studying biodiversity consequences of global climate change, and stress that tropical climate change impacts are likely to be context-dependent. PMID:25165769

  17. From global change to a butterfly flapping: biophysics and behaviour affect tropical climate change impacts

    PubMed Central

    Bonebrake, Timothy C.; Boggs, Carol L.; Stamberger, Jeannie A.; Deutsch, Curtis A.; Ehrlich, Paul R.

    2014-01-01

    Difficulty in characterizing the relationship between climatic variability and climate change vulnerability arises when we consider the multiple scales at which this variation occurs, be it temporal (from minute to annual) or spatial (from centimetres to kilometres). We studied populations of a single widely distributed butterfly species, Chlosyne lacinia, to examine the physiological, morphological, thermoregulatory and biophysical underpinnings of adaptation to tropical and temperate climates. Microclimatic and morphological data along with a biophysical model documented the importance of solar radiation in predicting butterfly body temperature. We also integrated the biophysics with a physiologically based insect fitness model to quantify the influence of solar radiation, morphology and behaviour on warming impact projections. While warming is projected to have some detrimental impacts on tropical ectotherms, fitness impacts in this study are not as negative as models that assume body and air temperature equivalence would suggest. We additionally show that behavioural thermoregulation can diminish direct warming impacts, though indirect thermoregulatory consequences could further complicate predictions. With these results, at multiple spatial and temporal scales, we show the importance of biophysics and behaviour for studying biodiversity consequences of global climate change, and stress that tropical climate change impacts are likely to be context-dependent. PMID:25165769

  18. Mild mutations in the pan neural gene prospero affect male-specific behaviour in Drosophila melanogaster.

    PubMed

    Grosjean, Yaël; Savy, Mathilde; Soichot, Julien; Everaerts, Claude; Cézilly, Frank; Ferveur, Jean François

    2004-01-30

    The fruitfly Drosophila melanogaster is one of the most appropriate model organisms to study the genetics of behaviour. Here, we focus on prospero (pros), a key gene for the development of the nervous system which specifies multiple aspects from the early formation of the embryonic central nervous system to the formation of larval and adult sensory organs. We studied the effects on locomotion, courtship and mating behaviour of three mild pros mutations. These newly isolated pros mutations were induced after the incomplete excision of a transposable genomic element that, before excision, caused a lethal phenotype during larval development. Strikingly, these mutant strains, but not the strains with a clean excision, produced a high frequency of heterozygous flies, after more than 50 generations in the lab. We investigated the factors that could decrease the fitness of homozygotes relatively to heterozygous pros mutant flies. Flies of both genotypes had slightly different levels of fertility. More strikingly, homozygous mutant males had a lower sexual activity than heterozygous males and failed to mate in a competitive situation. No similar effect was detected in mutant females. These findings suggest that mild mutations in pros did not alter vital functions during development but drastically changed adult male behaviour and reproductive fitness. PMID:14744542

  19. More support for mothers: a qualitative study on factors affecting immunisation behaviour in Kampala, Uganda

    PubMed Central

    2011-01-01

    Background The proportion of Ugandan children who are fully vaccinated has varied over the years. Understanding vaccination behaviour is important for the success of the immunisation programme. This study examined influences on immunisation behaviour using the attitude-social influence-self efficacy model. Methods We conducted nine focus group discussions (FGDs) with mothers and fathers. Eight key informant interviews (KIIs) were held with those in charge of community mobilisation for immunisation, fathers and mothers. Data was analysed using content analysis. Results Influences on the mother's immunisation behaviour ranged from the non-supportive role of male partners sometimes resulting into intimate partner violence, lack of presentable clothing which made mothers vulnerable to bullying, inconvenient schedules and time constraints, to suspicion against immunisation such as vaccines cause physical disability and/or death. Conclusions Immunisation programmes should position themselves to address social contexts. A community programme that empowers women economically and helps men recognise the role of women in decision making for child health is needed. Increasing male involvement and knowledge of immunisation concepts among caretakers could improve immunisation. PMID:21942999

  20. p38 MAPK Signaling in Osteoblast Differentiation

    PubMed Central

    Rodríguez-Carballo, Eddie; Gámez, Beatriz; Ventura, Francesc

    2016-01-01

    The skeleton is a highly dynamic tissue whose structure relies on the balance between bone deposition and resorption. This equilibrium, which depends on osteoblast and osteoclast functions, is controlled by multiple factors that can be modulated post-translationally. Some of the modulators are Mitogen-activated kinases (MAPKs), whose role has been studied in vivo and in vitro. p38-MAPK modifies the transactivation ability of some key transcription factors in chondrocytes, osteoblasts and osteoclasts, which affects their differentiation and function. Several commercially available inhibitors have helped to determine p38 action on these processes. Although it is frequently mentioned in the literature, this chemical approach is not always as accurate as it should be. Conditional knockouts are a useful genetic tool that could unravel the role of p38 in shaping the skeleton. In this review, we will summarize the state of the art on p38 activity during osteoblast differentiation and function, and emphasize the triggers of this MAPK. PMID:27200351

  1. ACTIVATION OF NFATC2 IN OSTEOBLASTS CAUSES OSTEOPENIA*

    PubMed Central

    Zanotti, Stefano; Canalis, Ernesto

    2015-01-01

    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 kilobase 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

  2. Activation of Nfatc2 in osteoblasts causes osteopenia.

    PubMed

    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

  3. Reproductive state affects hiding behaviour under risk of predation but not exploratory activity of female Spanish terrapins.

    PubMed

    Ibáñez, Alejandro; Marzal, Alfonso; López, Pilar; Martín, José

    2015-02-01

    Female investment during reproduction may reduce survivorship due to increased predation risk. During pregnancy, the locomotor performance of gravid females might be diminished due to the additional weight acquired. In addition, egg production may also increase thermoregulatory, metabolic and physiological costs. Also, pregnant females have greater potential fitness and should take fewer risks. Thus, females should ponder their reproductive state when considering their behavioural responses under risky situations. Here, we examine how reproductive state influence risk-taking behaviour in different contexts in female Spanish terrapins (Mauremys leprosa). We simulated predator attacks of different risk levels and measured the time that the turtles spent hiding entirely inside their own shells (i.e. appearance times). We also assessed the subsequent time after emergence from the shell that the turtles spent immobile monitoring for predators before starting to escape actively (i.e. waiting times). Likewise, we performed a novel-environment test and measured the exploratory activity of turtles. We found no correlations between appearance time, waiting time or exploratory activity, but appearance times were correlated across different risk levels. Only appearance time was affected by the reproductive state, where gravid females reappeared relatively later from their shells after a predator attack than non-gravid ones. Moreover, among gravid females, those carrying greater clutches tended to have longer appearance times. This suggests that only larger clutches could affect hiding behaviour in risky contexts. In contrast, waiting time spent scanning for predators and exploratory activity were not affected by the reproductive state. These differences between gravid and non-gravid females might be explained by the metabolic-physiological costs associated with egg production and embryo maintenance, as well as by the relatively higher potential fitness of gravid females. PMID

  4. How diabetic patients' ideas of illness course affect non-adherent behaviour: a qualitative study

    PubMed Central

    Lai, Wen An; Chie, Wei-Chu; Lew-Ting, Chih-Yin

    2007-01-01

    Background Type 2 diabetes is becoming more prevalent and its successful management relies on patients' self-care behaviours. Measures focusing on patients' perceptions can be effective behavioural interventions. Aim To gain insight into the perceptions of patients with diabetes, especially ideas of the illness course and perceived severity, and their impacts on self-care behaviour. Design of study Qualitative approach with in-depth patient interviews (n = 22) and seven focus groups (n = 53). Setting A rural town in Taiwan. Method The interview protocol was mainly derived from Kleinman's explanatory model. Purposive sampling strategies of maximum variation were used. The transcript of the interviews was analysed with editing and immersion/crystallisation styles. Results Diabetes is regarded as an incurable, inevitably deteriorating disorder of sugar metabolism with many chronic complications. Patients thought that renal injury, followed by blindness, leg amputation, and poor peripheral circulation, were the most frequent complications. They also assessed their perceived severity of the disease at specific points in time through different indicators in their daily lives, such as sugar level, presence of complications, and medications used. Patients felt that these aspects progressed concurrently and that the illness course followed a unidimensional process. The ever-increasing doses of medication was considered by these patients to be a side-effect of the drugs taken. Conclusion Physicians should clarify with their patients that the risks of uraemia, blindness, and leg amputation are less prevalent than expected and that patients should pay more attention to cardiovascular complications. Certain oral hypoglycaemic agents may not cause a vicious cycle of ever-increasing doses of medication and the drugs that need to be taken should not be seen as indicators of severity but, rather, measures taken to prevent the diabetes becoming severe in the future. PMID:17394733

  5. Environmental manipulation affects depressive-like behaviours in female Wistar-Kyoto rats.

    PubMed

    Mileva, Guergana R; Bielajew, Catherine

    2015-10-15

    While the efficacy of pharmacological interventions to treat depression has been well-studied in animal models, much less work has been done to shed light on how changes in the immediate environment can impact behaviour. Furthermore, most studies have focused on male rodents despite the prevalence of mood disorders in women. In this study, 36 Wistar Kyoto (validated animal model of depression) and 36 Wistar (control) female rats were used to examine the effects of environmental manipulation on depressive- and anxiety-like behaviours. Animals were assigned to one of three groups: standard (3 rats/cage), enriched (6 rats/cage plus physical enrichment), and isolation (1 rat/cage) housing. The elevated plus maze (EPM) and forced swim test (FST) were conducted prior to, and four weeks after environmental assignment to measure anxiety-like and depressive-like behaviours, respectively. Sucrose preference assessed anhedonia both before and after environmental assignment. Weight was measured every week to monitor weight-gain over time. Post-environment sucrose preference was significantly increased in animals in enriched housing as compared to those in isolated housing in both strains. While there were significant differences between strains in measures of open arm duration in the EPM and immobility in the FST, there appeared to be no differences between environmental groups. The results of this study highlight the importance of environmental factors in the expression of anhedonia. Enrichment appears to reduce anhedonia while isolation increases anhedonia. These effects should be studied further to assess whether longer periods of social and physical enrichment alleviate other symptoms of depression. PMID:26215574

  6. Does participation in an HIV vaccine efficacy trial affect risk behaviour in South Africa?

    PubMed Central

    Gray, GE; Metch, B; Churchyard, G; Mlisana, K; Nchabeleng, M; Allen, M; Moodie, Z; Kublin, J; Bekker, L-G

    2014-01-01

    Background Increased sexual risk behaviour in participants enrolled in HIV prevention trials has been a concern. The HVTN 503/Phambili study, a phase 2B study of the Merck Ad-5 multiclade HIV vaccine in South Africa, suspended enrollment and vaccinations following the results of the Step study. Participants were notified of their treatment allocation and continue to be followed. We investigated changes in risk behaviour over time and assessed the impact of study unblinding. Methods 801 participants were enrolled. Risk behaviors were assessed with an interviewer-administered questionnaire at 6-month intervals. We assessed change from enrolment to the first 6-month assessment pre-unblinding and between enrolment and at least 6 months post-unblinding on all participants with comparable data. A one-time unblinding risk perception questionnaire was administered post-unblinding. Results A decrease in participants reporting unprotected sex was observed in both measured time periods for men and women, with no differences by treatment arm. At 6 months (pre-unblinding), 29.6% of men and 35.8% of women reported changing from unprotected to protected sex (p <0.0001 for each).Men (22%) were more likely than women (14%) to report behavior change after unblinding (p=0.009). Post-enrolment, 142 (45%) of 313 previously uncircumcised men underwent medical circumcision. 663 participants completed the unblinding questionnaire. More vaccine (24.6%) as compared to placebo recipients (12.0%) agreed that they were more likely to get HIV than most people (p<0.0001), and attributed this to receiving the vaccine. Conclusion We did not find evidence of risk compensation during this clinical trial. Some risk behaviour reductions including male circumcision were noted irrespective of treatment allocation. PMID:23370155

  7. Acupuncture Affects Autonomic and Endocrine but Not Behavioural Responses Induced by Startle in Horses

    PubMed Central

    Villas-Boas, Julia Dias; Dias, Daniel Penteado Martins; Trigo, Pablo Ignacio; Almeida, Norma Aparecida dos Santos; de Almeida, Fernando Queiroz; de Medeiros, Magda Alves

    2015-01-01

    Startle is a fast response elicited by sudden acoustic, tactile, or visual stimuli in a variety of animals and in humans. As the magnitude of startle response can be modulated by external and internal variables, it can be a useful tool to study reaction to stress. Our study evaluated whether acupuncture can change cardiac autonomic modulation (heart rate variability); and behavioural (reactivity) and endocrine (cortisol levels) parameters in response to startle. Brazilian Sport horses (n = 6) were subjected to a model of startle in which an umbrella was abruptly opened near the horse. Before startle, the horses were subjected to a 20-minute session of acupuncture in acupoints GV1, HT7, GV20, and BL52 (ACUP) and in nonpoints (NP) or left undisturbed (CTL). For analysis of the heart rate variability, ultrashort-term (64 s) heart rate series were interpolated (4 Hz) and divided into 256-point segments and the spectra integrated into low (LF; 0.01–0.07 Hz; index of sympathetic modulation) and high (HF; 0.07–0.50 Hz; index of parasympathetic modulation) frequency bands. Acupuncture (ACUP) changed the sympathovagal balance with a shift towards parasympathetic modulation, reducing the prompt startle-induced increase in LF/HF and reducing cortisol levels 30 min after startle. However, acupuncture elicited no changes in behavioural parameters. PMID:26413116

  8. Putting Up a Big Front: Car Design and Size Affect Road-Crossing Behaviour.

    PubMed

    Klatt, Wilhelm K; Chesham, Alvin; Lobmaier, Janek S

    2016-01-01

    Previous research suggests that people tend to see faces in car fronts and that they attribute personality characteristics to car faces. In the present study we investigated whether car design influences pedestrian road-crossing behaviour. An immersive virtual reality environment with a zebra crossing scenario was used to determine a) whether the minimum accepted distance for crossing the street is larger for cars with a dominant appearance than for cars with a friendly appearance and b) whether the speed of dominant-looking cars is overestimated as compared to friendly-looking cars. Participants completed both tasks while either standing on the pavement or on the centre island. We found that people started to cross the road later in front of friendly-looking low-power cars compared to dominant-looking high-power cars, but only if the cars were relatively large in size. For small cars we found no effect of power. The speed of smaller cars was estimated to be higher compared to large cars (size-speed bias). Furthermore, there was an effect of starting position: From the centre island, participants entered the road significantly later (i. e. closer to the approaching car) and left the road later than when starting from the pavement. Similarly, the speed of the cars was estimated significantly lower when standing on the centre island compared to the pavement. To our knowledge, this is the first study to show that car fronts elicit responses on a behavioural level. PMID:27434187

  9. Putting Up a Big Front: Car Design and Size Affect Road-Crossing Behaviour

    PubMed Central

    Klatt, Wilhelm K.; Chesham, Alvin; Lobmaier, Janek S.

    2016-01-01

    Previous research suggests that people tend to see faces in car fronts and that they attribute personality characteristics to car faces. In the present study we investigated whether car design influences pedestrian road-crossing behaviour. An immersive virtual reality environment with a zebra crossing scenario was used to determine a) whether the minimum accepted distance for crossing the street is larger for cars with a dominant appearance than for cars with a friendly appearance and b) whether the speed of dominant-looking cars is overestimated as compared to friendly-looking cars. Participants completed both tasks while either standing on the pavement or on the centre island. We found that people started to cross the road later in front of friendly-looking low-power cars compared to dominant-looking high-power cars, but only if the cars were relatively large in size. For small cars we found no effect of power. The speed of smaller cars was estimated to be higher compared to large cars (size-speed bias). Furthermore, there was an effect of starting position: From the centre island, participants entered the road significantly later (i. e. closer to the approaching car) and left the road later than when starting from the pavement. Similarly, the speed of the cars was estimated significantly lower when standing on the centre island compared to the pavement. To our knowledge, this is the first study to show that car fronts elicit responses on a behavioural level. PMID:27434187

  10. Chronic exposure to mercuric chloride during gestation affects sensorimotor development and later behaviour in rats.

    PubMed

    Chehimi, Latifa; Roy, Vincent; Jeljeli, Mustapha; Sakly, Mohsen

    2012-09-01

    The current study was performed to assess the effects of inorganic mercury (mercuric chloride - HgCl(2)) on the development of offsprings from intoxicated-mother during pregnancy. In this respect, pregnant rats were chronically treated with HgCl(2) at 50 ppm (Hg50) and 100 ppm (Hg100) in drinking water. After parturition, maternal behaviour was recorded during 30 min at 1st to 6th postnatal day (Pnd). The development of their offspring was studied during the first 17 days after birth. Sensorimotor development of pups was measured by different tests: rooting reflex, vibrissae placing response, righting reflex, negative geotaxis, suspension test and rotating grid. Two month after birth, the anxiety of offspring was tested using the elevated plus maze test. Our results indicate that mercury treatment significantly reduced the nursing and increased the time out the nest or drinking and eating. We also showed that prenatal exposure to HgCl(2) decreased weight gain. Importantly, the rooting reflex, the development of the vibrissae placing response, the righting reflex, the grip strength and the negative geotaxis behaviour were delayed in the offspring of dams treated with Hg50, the delay being more severe with Hg100. We also found a decrease in anxiety in adulthood. Cross-fostering test support the direct toxic effects of mercury. PMID:22705860

  11. Does underwater flash photography affect the behaviour, movement and site persistence of seahorses?

    PubMed

    Harasti, D; Gladstone, W

    2013-11-01

    The effect of flash photography on seahorse species has never been tested. An experiment was established to test the effect of flash photography and the handling of Hippocampus whitei, a medium-sized seahorse species endemic to Australia, on their behavioural responses, movements and site persistence. A total of 24 H. whitei were utilized in the experiment with eight in each of the three treatments (flash photography, handling and control). The effect of underwater flash photography on H. whitei movements was not significant; however, the effect of handling H. whitei to take a photograph had a significant effect on their short-term behavioural responses to the photographer. Kaplan-Meier log-rank test revealed that there was no significant difference in site persistence of H. whitei from each of the three treatments and that flash photography had no long-term effects on their site persistence. It is concluded that the use of flash photography by divers is a safe and viable technique with H. whitei, particularly if photographs can be used for individual identification purposes. PMID:24131331

  12. How does the composition affect the mechanical behaviour of simulated clay-rich fault gouges?

    NASA Astrophysics Data System (ADS)

    Bakker, Elisenda; Spiers, Christopher J.; Hangx, Suzanne J. T.

    2014-05-01

    CO2 capture and storage (CCS) in depleted oil and gas reservoirs is seen as one of the most promising large-scale CO2-mitigation strategies. Prediction of the effect of fluid-rock interaction on the mechanical integrity and sealing capacity of a reservoir-seal system, on timescales of the order of 1,000 or 10,000 years, is important to ensure the safety and containment of a reservoir in relation to long-term CO2 storage. However, most chemical reactions in rock/CO2/brine systems are slow, which means that long-term effects of fluids on rock composition, microstructure, mechanical properties and transport properties cannot be easily reproduced under laboratory conditions. One way to overcome this problem is to use simulated fault gouges in experiments, investigating a range of possible mineralogical compositions resulting from CO2-exposure. Previous studies have shown that the mechanical and transport properties of clay-rich fault gouges are significantly influenced by the mineralogy, particularly by the presence and relative amount of secondary phases, such as quartz and/or carbonate. In CCS settings, where dissolution and/or precipitation of carbonates may play an important role, the carbonate:clay ratio is expected to influence fault frictional behaviour. This is supported by the different behaviour of phyllosilicates, which generally show stable slip behaviour (aseismic), compared to carbonates, which have shown to become prone to unstable slip (potentially seismic) with increasing temperature. However, little is known about the mechanical and transport properties of carbonate/clay mixtures. We investigated the effect of the carbonate:clay ratio on fault friction, fault reactivation potential and slip stability, i.e. seismic vs. aseismic behaviour, as well as transmissivity evolution during and after fault reactivation. We used two types of starting material, derived from crushed Opalinus Claystone (Mont Terri, Switzerland): i) untreated samples consisting

  13. MEK5 suppresses osteoblastic differentiation.

    PubMed

    Kaneshiro, Shoichi; Otsuki, Dai; Yoshida, Kiyoshi; Yoshikawa, Hideki; Higuchi, Chikahisa

    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. PMID:25998381

  14. How the flow affects the phase behaviour and microstructure of polymer nanocomposites

    SciTech Connect

    Stephanou, Pavlos S.

    2015-02-14

    We address the issue of flow effects on the phase behaviour of polymer nanocomposite melts by making use of a recently reported Hamiltonian set of evolution equations developed on principles of non-equilibrium thermodynamics. To this end, we calculate the spinodal curve, by computing values for the nanoparticle radius as a function of the polymer radius-of-gyration for which the second derivative of the generalized free energy of the system becomes zero. Under equilibrium conditions, we recover the phase diagram predicted by Mackay et al. [Science 311, 1740 (2006)]. Under non-equilibrium conditions, we account for the extra terms in the free energy due to changes in the conformations of polymer chains by the shear flow. Overall, our model predicts that flow enhances miscibility, since the corresponding miscibility window opens up for non-zero shear rate values.

  15. Supplementary feeding affects the breeding behaviour of male European treefrogs (Hyla arborea)

    PubMed Central

    Meuche, Ivonne; Grafe, T Ulmar

    2009-01-01

    Background We investigated the effects of energetic constraints on the breeding behaviour of male European treefrogs Hyla arborea and how calling males allocated additional energy supplied by feeding experiments. Results Presence in the chorus was energetically costly indicated by both fed and unfed males losing weight. Males that were supplied with additional energy did not show longer chorus tenure. Instead, fed males returned sooner to the chorus. Additionally, fed males called more often than control males, a novel response for anurans. A significantly higher calling rate was noted from males even 31 nights after supplementary feeding. Conclusion This strategy of allocating additional energy reserves to increasing calling rate is beneficial given the preference of female hylids for males calling at high rates and a female's ability to detect small incremental increases in calling rate. PMID:19128468

  16. Conditioned water affects pair formation behaviour in the marine polychaete Neanthes (Nereis) acuminata.

    PubMed

    Storey, Ellen J; Reish, Don J; Hardege, Jörg D

    2013-01-01

    Assessing cues from conspecifics is paramount during mate choice decisions. Neanthes acuminata is a marine polychaete with a unique life cycle: pair formation, female death following reproduction, male parental care and male ability to mate again after egg care. Males completing such egg care are 'experienced'. Females have been shown to prefer experienced males over all others, including aggressively dominant males. As the female dies following reproduction, the reproductive success of her offspring depends upon successful parental care by the male. It is therefore vital that the female makes a good mate choice decision. This paper shows that the use of conditioned water from males caring for eggs and newly experienced males caused the female to alter her choice to a previously undesired male. However, conditioned water from males, which had reproduced but were isolated for 2 weeks, did not have the same effect on pairing behaviour. This indicates that the smell of experience is short lived. PMID:22941305

  17. Plasma deposited composite coatings to control biological response of osteoblast-like MG-63 cells

    NASA Astrophysics Data System (ADS)

    Keremidarska, M.; Radeva, E.; Eleršič, K.; Iglič, A.; Pramatarova, L.; Krasteva, N.

    2014-12-01

    The successful osseointegration of a bone implant is greatly dependent on its ability to support cellular adhesion and functions. Deposition of thin composite coatings onto the implant surface is a promising approach to improve interactions with cells without compromising implant bulk properties. In this work, we have developed composite coatings, based on hexamethyldisiloxane (HMDS) and detonation nanodiamond (DND) particles and have studied adhesion, growth and function of osteoblast-like MG-63 cells. PPHMDS/DND composites are of interest for orthopedics because they combine superior mechanical properties and good biocompatibility of DND with high adherence of HMDS to different substrata including glass, metals and plastics. We have used two approaches of the implementation of DND particles into a polymer matrix: pre-mixture of both components followed by plasma polymerization and layer-by-layer deposition of HMDS and DND particles and found that the deposition approach affects significantly the surface properties of the resulting layers and cell behaviour. The composite, prepared by subsequent deposition of monomer and DND particles was hydrophilic, with a rougher surface and MG-63 cells demonstrated better spreading, growth and function compared to the other composite which was hydrophobic with a smooth surface similarly to unmodified polymer. Thus, by varying the deposition approach, different PPHMDS/DND composite coatings, enhancing or inhibiting osteoblast adhesion and functions, can be obtained. In addition, the effect of fibronectin pre-adsorption was studied and was found to increase greatly MG-63 cell spreading.

  18. More than a Signature: How Advisor Choice and Advisor Behaviour Affect Doctoral Student Satisfaction

    ERIC Educational Resources Information Center

    Zhao, Chun-Mei; Golde, Chris M.; McCormick, Alexander C.

    2007-01-01

    A satisfactory relationship between doctoral students and their advisors is an essential component of successful doctoral training. Using responses to a national survey of doctoral students in the US from 27 universities and 11 disciplines, this paper explores factors affecting students' satisfaction with the advising relationship. We find that…

  19. Mother-Toddler Affect Exchanges and Children's Mastery Behaviours during Preschool Years

    ERIC Educational Resources Information Center

    Wang, Jun; Morgan, George A.; Biringen, Zeynep

    2014-01-01

    This study examined the longitudinal relations of mother-child affect exchanges at 18?months with children's mastery motivation at 39?months. Observation and questionnaire data were collected from mother-child dyads when children were 18?months; 43 mothers again rated their children's mastery motivation at 39?months. Results suggested…

  20. Studying User Income through Language, Behaviour and Affect in Social Media

    PubMed Central

    Preoţiuc-Pietro, Daniel; Volkova, Svitlana; Lampos, Vasileios; Bachrach, Yoram; Aletras, Nikolaos

    2015-01-01

    Automatically inferring user demographics from social media posts is useful for both social science research and a range of downstream applications in marketing and politics. We present the first extensive study where user behaviour on Twitter is used to build a predictive model of income. We apply non-linear methods for regression, i.e. Gaussian Processes, achieving strong correlation between predicted and actual user income. This allows us to shed light on the factors that characterise income on Twitter and analyse their interplay with user emotions and sentiment, perceived psycho-demographics and language use expressed through the topics of their posts. Our analysis uncovers correlations between different feature categories and income, some of which reflect common belief e.g. higher perceived education and intelligence indicates higher earnings, known differences e.g. gender and age differences, however, others show novel findings e.g. higher income users express more fear and anger, whereas lower income users express more of the time emotion and opinions. PMID:26394145

  1. Social bonds affect anti-predator behaviour in a tolerant species of macaque, Macaca nigra.

    PubMed

    Micheletta, Jérôme; Waller, Bridget M; Panggur, Maria R; Neumann, Christof; Duboscq, Julie; Agil, Muhammad; Engelhardt, Antje

    2012-10-01

    Enduring positive social bonds between individuals are crucial for humans' health and well being. Similar bonds can be found in a wide range of taxa, revealing the evolutionary origins of humans' social bonds. Evidence suggests that these strong social bonds can function to buffer the negative effects of living in groups, but it is not known whether they also function to minimize predation risk. Here, we show that crested macaques (Macaca nigra) react more strongly to playbacks of recruitment alarm calls (i.e. calls signalling the presence of a predator and eliciting cooperative mobbing behaviour) if they were produced by an individual with whom they share a strong social bond. Dominance relationships between caller and listener had no effect on the reaction of the listener. Thus, strong social bonds may improve the coordination and efficiency of cooperative defence against predators, and therefore increase chances of survival. This result broadens our understanding of the evolution and function of social bonds by highlighting their importance in the anti-predator context. PMID:22859593

  2. Oceanographic and behavioural processes affecting invertebrate larval dispersal and supply in the western Iberia upwelling ecosystem

    NASA Astrophysics Data System (ADS)

    Queiroga, Henrique; Cruz, Teresa; dos Santos, Antonina; Dubert, Jesus; González-Gordillo, Juan Ignácio; Paula, José; Peliz, Álvaro; Santos, A. Miguel P.

    2007-08-01

    The present review addresses recent findings made in the western Iberia ecosystem on the behavioural and physical interactions that regulate dispersal, supply to coastal habitats and settlement of invertebrate larvae. These studies used the barnacle Chthamalus spp. and the crab Carcinus maenas as model organisms. The observations made on the Iberian shelf showed extensive diel vertical migrations along the water column by representatives of both groups that have never been reported before. The interaction of the diel vertical migration with the two-layer flow structure of upwelling/downwelling circulation suggests a mechanism that may help to retain larvae in shelf waters during upwelling conditions. Measurements of daily supply of C. maenas megalopae to estuaries separated by 500 km disclosed a semilunar pattern, with highest supply around highest amplitude tides, indicating that supply of megalopae to estuaries is accomplished by selective tidal stream transport. Relaxation of equatorward winds also played a role in supply, by enhancing translocation of megalopae to the nearshore. Concerning Chthamalus larvae, the observations on daily settlement made at rocky shores also separated by 500 km showed unclear patterns between locations and years. The relationship of settlement with water temperature, tidal range and upwelling indices indicated that supply of barnacle cyprids may be controlled by multiple mechanisms, viz. upwelling/downwelling circulation, internal tidal bores and sea breezes.

  3. Studying User Income through Language, Behaviour and Affect in Social Media.

    PubMed

    Preoţiuc-Pietro, Daniel; Volkova, Svitlana; Lampos, Vasileios; Bachrach, Yoram; Aletras, Nikolaos

    2015-01-01

    Automatically inferring user demographics from social media posts is useful for both social science research and a range of downstream applications in marketing and politics. We present the first extensive study where user behaviour on Twitter is used to build a predictive model of income. We apply non-linear methods for regression, i.e. Gaussian Processes, achieving strong correlation between predicted and actual user income. This allows us to shed light on the factors that characterise income on Twitter and analyse their interplay with user emotions and sentiment, perceived psycho-demographics and language use expressed through the topics of their posts. Our analysis uncovers correlations between different feature categories and income, some of which reflect common belief e.g. higher perceived education and intelligence indicates higher earnings, known differences e.g. gender and age differences, however, others show novel findings e.g. higher income users express more fear and anger, whereas lower income users express more of the time emotion and opinions. PMID:26394145

  4. Changes in osteoblastic activity due to simulated weightless conditions

    NASA Technical Reports Server (NTRS)

    Doty, S. B.; Morey-Holton, E. R.

    1982-01-01

    Using histochemistry and electron microscopy, the reduced bone formation which occurs in the hypokinetic, orthostatically treated adult rat has been studied. The two major changes noted occurred in the osteoblast population, indicated by a reduced alkaline phosphatase activity and reduced numbers of gap junctions between cells. These results were most noticeable in the periosteum and endosteum of the long bones. Changes in osteoblasts lining the surface of trabecular bone were not as evident. These results indicate that the cells lining the surfaces of weight bearing bones are most affected by hypokinesia and this reduction in cellular activity may be a mechanically induced effect.

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

    SciTech Connect

    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

  6. Baicalin, a Flavone, Induces the Differentiation of Cultured Osteoblasts

    PubMed Central

    Guo, Ava J. Y.; Choi, Roy C. Y.; Cheung, Anna W. H.; Chen, Vicky P.; Xu, Sherry L.; Dong, Tina T. X.; Chen, Ji J.; Tsim, Karl W. K.

    2011-01-01

    Flavonoids, a group of natural compounds found in a variety of vegetables and herbal medicines, have been intensively reported on regarding their estrogen-like activities and particularly their ability to affect bone metabolism. Here, different subclasses of flavonoids were screened for their osteogenic properties by measuring alkaline phosphatase activity in cultured rat osteoblasts. The flavone baicalin derived mainly from the roots of Scutellaria baicalensis showed the strongest induction of alkaline phosphatase activity. In cultured osteoblasts, application of baicalin increased significantly the osteoblastic mineralization and the levels of mRNAs encoding the bone differentiation markers, including osteonectin, osteocalcin, and collagen type 1α1. Interestingly, the osteogenic effect of baicalin was not mediated by its estrogenic activity. In contrast, baicalin promoted osteoblastic differentiation via the activation of the Wnt/β-catenin signaling pathway; the activation resulted in the phosphorylation of glycogen synthase kinase 3β and, subsequently, induced the nuclear accumulation of the β-catenin, leading to the transcription activation of Wnt-targeted genes for osteogenesis. The baicalin-induced osteogenic effects were fully abolished by DKK-1, a blocker of Wnt/β-catenin receptor. Moreover, baicalin also enhanced the mRNA expression of osteoprotegerin, which could regulate indirectly the activation of osteoclasts. Taken together, our results suggested that baicalin could act via Wnt/β-catenin signaling to promote osteoblastic differentiation. The osteogenic flavonoids could be very useful in finding potential drugs, or food supplements, for treating post-menopausal osteoporosis. PMID:21652696

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

    PubMed

    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

  8. Synergistic activity of polarised osteoblasts inside condensations cause their differentiation

    PubMed Central

    Kaul, Himanshu; Hall, Brian K.; Newby, Chris; Ventikos, Yiannis

    2015-01-01

    Condensation of pre-osteogenic, or pre-chondrogenic, cells is the first of a series of processes that initiate skeletal development. We present a validated, novel, three-dimensional agent-based model of in vitro intramembranous osteogenic condensation. The model, informed by system heterogeneity and relying on an interaction-reliant strategy, is shown to be sensitive to ‘rules’ capturing condensation growth and can be employed to track activity of individual cells to observe their macroscopic impact. It, therefore, makes available previously inaccessible data, offering new insights and providing a new context for exploring the emergence, as well as normal and abnormal development, of osteogenic structures. Of the several stages of condensation we investigate osteoblast ‘burial’ within the osteoid they deposit. The mechanisms underlying entrapment – required for osteoblasts to differentiate into osteocytes – remain a matter of conjecture with several hypotheses claiming to capture this important transition. Computational examination of this transition indicates that osteoblasts neither turn off nor slow down their matrix secreting genes – a widely held view; nor do they secrete matrix randomly. The model further reveals that osteoblasts display polarised behaviour to deposit osteoid. This is both an important addition to our understanding of condensation and an important validation of the model’s utility. PMID:26146365

  9. Mutual enhancement of differentiation of osteoblasts and osteocytes occurs through direct cell-cell contact

    PubMed Central

    Fujita, Koji; Xing, Qian; Khosla, Sundeep; Monroe, David G.

    2014-01-01

    There is increasing evidence that osteocytes regulate multiple aspects of bone remodeling through bi-directional communication with osteoblasts. This is potentially mediated through cell-cell contact via osteocytic dendritic processes, through the activity of secreted factors, or both. To test whether cell-cell contact affects gene expression patterns in osteoblasts and osteocytes, we used a co-culture system where calvarial osteoblasts and IDG-SW3 osteocytes were allowed to touch through a porous membrane, while still being physically separated to allow for phenotypic characterization. Osteoblast/osteocyte cell-contact resulted in up-regulation of osteoblast differentiation genes in the osteoblasts, when compared to wells where no cell contact was allowed. Examination of osteocyte gene expression when in direct contact with osteoblasts also revealed increased expression of osteocyte-specific genes. These data suggest that physical contact mutually enhances both the osteoblastic and osteocytic character of each respective cell type. Interestingly, Gja1 (a gap junction protein) was increased in the osteoblasts only when in direct contact with the osteocytes, suggesting that Gja1 may mediate some of the effects of direct cell contact. To test this hypothesis, we treated the direct contact system with the gap junction inhibitor 18-alpha-glycyrrhetinic acid and found that Bglap expression was significantly inhibited. This suggests that osteocytes may regulate late osteoblast differentiation at least in part through Gja1. Identification of the specific factors involved in the enhancement of differentiation of both osteoblasts and osteocytes when in direct contact will uncover new biology concerning how these bone cells communicate. PMID:25043105

  10. Prawn Shell Chitosan Exhibits Anti-Obesogenic Potential through Alterations to Appetite, Affecting Feeding Behaviour and Satiety Signals In Vivo.

    PubMed

    Egan, Áine M; O'Doherty, John V; Vigors, Stafford; Sweeney, Torres

    2016-01-01

    The crustacean shells-derived polysaccharide chitosan has received much attention for its anti-obesity potential. Dietary supplementation of chitosan has been linked with reductions in feed intake, suggesting a potential link between chitosan and appetite control. Hence the objective of this experiment was to investigate the appetite suppressing potential of prawn shell derived chitosan in a pig model. Pigs (70 ± 0.90 kg, 125 days of age, SD 2.0) were fed either T1) basal diet or T2) basal diet plus 1000 ppm chitosan (n = 20 gilts per group) for 63 days. The parameter categories which were assessed included performance, feeding behaviour, serum leptin concentrations and expression of genes influencing feeding behaviour in the small intestine, hypothalamus and adipose tissue. Pigs offered chitosan visited the feeder less times per day (P<0.001), had lower intake per visit (P<0.001), spent less time eating per day (P<0.001), had a lower eating rate (P<0.01) and had reduced feed intake and final body weight (P< 0.001) compared to animals offered the basal diet. There was a treatment (P<0.05) and time effect (P<0.05) on serum leptin concentrations in animals offered the chitosan diet compared to animals offered the basal diet. Pigs receiving dietary chitosan had an up-regulation in gene expression of growth hormone receptor (P<0.05), Peroxisome proliferator activated receptor gamma (P<0.01), neuromedin B (P<0.05), neuropeptide Y receptor 5 (P<0.05) in hypothalamic nuclei and neuropeptide Y (P<0.05) in the jejunum. Animals consuming chitosan had increased leptin expression in adipose tissue compared to pigs offered the basal diet (P<0.05). In conclusion, these data support the hypothesis that dietary prawn shell chitosan exhibits anti-obesogenic potential through alterations to appetite, and feeding behaviour affecting satiety signals in vivo. PMID:26901760

  11. Prawn Shell Chitosan Exhibits Anti-Obesogenic Potential through Alterations to Appetite, Affecting Feeding Behaviour and Satiety Signals In Vivo

    PubMed Central

    Egan, Áine M.; O’Doherty, John V.; Vigors, Stafford; Sweeney, Torres

    2016-01-01

    The crustacean shells-derived polysaccharide chitosan has received much attention for its anti-obesity potential. Dietary supplementation of chitosan has been linked with reductions in feed intake, suggesting a potential link between chitosan and appetite control. Hence the objective of this experiment was to investigate the appetite suppressing potential of prawn shell derived chitosan in a pig model. Pigs (70 ± 0.90 kg, 125 days of age, SD 2.0) were fed either T1) basal diet or T2) basal diet plus 1000 ppm chitosan (n = 20 gilts per group) for 63 days. The parameter categories which were assessed included performance, feeding behaviour, serum leptin concentrations and expression of genes influencing feeding behaviour in the small intestine, hypothalamus and adipose tissue. Pigs offered chitosan visited the feeder less times per day (P<0.001), had lower intake per visit (P<0.001), spent less time eating per day (P<0.001), had a lower eating rate (P<0.01) and had reduced feed intake and final body weight (P< 0.001) compared to animals offered the basal diet. There was a treatment (P<0.05) and time effect (P<0.05) on serum leptin concentrations in animals offered the chitosan diet compared to animals offered the basal diet. Pigs receiving dietary chitosan had an up-regulation in gene expression of growth hormone receptor (P<0.05), Peroxisome proliferator activated receptor gamma (P<0.01), neuromedin B (P<0.05), neuropeptide Y receptor 5 (P<0.05) in hypothalamic nuclei and neuropeptide Y (P<0.05) in the jejunum. Animals consuming chitosan had increased leptin expression in adipose tissue compared to pigs offered the basal diet (P<0.05). In conclusion, these data support the hypothesis that dietary prawn shell chitosan exhibits anti-obesogenic potential through alterations to appetite, and feeding behaviour affecting satiety signals in vivo. PMID:26901760

  12. Phenotypic and Evolutionary Consequences of Social Behaviours: Interactions among Individuals Affect Direct Genetic Effects

    PubMed Central

    Trubenová, Barbora; Hager, Reinmar

    2012-01-01

    Traditional quantitative genetics assumes that an individual's phenotype is determined by both genetic and environmental factors. For many animals, part of the environment is social and provided by parents and other interacting partners. When expression of genes in social partners affects trait expression in a focal individual, indirect genetic effects occur. In this study, we explore the effects of indirect genetic effects on the magnitude and range of phenotypic values in a focal individual in a multi-member model analyzing three possible classes of interactions between individuals. We show that social interactions may not only cause indirect genetic effects but can also modify direct genetic effects. Furthermore, we demonstrate that both direct and indirect genetic effects substantially alter the range of phenotypic values, particularly when a focal trait can influence its own expression via interactions with traits in other individuals. We derive a function predicting the relative importance of direct versus indirect genetic effects. Our model reveals that both direct and indirect genetic effects can depend to a large extent on both group size and interaction strength, altering group mean phenotype and variance. This may lead to scenarios where between group variation is much higher than within group variation despite similar underlying genetic properties, potentially affecting the level of selection. Our analysis highlights key properties of indirect genetic effects with important consequences for trait evolution, the level of selection and potentially speciation. PMID:23226195

  13. Does influenza A virus infection affect movement behaviour during stopover in its wild reservoir host?

    PubMed Central

    Bengtsson, Daniel; Safi, Kamran; Avril, Alexis; Fiedler, Wolfgang; Wikelski, Martin; Gunnarsson, Gunnar; Elmberg, Johan; Tolf, Conny; Olsen, Björn; Waldenström, Jonas

    2016-01-01

    The last decade has seen a surge in research on avian influenza A viruses (IAVs), in part fuelled by the emergence, spread and potential zoonotic importance of highly pathogenic virus subtypes. The mallard (Anas platyrhynchos) is the most numerous and widespread dabbling duck in the world, and one of the most important natural hosts for studying IAV transmission dynamics. In order to predict the likelihood of IAV transmission between individual ducks and to other hosts, as well as between geographical regions, it is important to understand how IAV infection affects the host. In this study, we analysed the movements of 40 mallards equipped with GPS transmitters and three-dimensional accelerometers, of which 20 were naturally infected with low pathogenic avian influenza virus (LPAIV), at a major stopover site in the Northwest European flyway. Movements differed substantially between day and night, as well as between mallards returning to the capture site and those feeding in natural habitats. However, movement patterns did not differ between LPAIV infected and uninfected birds. Hence, LPAIV infection probably does not affect mallard movements during stopover, with high possibility of virus spread along the migration route as a consequence. PMID:26998334

  14. Coping behaviours and post-traumatic stress in war-affected eastern Congolese adolescents.

    PubMed

    Mels, Cindy; Derluyn, Ilse; Broekaert, Eric; García-Pérez, Coral

    2015-02-01

    This study explores coping strategies used by war-affected eastern Congolese adolescents across age and sex, and the association between post-traumatic stress symptoms and engagement and disengagement coping. Cross-sectional data were collected in 11 secondary schools across four areas in the Ituri province, Democratic Republic of Congo. A total of 952 pupils (45.3% girls, 54.7% boys) aged 13-21 years (M = 15.83, standard deviation = 1.81) participated in self-report assessment, using instruments that were either specifically developed (Adolescent Complex Emergency Exposure Scale, assessing traumatic exposure), validated (Impact of Event Scale Revised, assessing post-traumatic stress symptoms) or reviewed (Kidcope, assessing coping strategies) for the study population. Reported coping strategies varied with age, and boys more frequently reported problem solving and resignation as compared with girls. Disengagement coping was associated with lower symptom scores in younger adolescent girls, as was the interaction effect between engagement and disengagement coping. We conclude that disengagement coping is not necessarily a maladaptive reaction to stressful events in war-affected situations and that future research should aim to better understand the heterogeneous patterns of stress and coping responses, including the role of factors such as the nature and appraisal of stressors, available resources for coping and cultural preferences. PMID:24130163

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

    SciTech Connect

    Vezeridis, Peter S.; Chen Qian . E-mail: j.kleinnulend@vumc.nl

    2006-09-29

    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.

  16. Fibronectin regulates calvarial osteoblast differentiation

    NASA Technical Reports Server (NTRS)

    Moursi, A. M.; Damsky, C. H.; Lull, J.; Zimmerman, D.; Doty, S. B.; Aota, S.; Globus, R. K.

    1996-01-01

    The secretion of fibronectin by differentiating osteoblasts and its accumulation at sites of osteogenesis suggest that fibronectin participates in bone formation. To test this directly, we determined whether fibronectin-cell interactions regulate progressive differentiation of cultured fetal rat calvarial osteoblasts. Spatial distributions of alpha 5 integrin subunit, fibronectin, osteopontin (bone sialoprotein I) and osteocalcin (bone Gla-protein) were similar in fetal rat calvaria and mineralized, bone-like nodules formed by cultured osteoblasts. Addition of anti-fibronectin antibodies to cultures at confluence reduced subsequent formation of nodules to less than 10% of control values, showing that fibronectin is required for normal nodule morphogenesis. Anti-fibronectin antibodies selectively inhibited steady-state expression of mRNA for genes associated with osteoblast differentiation; mRNA levels for alkaline phosphatase and osteocalcin were suppressed, whereas fibronectin, type I collagen and osteopontin were unaffected. To identify functionally relevant domains of fibronectin, we treated cells with soluble fibronectin fragments and peptides. Cell-binding fibronectin fragments (type III repeats 6-10) containing the Arg-Gly-Asp (RGD) sequence blocked both nodule initiation and maturation, whether or not they contained a functional synergy site. In contrast, addition of the RGD-containing peptide GRGDSPK alone did not inhibit nodule initiation, although it did block nodule maturation. Thus, in addition to the RGD sequence, other features of the large cell-binding fragments contribute to the full osteogenic effects of fibronectin. Nodule formation and osteoblast differentiation resumed after anti-fibronectin antibodies or GRGDSPK peptides were omitted from the media, showing that the inhibition was reversible and the treatments were not cytotoxic. Outside the central cell-binding domain, peptides from the IIICS region and antibodies to the N terminus did not

  17. Evidence that the type of person affects the strength of the perceived behavioural control-intention relationship.

    PubMed

    Sheeran, Paschal; Trafimow, David; Finlay, Krystina A; Norman, Paul

    2002-06-01

    This study examined the role of person type in explaining the relationship between perceived behavioural control and behavioural intentions. Participants (N = 187) completed measures of the theory of planned behaviour (Ajzen, 1991) variables regarding 30 behaviours. Within-participants analyses demonstrated that intentions were more strongly predicted by perceived behavioural control (PBC) than a combination of attitudes and subjective norms among a minority of the sample. When these 'PBC controlled' participants were considered separately, the effects for perceived behavioural control obtained in previous between-participants analyses were augmented. Conversely, when these participants were excluded from the sample, the effects of perceived behavioural control were reduced. PBC control was also modestly associated with dispositional measures of perceived controllability. Overall, the findings indicate that the strength of the perceived behavioural control-intention relationship depends not only on the type of behaviour but also on the type of person. PMID:12133227

  18. Predicting intentions to purchase organic food: the role of affective and moral attitudes in the Theory of Planned Behaviour.

    PubMed

    Arvola, A; Vassallo, M; Dean, M; Lampila, P; Saba, A; Lähteenmäki, L; Shepherd, R

    2008-01-01

    This study examined the usefulness of integrating measures of affective and moral attitudes into the Theory of Planned Behaviour (TPB)-model in predicting purchase intentions of organic foods. Moral attitude was operationalised as positive self-rewarding feelings of doing the right thing. Questionnaire data were gathered in three countries: Italy (N=202), Finland (N=270) and UK (N=200) in March 2004. Questions focussed on intentions to purchase organic apples and organic ready-to-cook pizza instead of their conventional alternatives. Data were analysed using Structural Equation Modelling by simultaneous multi-group analysis of the three countries. Along with attitudes, moral attitude and subjective norms explained considerable shares of variances in intentions. The relative influences of these variables varied between the countries, such that in the UK and Italy moral attitude rather than subjective norms had stronger explanatory power. In Finland it was other way around. Inclusion of moral attitude improved the model fit and predictive ability of the model, although only marginally in Finland. Thus the results partially support the usefulness of incorporating moral measures as well as affective items for attitude into the framework of TPB. PMID:18036702

  19. Does mating behaviour affect connectivity in marine fishes? Comparative population genetics of two protogynous groupers (Family Serranidae).

    PubMed

    Portnoy, D S; Hollenbeck, C M; Renshaw, M A; Cummings, N J; Gold, J R

    2013-01-01

    Pelagic larval duration (PLD) has been hypothesized to be the primary predictor of connectivity in marine fishes; however, few studies have examined the effects that adult reproductive behaviour may have on realized dispersal. We assessed gene flow (connectivity) by documenting variation in microsatellites and mitochondrial DNA sequences in two protogynous species of groupers, the aggregate spawning red hind, Epinephelus guttatus, and the single-male, harem-spawning coney, Cephalopholis fulva, to ask whether reproductive strategy affects connectivity. Samples of both species were obtained from waters off three islands (Puerto Rico, St. Thomas and St. Croix) in the Caribbean Sea. Despite the notion that aggregate spawning of red hind may facilitate larval retention, stronger signals of population structure were detected in the harem-spawning coney. Heterogeneity and/or inferred barriers, based on microsatellites, involved St. Croix (red hind and coney) and the west coast of Puerto Rico (coney). Heterogeneity and/or inferred barriers, based on mitochondrial DNA, involved St. Croix (coney only). Genetic divergence in both species was stronger for microsatellites than for mitochondrial DNA, suggesting sex-biased dispersal in both species. Long-term migration rates, based on microsatellites, indicated asymmetric gene flow for both species in the same direction as mean surface currents in the region. Red hind had higher levels of variation in microsatellites and lower levels of variation in mitochondrial DNA. Long-term effective size and effective number of breeders were greater for red hind; estimates of θ(f) , a proxy for long-term effective female size, were the same in both species. Patterns of gene flow in both species appear to stem in part from shared aspects of larval and adult biology, local bathymetry and surface current patterns. Differences in connectivity and levels of genetic variation between the species, however, likely stem from differences in behaviour

  20. Harmine promotes osteoblast differentiation through bone morphogenetic protein signaling

    SciTech Connect

    Yonezawa, Takayuki; Lee, Ji-Won; Hibino, Ayaka; Asai, Midori; Hojo, Hironori; Cha, Byung-Yoon; Teruya, Toshiaki; Nagai, Kazuo; Chung, Ung-Il; Yagasaki, Kazumi; and others

    2011-06-03

    Highlights: {yields} Harmine promotes the activity and mRNA expression of ALP. {yields} Harmine enhances the expressions of osteocalcin mRNA and protein. {yields} Harmine induces osteoblastic mineralization. {yields} Harmine upregulates the mRNA expressions of BMPs, Runx2 and Osterix. {yields} 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 {beta}-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 {beta}-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

  1. Innate defensive behaviour and panic-like reactions evoked by rodents during aggressive encounters with Brazilian constrictor snakes in a complex labyrinth: behavioural validation of a new model to study affective and agonistic reactions in a prey versus predator paradigm.

    PubMed

    Guimarães-Costa, Raquel; Guimarães-Costa, Maria Beatriz; Pippa-Gadioli, Leonardo; Weltson, Alfredo; Ubiali, Walter Adriano; Paschoalin-Maurin, Tatiana; Felippotti, Tatiana Tocchini; Elias-Filho, Daoud Hibrahim; Laure, Carlos Júlio; Coimbra, Norberto Cysne

    2007-09-15

    Defensive behaviour has been extensively studied, and non-invasive methodologies may be interesting approaches to analyzing the limbic system function as a whole. Using experimental models of animals in the state of anxiety has been fundamental in the search for new anxiolytic and antipanic compounds. The aim of this present work is to examine a new model for the study of affective behaviour, using a complex labyrinth consisting of an arena and galleries forming a maze. Furthermore, it aims to compare the defensive behaviour of Wistar rats, Mongolian gerbils and golden hamsters in a complex labyrinth, as well as the defensive behaviour of Meriones unguiculatus in aggressive encounters with either Epicrates cenchria assisi or Boa constrictor amarali in this same model. Among species presently studied, the Mongolian gerbils showed better performance in the exploration of both arena and galleries of the labyrinth, also demonstrating less latency in finding exits of the galleries. This increases the possibility of survival, as well as optimizes the events of encounter with the predator. The duration of alertness and freezing increased during confrontation with living Epicrates, as well as the duration of exploratory behaviour in the labyrinth. There was an increase in the number of freezing and alertness behaviours, as well as in duration of alertness during confrontations involving E.c. assisi, compared with behavioural reactions elicited by jirds in presence of B.c. amarali. Interestingly, the aggressive behaviour of Mongolian gerbils was more prominent against B.c. amarali compared with the other Boidae snake. E.c. assisi elicited more offensive attacks and exhibited a greater time period of body movement than B.c. amarali, which spent more time in the arena and in defensive immobility than the E.c. assisi. Considering that jirds evoked more fear-like reaction in contact with E.c. assisi, a fixed E.c. assisi kept in a hermetically closed acrylic box was used as

  2. Vinpocetine Attenuates the Osteoblastic Differentiation of Vascular Smooth Muscle Cells.

    PubMed

    Ma, Yun-Yun; Sun, Lin; Chen, Xiu-Juan; Wang, Na; Yi, Peng-Fei; Song, Min; Zhang, Bo; Wang, Yu-Zhong; Liang, Qiu-Hua

    2016-01-01

    Vascular calcification is an active process of osteoblastic differentiation of vascular smooth muscle cells; however, its definite mechanism remains unknown. Vinpocetine, a derivative of the alkaloid vincamine, has been demonstrated to inhibit the high glucose-induced proliferation of vascular smooth muscle cells; however, it remains unknown whether vinpocetine can affect the osteoblastic differentiation of vascular smooth muscle cells. We hereby investigated the effect of vinpocetine on vascular calcification using a beta-glycerophosphate-induced cell model. Our results showed that vinpocetine significantly reduced the osteoblast-like phenotypes of vascular smooth muscle cells including ALP activity, osteocalcin, collagen type I, Runx2 and BMP-2 expression as well as the formation of mineralized nodule. Vinpocetine, binding to translocation protein, induced phosphorylation of extracellular signal-related kinase and Akt and thus inhibited the translocation of nuclear factor-kappa B into the nucleus. Silencing of translocator protein significantly attenuated the inhibitory effect of vinpocetine on osteoblastic differentiation of vascular smooth muscle cells. Taken together, vinpocetine may be a promising candidate for the clinical therapy of vascular calcification. PMID:27589055

  3. Allopurinol and oxypurinol promote osteoblast differentiation and increase bone formation

    PubMed Central

    Orriss, Isabel R.; Arnett, Timothy R.; George, Jacob; Witham, Miles D.

    2016-01-01

    Allopurinol and its active metabolite, oxypurinol are widely used in the treatment of gout and hyperuricemia. They inhibit xanthine oxidase (XO) an enzyme in the purine degradation pathway that converts xanthine to uric acid. This investigation examined the effect of allopurinol and oxypurinol on bone formation, cell number and viability, gene expression and enzyme activity in differentiating and mature, bone-forming osteoblasts. Although mRNA expression remained relatively constant, XO activity decreased over time with mature osteoblasts displaying reduced levels of uric acid (20% decrease). Treatment with allopurinol and oxypurinol (0.1–1 µM) reduced XO activity by up to 30%. At these concentrations, allopurinol and oxypurinol increased bone formation by osteoblasts ~4-fold and ~3-fold, respectively. Cell number and viability were unaffected. Both drugs increased tissue non-specific alkaline phosphatase (TNAP) activity up to 65%. Osteocalcin and TNAP mRNA expression was increased, 5-fold and 2-fold, respectively. Expression of NPP1, the enzyme responsible for generating the mineralisation inhibitor, pyrophosphate, was decreased 5-fold. Col1α1 mRNA expression and soluble collagen levels were unchanged. Osteoclast formation and resorptive activity were not affected by treatment with allopurinol or oxypurinol. Our data suggest that inhibition of XO activity promotes osteoblast differentiation, leading to increased bone formation in vitro. PMID:26968635

  4. Leukemogenesis Induced by an Activating β-catenin mutation in Osteoblasts

    PubMed Central

    Kode, Aruna; Manavalan, John S.; Mosialou, Ioanna; Bhagat, Govind; Rathinam, Chozha V.; Luo, Na; Khiabanian, Hossein; Lee, Albert; Vundavalli, Murty; Friedman, Richard; Brum, Andrea; Park, David; Galili, Naomi; Mukherjee, Siddhartha; Teruya-Feldstein, Julie; Raza, Azra; Rabadan, Raul; Berman, Ellin; Kousteni, Stavroula

    2014-01-01

    Summary Cells of the osteoblast lineage affect homing, 1, 2 number of long term repopulating hematopoietic stem cells (HSCs) 3, 4, HSC mobilization and lineage determination and B lymphopoiesis 5-8. More recently osteoblasts were implicated in pre-leukemic conditions in mice 9, 10. Yet, it has not been shown that a single genetic event taking place in osteoblasts can induce leukemogenesis. We show here that in mice, an activating mutation of β-catenin in osteoblasts alters the differentiation potential of myeloid and lymphoid progenitors leading to development of acute myeloid leukemia (AML) with common chromosomal aberrations and cell autonomous progression. Activated β-catenin stimulates expression of the Notch ligand Jagged-1 in osteoblasts. Subsequent activation of Notch signaling in HSC progenitors induces the malignant changes. Demonstrating the pathogenetic role of the Notch pathway, genetic or pharmacological inhibition of Notch signaling ameliorates AML. Nuclear accumulation and increased β-catenin signaling in osteoblasts was also identified in 38% of patients with MDS/AML. These patients showed increased Notch signaling in hematopoietic cells. These findings demonstrate that genetic alterations in osteoblasts can induce AML, identify molecular signals leading to this transformation and suggest a potential novel pharmacotherapeutic approach to AML. PMID:24429522

  5. Contextual Variables Affecting Aggressive Behaviour in Individuals with Mild to Borderline Intellectual Disabilities Who Live in a Residential Facility

    ERIC Educational Resources Information Center

    Embregts, P. J. C. M.; Didden, R.; Huitink, C.; Schreuder, N.

    2009-01-01

    Background: Aggression is a common type of problem behaviour in clients with mild to borderline intellectual disability who live in a residential facility. We explored contextual events that elicit aggressive behaviour and variables that were associated with such events. Method: Respondents were 87 direct-care staff members of 87 clients with…

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

    PubMed

    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

  7. A SYNCHROTRON DIFFRACTION STUDY OF TRANSFORMATION BEHAVIOUR IN 9 CR STEELS USING SIMULATED WELD HEAT-AFFECTED ZONE CONDITIONS

    SciTech Connect

    Santella, Michael L; Specht, Eliot D; Shingledecker, John P; Abe, Fujio

    2007-01-01

    Synchrotron diffraction experiments were conducted to examine the real-time transformation behaviours of an ex-perimental 9Cr-3W-3Co-NbV steel with high B and low N (N130B), and the commercial P92 steel under simulated weld heat-affected zone thermal cycles. When heated to peak temperatures near 1100 C, both steels rapidly trans-formed from ferrite to 100% austenite. During cooling, both transformed to martensite near 400 C. Both steels also retained untransformed austenite: 1.7% in N130B, and 5.8% in P92. The N130B was also heated to about 60 C above its A3 of 847 C. About 56% of the original ferrite never transformed to austenite. During cooling an additional 21% of ferrite and 23% of martensite formed. It retained no austenite. The P92 was heated to just above its A3 of 889 C. About 15% of the original ferrite never transformed to austenite. During cooling an additional 22% of ferrite and 60% of martensite formed. This steel retained about 2.3% austenite. Metallographic examina-tions indicated that the M23C6 in N130B was much more stable than that in P92 for heating to the lower peak tem-peratures. Analysis using equilibrium thermodynamics suggested that the more stable M23C6 in N130B could raise its apparent A3 by sequestering C. This could cause the ferrite-austenite transformation to appear sluggish. Ther-modynamic analysis also indicated that the M23C6 in N130B contained about 3.9 at% B compared to about 0.08 at% B in that of P92. In contrast, the refractory metal element content of the M23C6 was predicted to be higher in P92.

  8. Do health literacy and patient empowerment affect self-care behaviour? A survey study among Turkish patients with diabetes

    PubMed Central

    Eyüboğlu, Ezgi; Schulz, Peter J

    2016-01-01

    Objective This study aimed to assess the impact of health literacy and patient empowerment on diabetes self-care behaviour in patients in metropolitan Turkish diabetes centres. The conceptual background is provided by the psychological health empowerment model, which holds that health literacy without patient empowerment comes down to wasting health resources, while empowerment without health literacy can lead to dangerous or suboptimal health behaviour. Design, setting and participants A cross-sectional study was conducted with 167 patients over the age of 18 from one of two diabetes clinics in a major Turkish City. Self-administered questionnaires were distributed to eligible outpatients who had an appointment in one of the clinics. Health literacy was measured by a newly translated Turkish version of the Short Test of Functional Health Literacy in Adults (S-TOFHLA) and the Chew self-report scale. Patient empowerment was measured by a 12-item scale based on Spreitzer's conceptualisation of psychological empowerment in the workplace. Self-care behaviour was measured by the Self-care behaviours were measured by the Summary of Diabetes Self-Care Activities Measure (SDSCA). Level of diabetes knowledge was measured by Diabetes Knowledge Test. Results Two subscales of empowerment, impact and self-determination, predicted self-reported frequency of self-care behaviours. Neither health literacy nor diabetes knowledge had an effect on self-care behaviours. Conclusions Health literacy might be more effective in clinical decisions while empowerment might exert a stronger influence on habitual health behaviours. PMID:26975936

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

    SciTech Connect

    Tang Lin; Lin Zhu; Li Yongming . E-mail: liyongming@fmmu.edu.cn

    2006-05-26

    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-{kappa}B 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. Dexamethasone suppresses Smad3 pathway in osteoblastic cells.

    PubMed

    Iu, Mei-Fway; Kaji, Hiroshi; Sowa, Hideaki; Naito, Junko; Sugimoto, Toshitsugu; Chihara, Kazuo

    2005-04-01

    Central in the pathogenesis of glucocorticoid (GC)-induced osteoporosis is the effects of GC on bone formation. However, the mechanism of GC-inhibited bone formation is not well known. Transforming growth factor (TGF)-beta is most abundant in bone matrix compared with other tissues, and we have recently proposed that Smad3, a TGF-beta signaling molecule, is important for promoting bone formation. However, no reports have been available about the effects of GC on Smad3 in osteoblasts. In the present study, we investigated whether dexamethasone (Dex), an active GC analog, would affect the expression and activity of Smad3 in mouse osteoblastic MC3T3-E1 and rat osteoblastic UMR-106 cells. Dex significantly suppressed Smad3-stimulated alkaline phosphatase (ALP) activity, although it did not affect TGF-beta-inhibited ALP activity in MC3T3-E1 cells. Moreover, pretreatment with Dex suppressed TGF-beta-enhanced expression of type I collagen in MC3T3-E1 and UMR-106 cells. In the luciferase assay using p3TP-Lux with a Smad3-specific response element, Dex significantly suppressed the transcriptional activity induced by TGF-beta as well as Smad3. However, Dex did not affect the expression of Smad3 in these cells at both mRNA and protein levels. In conclusion, the present study indicates that Dex inhibits ALP activity and type I collagen expression, presumably by suppressing Smad3-induced transcriptional activity but not by modulating Smad3 expression in osteoblastic cells. PMID:15817834

  11. DBI/ACBP loss-of-function does not affect anxiety-like behaviour but reduces anxiolytic responses to diazepam in mice.

    PubMed

    Budry, Lionel; Bouyakdan, Khalil; Tobin, Stephanie; Rodaros, Demetra; Marcher, Ann-Britt; Mandrup, Susanne; Fulton, Stephanie; Alquier, Thierry

    2016-10-15

    Diazepam is well known for its anxiolytic properties, which are mediated via activation of the GABAA receptor. Diazepam Binding Inhibitor (DBI), also called acyl-CoA binding protein (ACBP), is a ubiquitously expressed protein originally identified based on its ability to displace diazepam from its binding site on the GABAA receptor. Central administration of ACBP or its cleaved fragment, commonly referred to as endozepines, induces proconflict and anxiety-like behaviour in rodents. For this reason, ACBP is known as an anxiogenic peptide. However, the role of endogenous ACBP in anxiety-like behaviour and anxiolytic responses to diazepam has not been investigated. To address this question, we assessed anxiety behaviour and anxiolytic responses to diazepam in two complementary loss-of-function mouse models including astrocyte-specific ACBP KO (ACBP(GFAP) KO) and whole-body KO (ACBP KO) mice. Male and female ACBP(GFAP) KO and ACBP KO mice do not show significant changes in anxiety-like behaviour compared to control littermates during elevated plus maze (EPM) and open field (OF) tests. Surprisingly, ACBP(GFAP) KO and ACBP KO mice were unresponsive to the anxiolytic effect of a low dose of diazepam during EPM tests. In conclusion, our experiments using genetic ACBP loss-of-function models suggest that endozepines deficiency does not affect anxiety-like behaviour in mice and impairs the anxiolytic action of diazepam. PMID:27363924

  12. Analysis of the factors that affect dental health behaviour and attendance at scheduled dental check-ups using the PRECEDE-PROCEED Model.

    PubMed

    Sato, Kimiko; Oda, Megumi

    2011-04-01

    A questionnaire survey was administered to 317 parents who attended infant health check-ups in City B, Okayama Prefecture between October, 2008 and March, 2009. The questionnaire survey studied 7 factors based on the PRECEDE-PROCEED Model. We analysed factors that affected oral health behaviour and attendance at scheduled dental health check-ups. The survey containing 22 items concerning matters such as 'QOL' and 'health problems' was posted to parents and guardians in advance, and then collected on the day of the medical check-up. The collected data was analysed using the t-test and Pearson's correlation coefficient, following which we conducted a covariance structure analysis. The results showed that dental health behaviour was directly affected by reinforcing factors, and indirectly associated with enabling and predisposing factors influenced by reinforcing factors. It was also shown that predisposing factors and oral health behaviour were associated with attendance at scheduled oral health check-ups. The results indicated that strengthening oral health education by sharing knowledge that acts as predisposing factors and introducing adaptations of oral health behaviour that that fit individual lives will lead to improved attendance at scheduled dental health check-ups. PMID:21519364

  13. Osteoblastic Osteosarcoma in a Rabbit

    PubMed Central

    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 osteoblasic osteosarcoma is the most common bone tumor in rabbits, with half of all cases developing in the skull or facial bones. PMID:22546918

  14. Differential Responses of Osteoblast Lineage Cells to Nanotopographically-Modified, Microroughened Titanium-Aluminum-Vanadium Alloy Surfaces

    PubMed Central

    Gittens, Rolando A.; Olivares-Navarrete, Rene; McLachlan, Taylor; Cai, Ye; Hyzy, Sharon L.; Schneider, Jennifer M.; Schwartz, Zvi; Sandhage, Kenneth H.; Boyan, Barbara D.

    2013-01-01

    Surface structural modifications at the micrometer and nanometer scales have driven improved success rates of dental and orthopaedic implants by mimicking the hierarchical structure of bone. However, how initial osteoblast-lineage cells populating an implant surface respond to different hierarchical surface topographical cues remains to be elucidated, with bone marrow mesenchymal stem cells (MSCs) or immature osteoblasts as possible initial colonizers. Here we show that in the absence of any exogenous soluble factors, osteoblastic maturation of primary human osteoblasts (HOBs) but not osteoblastic differentiation of MSCs is strongly influenced by nanostructures superimposed onto a microrough Ti6Al4V (TiAlV) alloy. The sensitivity of osteoblasts to both surface microroughness and nanostructures led to a synergistic effect on maturation and local factor production. Osteoblastic differentiation of MSCs was sensitive to TiAlV surface microroughness with respect to production of differentiation markers, but no further enhancement was found when cultured on micro/nanostructured surfaces. Superposition of nanostructures to microroughened surfaces affected final MSC numbers and enhanced production of vascular endothelial growth factor (VEGF) but the magnitude of the response was lower than for HOB cultures. Our results suggest that the differentiation state of osteoblast-lineage cells determines the recognition of surface nanostructures and subsequent cell response, which has implications for clinical evaluation of new implant surface nanomodifications. PMID:22989383

  15. Interaction of the neuropeptide S receptor gene Asn¹⁰⁷Ile variant and environment: contribution to affective and anxiety disorders, and suicidal behaviour.

    PubMed

    Laas, Kariina; Reif, Andreas; Akkermann, Kirsti; Kiive, Evelyn; Domschke, Katharina; Lesch, Klaus-Peter; Veidebaum, Toomas; Harro, Jaanus

    2014-04-01

    Neuropeptide S is involved in anxiety and arousal modulation, and the functional polymorphism Asn107Ile (rs324981, A > T) of the neuropeptide S receptor gene (NPSR1) is associated with panic disorder and anxiety/fear-related traits. NPSR1 also interacts with the environment in shaping personality and impulsivity. We therefore examined whether the NPSR1 A/T polymorphism is associated with affective and anxiety disorders in a population-representative sample. Lifetime psychiatric disorders were assessed by MINI interview (n = 501) in the older cohort of the longitudinal Estonian Children Personality, Behaviour and Health Study (ECPBHS). Anxiety (STAI), self-esteem (RSES), depression (MÅDRS), suicide attempts and environmental factors were self-reported in both the younger (original n = 583) and the older cohort (original n = 593). Most of the NPSR1 effects were sex-specific and depended on environmental factors. Females with the functionally least active NPSR1 AA genotype and exposed to environmental adversity had affective/anxiety disorders more frequently; they also exhibited higher anxiety and depressiveness, and lower self-esteem. Female AA homozygotes also reported suicidal behaviour more frequently, and this was further accentuated by adverse family environment. In the general population, the NPSR1 A/T polymorphism together with environmental factors is associated with anxious, depressive and activity-related traits, increased prevalence of affective/anxiety disorders and a higher likelihood of suicidal behaviour. PMID:24331455

  16. Dietary protein ingested before and during short photoperiods makes an impact on affect-related behaviours and plasma composition of amino acids in mice.

    PubMed

    Otsuka, Tsuyoshi; Goda, Ryosei; Iwamoto, Ayaka; Kawai, Misato; Shibata, Satomi; Oka, Yoshiaki; Mizunoya, Wataru; Furuse, Mitsuhiro; Yasuo, Shinobu

    2015-11-28

    In mammals, short photoperiod is associated with high depression- and anxiety-like behaviours with low levels of the brain serotonin and its precursor tryptophan (Trp). Because the brain Trp levels are regulated by its ratio to large neutral amino acids (Trp:LNAA) in circulation, this study elucidated whether diets of various protein sources that contain different Trp:LNAA affect depression- and anxiety-like behaviours in C57BL/6J mice under short-day conditions (SD). In the control mice on a casein diet, time spent in the central area in the open field test (OFT) was lower in the mice under SD than in those under long-day conditions (LD), indicating that SD exposure induces anxiety-like behaviour. The SD-induced anxiety-like behaviour was countered by an α-lactalbumin diet given under SD. In the mice that were on a gluten diet before transition to SD, the time spent in the central area in the OFT under SD was higher than that in the SD control mice. Alternatively, mice that ingested soya protein before the transition to SD had lower immobility in the forced swim test, a depression-like behaviour, compared with the SD control. Analysis of Trp:LNAA revealed lower Trp:LNAA in the SD control compared with the LD control, which was counteracted by an α-lactalbumin diet under SD. Furthermore, mice on gluten or soya protein diets before transition to SD exhibited high Trp:LNAA levels in plasma under SD. In conclusion, ingestion of specific proteins at different times relative to photoperiodic transition may modulate anxiety- and/or depression-like behaviours, partially through changes in plasma Trp:LNAA. PMID:26370332

  17. FoxO1 expression in osteoblasts regulates glucose homeostasis through regulation of osteocalcin in mice

    PubMed Central

    Rached, Marie-Therese; Kode, Aruna; Silva, Barbara C.; Jung, Dae Young; Gray, Susan; Ong, Helena; Paik, Ji-Hye; DePinho, Ronald A.; Kim, Jason K.; Karsenty, Gerard; Kousteni, Stavroula

    2009-01-01

    Osteoblasts have recently been found to play a role in regulating glucose metabolism through secretion of osteocalcin. It is unknown, however, how this osteoblast function is regulated transcriptionally. As FoxO1 is a forkhead family transcription factor known to regulate several key aspects of glucose homeostasis, we investigated whether its expression in osteoblasts may contribute to its metabolic functions. Here we show that mice lacking Foxo1 only in osteoblasts had increased pancreatic β cell proliferation, insulin secretion, and insulin sensitivity. The ability of osteoblast-specific FoxO1 deficiency to affect metabolic homeostasis was due to increased osteocalcin expression and decreased expression of Esp, a gene that encodes a protein responsible for decreasing the bioactivity of osteocalcin. These results indicate that FoxO1 expression in osteoblasts contributes to FoxO1 control of glucose homeostasis and identify FoxO1 as a key modulator of the ability of the skeleton to function as an endocrine organ regulating glucose metabolism. PMID:20038793

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

    PubMed

    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

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

    PubMed

    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

  20. Does oral administration of the amino acid tyrosine affect oestradiol-17β concentration and sexual behaviour in the bitch?

    PubMed

    Spankowsky, S; Heuwieser, W; Arlt, S P

    2013-02-23

    The oral administration of the amino acid, tyrosine, has been for years recommended in order to improve fertility, especially to improve copulation behaviour in female dogs. However, evidence is comparatively poor. The objective of our study was to determine whether oral administration of tyrosine has an effect on oestradiol-17β concentrations and the oestrous behaviour in the bitch. Fifty bitches were randomly allocated to one of two treatment groups in which each dog received 100mg/kg/day of either tyrosine or milk sugar orally between Day 3 and Day 9 of heat. Every two to three days, a gynaecological examination was performed and blood samples were taken to determine oestradiol-17β and progesterone concentrations. The day of ovulation was estimated by clinical findings, and according to the specifications of the laboratory, once progesterone values exceeded 12.7nmol/l. The observed copulation behaviour was not different between the groups. No differences in volume and visual nature of vaginal discharge were observed. At the day of ovulation, mean oestradiol-17β concentration in the treated group was 163.4pmol/l and 162.2pmol/l in the placebo group, respectively. In conclusion, feeding tyrosine to female dogs between Day 3 and Day 9 of heat did not alter visual signs of heat or copulation behaviour, and did not alter oestradiol-17β concentration. PMID:23315766

  1. Group housing during gestation affects the behaviour of sows and the physiological indices of offspring piglets at weaning

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In order to compare the behaviour of sows in stalls and group housing systems, and the physiological indices of their offspring, 28 sows were randomly distributed into 2 systems with 16 sows in stalls, and the other 12 sows were divided into 3 groups with 4 sows per pen. The area per sow in stalls a...

  2. A Structural Equation Model of the Factors Affecting Filipino University Students' Shadow Education Satisfaction and Behavioural Intentions

    ERIC Educational Resources Information Center

    de Castro, Belinda V.; de Guzman, Allan B.

    2014-01-01

    The overall intent of this exploratory study is to test a model that considers demographic characteristics, attitudes toward shadow education and shadow education institutions' service attributes as antecedents of satisfaction level and behavioural intentions among a select group of Filipino university students. To test the seven hypotheses…

  3. Home tank water versus novel water differentially affect alcohol-induced locomotor activity and anxiety related behaviours in zebrafish.

    PubMed

    Tran, Steven; Facciol, Amanda; Gerlai, Robert

    2016-05-01

    The zebrafish may be uniquely well suited for studying alcohol's mechanisms of action in vivo, since alcohol can be administered via immersion in a non-invasive manner. Despite the robust behavioural effects of alcohol administration in mammals, studies reporting the locomotor stimulant and anxiolytic effects of alcohol in zebrafish have been inconsistent. In the current study, we examined whether differences in the type of water used for alcohol exposure and behavioural testing contribute to these inconsistencies. To answer this question, we exposed zebrafish to either home water from their housing tanks or novel water from an isolated reservoir (i.e. water lacking zebrafish chemosensory and olfactory cues) with 0% or 1% v/v alcohol for 30min, a 2×2 between subject experimental designs. Behavioural responses were quantified throughout the 30-minute exposure session via a video tracking system. Although control zebrafish exposed to home water and novel water were virtually indistinguishable in their behavioural responses, alcohol's effect on locomotor activity and anxiety-like behavioural responses were dependent on the type of water used for testing. Alcohol exposure in home tank water produced a mild anxiolytic and locomotor stimulant effect, whereas alcohol exposure in novel water produced an anxiogenic effect without altering locomotor activity. These results represent a dissociation between alcohol's effects on locomotor and anxiety related responses, and also illustrate how environmental factors, in this case familiarity with the water, may interact with such effects. In light of these findings, we urge researchers to explicitly state the type of water used. PMID:26921455

  4. Small Variations in Early-Life Environment Can Affect Coping Behaviour in Response to Foraging Challenge in the Three-Spined Stickleback

    PubMed Central

    2016-01-01

    Context An increasing concern in the face of human expansion throughout natural habitats is whether animal populations can respond adaptively when confronted with challenges like environmental change and novelty. Behavioural flexibility is an important factor in estimating the adaptive potential of both individuals and populations, and predicting the degree to which they can cope with change. Study Design This study on the three-spined stickleback (Gasterosteus aculeatus) is an empiric illustration of the degree of behavioural variation that can emerge between semi-natural systems within only a single generation. Wild-caught adult sticklebacks (P, N = 400) were randomly distributed in equal densities over 20 standardized semi-natural environments (ponds), and one year later offspring (F1, N = 652) were presented with repeated behavioural assays. Individuals were challenged to reach a food source through a novel transparent obstacle, during which exploration, activity, foraging, sociability and wall-biting behaviours were recorded through video observation. We found that coping responses of individuals from the first generation to this unfamiliar foraging challenge were related to even relatively small, naturally diversified variation in developmental environment. All measured behaviours were correlated with each other. Especially exploration, sociability and wall-biting were found to differ significantly between ponds. These differences could not be explained by stickleback density or the turbidity of the water. Findings Our findings show that a) differences in early-life environment appear to affect stickleback feeding behaviour later in life; b) this is the case even when the environmental differences are only small, within natural parameters and diversified gradually; and c) effects are present despite semi-natural conditions that fluctuate during the year. Therefore, in behaviourally plastic animals like the stickleback, the adaptive response to human

  5. Bruton tyrosine kinase (Btk) suppresses osteoblastic differentiation.

    PubMed

    Kaneshiro, Shoichi; Ebina, Kosuke; Shi, Kenrin; Yoshida, Kiyoshi; Otsuki, Dai; Yoshikawa, Hideki; Higuchi, Chikahisa

    2015-09-01

    The Tec family of nonreceptor tyrosine kinases has been shown to play a key role in inflammation and bone destruction. Bruton tyrosine kinase (Btk) has been the most widely studied because of its critical role in B cells. Furthermore, recent evidence has demonstrated that blocking Btk signaling is effective in ameliorating lymphoma progression and experimental arthritis. The role of Btk in osteoblastic differentiation has not been well elucidated. In this study, we demonstrated the role of Btk in osteoblastic differentiation and investigated the effects of a Btk inhibitor on osteoblastic differentiation in mouse preosteoblastic MC3T3-E1 cells, primary calvarial osteoblasts, and bone marrow stromal ST2 cells. Btk expression was detected in all three cell lines. Btk inhibition stimulated mRNA expression of osteoblastic markers (alkaline phosphatase, osteocalcin, and osterix) and promoted mineralization of the extracellular matrix. In addition, Btk knockdown caused increased mRNA expression of osteoblastic markers. Furthermore, Btk inhibition suppressed the phosphorylation of mitogen-activated protein kinase (MAPK), nuclear factor kappa B (NFκB), and protein kinase Cα (PKCα). Our results indicate that Btk may regulate osteoblastic differentiation through the MAPK, NFκB, and PKCα signaling pathways. PMID:25230818

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

    PubMed

    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 (<20, 40-80, or 80-200 microm). The smallest microparticles decreased the viability of both cell types as measured with LDH and methyl tetrazolium salt assays, and enhanced the secretion of pro-inflammatory cytokines (IL-6 and TNF-alpha) by macrophages after 24 h, as revealed by ELISA. Osteoblastic cells were then cultured for 96 h in the presence of these pro-inflammatory cytokines and their differentiation studied by RT-PCR. MC3T3-E1 cells cultured with TNF-alpha showed a decrease in osterix, PTH receptor (PTHR1), and osteocalcin gene expression. On the contrary, IL-6 enhanced the expression of osterix, Runx2, alkaline phosphatase, and osteocalcin compared with plastic. In conclusion, this study shows that the inflammatory response initiated by BCP microparticles may have both detrimental and beneficial effects on osteogenesis. PMID:20014296

  7. Interactions between integrin receptors and fibronectin are required for calvarial osteoblast differentiation in vitro

    NASA Technical Reports Server (NTRS)

    Moursi, A. M.; Globus, R. K.; Damsky, C. H.

    1997-01-01

    We previously showed that anti-fibronectin antibodies or soluble fibronectin fragments containing the central cell-binding domain inhibit formation of mineralized nodules by fetal calvarial osteoblasts in vitro. These findings suggest a critical role for fibronectin in osteoblast differentiation and morphogenesis. In this study we tested the hypothesis that fibronectin's effects on osteogenesis are mediated via direct interactions with integrin receptors for fibronectin on osteoblasts. Immunocytochemical analysis identified the integrin fibronectin receptor alpha5ss1 in fetal rat calvarial tissue and in cultured osteoblasts at all stages of differentiation. Three other integrins, alpha3ss1, alpha8ss1 and alphavss3, which can bind fibronectin, as well as other matrix components, were also identified in tissue and at all stages of cell culture. Immunoprecipitation data showed that alpha5ss1 levels are constant throughout osteoblast differentiation whereas levels of alpha3ss1 and alpha8ss1 decline in mature mineralized cultures. To determine whether integrin fibronectin receptors are required for osteoblast formation of mineralized nodules, we examined the extent of nodule formation in the presence and absence of function-perturbing anti-integrin antibodies. The antibodies were present continuously in cultures beginning at confluence (day 3), and nodule formation was measured at days 10 and 20. An anti-alpha5 integrin subunit antibody reduced nodule formation to less than 5% of control values at both time points. Inhibition of nodule formation was reversible and did not affect cell attachment and viability. Function-perturbing antibodies against alpha3ss1 and alpha8ss1 also reduced nodule formation, to less than 20% of control values. In contrast, function-perturbing antibodies to alphavss3 and alphavss5 did not affect nodule formation, indicating that the inhibitions noted were indeed specific. To determine the effect of antibody treatment on gene expression, steady

  8. Rebamipide Delivered by Brushite Cement Enhances Osteoblast and Macrophage Proliferation

    PubMed Central

    Pujari-Palmer, Michael; Pujari-Palmer, Shiuli; Engqvist, Håkan; Karlsson Ott, Marjam

    2015-01-01

    Many of the bioactive agents capable of stimulating osseous regeneration, such as bone morphogenetic protein-2 (BMP-2) or prostaglandin E2 (PGE2), are limited by rapid degradation, a short bioactive half-life at the target site in vivo, or are prohibitively expensive to obtain in large quantities. Rebamipide, an amino acid modified hydroxylquinoline, can alter the expression of key mediators of bone anabolism, cyclo-oxygenase 2 (COX-2), BMP-2 and vascular endothelial growth factor (VEGF), in diverse cell types such as mucosal and endothelial cells or chondrocytes. The present study investigates whether Rebamipide enhances proliferation and differentiation of osteoblasts when delivered from brushite cement. The reactive oxygen species (ROS) quenching ability of Rebampide was tested in macrophages as a measure of bioactivity following drug release incubation times, up to 14 days. Rebamipide release from brushite occurrs via non-fickian diffusion, with a rapid linear release of 9.70% ±0.37% of drug per day for the first 5 days, and an average of 0.5%-1% per day thereafter for 30 days. Rebamipide slows the initial and final cement setting time by up to 3 and 1 minute, respectively, but does not significantly reduce the mechanical strength below 4% (weight percentage). Pre-osteoblast proliferation increases by 24% upon exposure to 0.4uM Rebamipide, and by up to 73% when Rebamipide is delivered via brushite cement. Low doses of Rebamipide do not adversely affect peak alkaline phosphatase activity in differentiating pre-osteoblasts. Rebamipide weakly stimulates proliferation in macrophages at low concentrations (118 ±7.4% at 1uM), and quenches ROS by 40-60%. This is the first investigation of Rebamipide in osteoblasts. PMID:26023912

  9. Effects of broad frequency vibration on cultured osteoblasts

    NASA Technical Reports Server (NTRS)

    Tanaka, Shigeo M.; Li, Jiliang; Duncan, Randall L.; Yokota, Hiroki; Burr, David B.; Turner, Charles H.

    2003-01-01

    Bone is subjected in vivo to both high amplitude, low frequency strain, incurred by locomotion, and to low amplitude, broad frequency strain. The biological effects of low amplitude, broad frequency strain are poorly understood. To evaluate the effects of low amplitude strains ranging in frequency from 0 to 50 Hz on osteoblastic function, we seeded MC3T3-E1 cells into collagen gels and applied the following loading protocols for 3 min per day for either 3 or 7 days: (1) sinusoidal strain at 3 Hz, with 0-3000 microstrain peak-to-peak followed by 0.33 s resting time, (2) "broad frequency vibration" of low amplitude strain (standard deviation of 300 microstrain) including frequency components from 0 to 50 Hz, and (3) sinusoidal strain combined with broad frequency vibration (S + V). The cells were harvested on day 4 or 8. We found that the S + V stimulation significantly repressed cell proliferation by day 8. Osteocalcin mRNA was up-regulated 2.6-fold after 7 days of S + V stimulation, and MMP-9 mRNA was elevated 1.3-fold after 3 days of vibration alone. Sinusoidal stimulation alone did not affect the cell responses. No differences due to loading were observed in alkaline phosphatase activity and in mRNA levels of type I collagen, osteopontin, connexin 43, MMPs-1A, -3, -13. These results suggest that osteoblasts are more sensitive to low amplitude, broad frequency strain, and this kind of strain could sensitize osteoblasts to high amplitude, low frequency strain. This suggestion implies a potential contribution of stochastic resonance to the mechanical sensitivity of osteoblasts. Copyright 2002 Elsevier Science Ltd.

  10. Suicidal behaviour in first-episode non-affective psychosis: Specific risk periods and stage-related factors.

    PubMed

    Ayesa-Arriola, Rosa; Alcaraz, Elisa García; Hernández, Begoña Vicente; Pérez-Iglesias, Rocío; López Moríñigo, Javier David; Duta, Rina; David, Anthony S; Tabares-Seisdedos, Rafael; Crespo-Facorro, Benedicto

    2015-12-01

    Suicide is a major cause of premature death in psychosis. Earlier stages have been associated with higher risk. However, such risk periods have not been specifically determined and risk factors for suicidal behaviour may change over those periods, which may have crucial implications for suicide prevention. The aim of this study was to determine and characterize the highest risk period for suicide in a representative sample of first-episode psychosis (FEP) patients. Suicidal behaviour prior to first presentation of psychosis and during a 3-year follow-up was examined in a sample of 397 individuals. Risk factors for suicidal behaviour during specific time periods were investigated and compared. The greatest suicide risk was found during the month before and 2 months after first contact with psychiatric services (i.e., 'early' attempts). Severity of depressive symptoms and cannabis use emerged as predominant risk factors across time. 'Early' attempters were characterized as being male, living in urban areas, having poor premorbid adjustment, requiring hospitalization, scoring higher on anxiety measures and unusual thought content than non-attempters. Greater suspiciousness and more severe depressive symptoms distinguished the 'late' attempters. In conclusion, there is a specific high risk period for suicide in FEP around the time of the first presentation. Early intervention programmes targeting phase-specific risk factors, particularly psychotic symptoms management and secondary depression prevention strategies may be useful for suicide prevention in psychosis. PMID:26475577

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

    SciTech Connect

    Roforth, Matthew M. Khosla, Sundeep Monroe, David G.

    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.

  12. Perinatal Exposure to a Diet High in Saturated Fat, Refined Sugar and Cholesterol Affects Behaviour, Growth, and Feed Intake in Weaned Piglets.

    PubMed

    Clouard, Caroline; Gerrits, Walter J J; Kemp, Bas; Val-Laillet, David; Bolhuis, J Elizabeth

    2016-01-01

    The increased consumption of diets high in saturated fats and refined sugars is a major public health concern in Western human societies. Recent studies suggest that perinatal exposure to dietary fat and/or sugar may affect behavioural development. We thus investigated the effects of perinatal exposure to a high-fat high-sugar diet (HFS) on behavioural development and production performance of piglets. Thirty-two non-obese sows and their piglets were allocated to 1 of 4 treatments in a 2 × 2 factorial design, with 8-week prenatal (gestation) and 8-week postnatal (lactation and post-weaning) exposure to a HFS diet (12% saturated fat, 18.5% sucrose, 1% cholesterol) or control low-fat low-sugar high-starch diets as factors. From weaning onwards (4 weeks of age), piglets were housed in group of 3 littermates (n = 8 groups/treatment) and fed ad libitum. After the end of the dietary intervention (8 weeks of age), all the piglets were fed a standard commercial diet. Piglet behaviours in the home pens were scored, and skin lesions, growth, feed intake and feed efficiency were measured up to 8 weeks after the end of the dietary treatment, i.e. until 16 weeks of age. At the end of the dietary treatment (8 weeks of age), response to novelty was assessed in a combined open field and novel object test (OFT/NOT). During the weeks following weaning, piglets fed the postnatal HFS diet tended to be less aggressive (p = 0.06), but exhibited more oral manipulation of pen mates (p = 0.05) than controls. Compared to controls, piglets fed the prenatal or postnatal HFS diet walked more in the home pen (p ≤ 0.05), and tended to have fewer skin lesions (p < 0.10). Several behavioural effects of the postnatal HFS diet depended on the prenatal diet, with piglets subjected to a switch of diet at birth being more active, and exploring feeding materials, pen mates, and the environment more than piglets that remained on the same diet. Behaviours during the OFT/NOT were not affected by the

  13. Perinatal Exposure to a Diet High in Saturated Fat, Refined Sugar and Cholesterol Affects Behaviour, Growth, and Feed Intake in Weaned Piglets

    PubMed Central

    Gerrits, Walter J. J.; Kemp, Bas; Val-Laillet, David; Bolhuis, J. Elizabeth

    2016-01-01

    The increased consumption of diets high in saturated fats and refined sugars is a major public health concern in Western human societies. Recent studies suggest that perinatal exposure to dietary fat and/or sugar may affect behavioural development. We thus investigated the effects of perinatal exposure to a high-fat high-sugar diet (HFS) on behavioural development and production performance of piglets. Thirty-two non-obese sows and their piglets were allocated to 1 of 4 treatments in a 2 × 2 factorial design, with 8-week prenatal (gestation) and 8-week postnatal (lactation and post-weaning) exposure to a HFS diet (12% saturated fat, 18.5% sucrose, 1% cholesterol) or control low-fat low-sugar high-starch diets as factors. From weaning onwards (4 weeks of age), piglets were housed in group of 3 littermates (n = 8 groups/treatment) and fed ad libitum. After the end of the dietary intervention (8 weeks of age), all the piglets were fed a standard commercial diet. Piglet behaviours in the home pens were scored, and skin lesions, growth, feed intake and feed efficiency were measured up to 8 weeks after the end of the dietary treatment, i.e. until 16 weeks of age. At the end of the dietary treatment (8 weeks of age), response to novelty was assessed in a combined open field and novel object test (OFT/NOT). During the weeks following weaning, piglets fed the postnatal HFS diet tended to be less aggressive (p = 0.06), but exhibited more oral manipulation of pen mates (p = 0.05) than controls. Compared to controls, piglets fed the prenatal or postnatal HFS diet walked more in the home pen (p ≤ 0.05), and tended to have fewer skin lesions (p < 0.10). Several behavioural effects of the postnatal HFS diet depended on the prenatal diet, with piglets subjected to a switch of diet at birth being more active, and exploring feeding materials, pen mates, and the environment more than piglets that remained on the same diet. Behaviours during the OFT/NOT were not affected by the

  14. Behaviour Recovery. Second Edition

    ERIC Educational Resources Information Center

    Rogers, Bill

    2004-01-01

    This second edition of Behaviour Recovery puts emphasis on teaching behaviour concerning children with emotional and behavioural disorders (EBD). These children have many factors in their lives that affect their behaviour over which schools have limited control. This book acknowledges the challenge and explores the practical realities, options and…

  15. Generation of rodent and human osteoblasts

    PubMed Central

    Taylor, Sarah E B; Shah, Mittal; Orriss, Isabel R

    2014-01-01

    This paper describes the isolation, culture and staining of primary osteoblasts from neonatal rodents and human samples. The calvaria and long-bone assays allow direct measurement of bone matrix deposition and mineralisation, as well as producing osteoblasts at defined stages of differentiation for molecular and histological analysis. Culture of human osteoblasts enables cell function to be investigated in targeted patient groups. The described methods will provide a step-by-step guide of what to expect at each stage of the culture and highlight the varied tissue culture conditions required to successfully grow osteoblasts from different sources. A special focus of this paper is the methods used for analysis of bone mineralisation and how to ensure that nonspecific mineral deposition or staining is not quantified. PMID:25396049

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

    PubMed

    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

  17. Direct effects of caffeine on osteoblastic cells metabolism: the possible causal effect of caffeine on the formation of osteoporosis

    PubMed Central

    Tsuang, Yang-Hwei; Sun, Jui-Sheng; Chen, Li-Ting; Sun, Samuel Chung-Kai; Chen, San-Chi

    2006-01-01

    Background Caffeine consumption has been reported to decrease bone mineral density (BMD), increase the risk of hip fracture, and negatively influence calcium retention. In this study, we investigated the influence of caffeine on the osteoblasts behaviour. Method Osteoblasts derived from newborn Wistar-rat calvaria was used in this study. The effects of various concentrations of caffeine on bone cell activities were evaluated by using MTT assay. Alkaline phosphatase (ALP) staining, von Kossa staining and biochemical parameters including ALP, lactate dehydrogenase (LDH), prostaglandin E2 (PGE2) and total protein were performed at day 1, 3, and 7. DNA degradation analysis under the caffeine influence was also performed. Results and discussion The results showed that the viability of the osteoblasts, the formation of ALP positive staining colonies and mineralization nodules formation in the osteoblasts cultures decreased significantly in the presence of 10 mM caffeine. The intracellular LDH, ALP and PGE2 content decreased significantly, the LDH and PGE2 secreted into the medium increased significantly. The activation of an irreversible commitment to cell death by caffeine was clearly demonstrated by DNA ladder staining. Conclusion In summary, our results suggest that caffeine has potential deleterious effect on the osteoblasts viability, which may enhance the rate of osteoblasts apoptosis. PMID:17150127

  18. Velocity, safety, or both? How do balance and strength of goal conflicts affect drivers' behaviour, feelings and physiological responses?

    PubMed

    Schmidt-Daffy, Martin; Brandenburg, Stefan; Beliavski, Alina

    2013-06-01

    Motivational models of driving behaviour agree that choice of speed is modulated by drivers' goals. Whilst it is accepted that some goals favour fast driving and others favour safe driving, little is known about the interplay of these conflicting goals. In the present study, two aspects of this interplay are investigated: the balance of conflict and the strength of conflict. Thirty-two participants completed several simulated driving runs in which fast driving was rewarded with a monetary gain if the end of the track was reached. However, unpredictably, some runs ended with the appearance of a deer. In these runs, fast driving was punished with a monetary loss. The ratio between the magnitudes of gains and losses varied in order to manipulate the balance of conflict. The absolute magnitudes of both gains and losses altered the strength of conflict. Participants drove slower, reported an increase in anxiety-related feelings, and showed indications of physiological arousal if there was more money at stake. In contrast, only marginal effects of varying the ratio between gains and losses were observed. Results confirm that the strength of a safety-velocity conflict is an important determinant of drivers' behaviour, feelings, and physiological responses. The lack of evidence for the balance of conflict playing a role suggests that in each condition, participants subjectively weighted the loss higher than the gain (loss aversion). It is concluded that the interplay of the subjective values that drivers attribute to objective incentives for fast and safe driving is a promising field for future research. Incorporating this knowledge into motivational theories of driving behaviour might improve their contribution to the design of adequate road safety measures. PMID:23523895

  19. Suberoylanilide hydroxamic acid (SAHA; vorinostat) causes bone loss by inhibiting immature osteoblasts

    PubMed Central

    McGee-Lawrence, Meghan E.; McCleary-Wheeler, Angela L.; Secreto, Frank J.; Razidlo, David F.; Zhang, Minzhi; Stensgard, Bridget A.; Li, Xiaodong; Stein, Gary S.; Lian, Jane B.; Westendorf, Jennifer J.

    2011-01-01

    Histone deacetylase (Hdac) inhibitors are used clinically to treat cancer and epilepsy. Although Hdac inhibition accelerates osteoblast maturation and suppresses osteoclast maturation in vitro, the effects of Hdac inhibitors on the skeleton are not understood. The purpose of this study was to determine how the pan-Hdac inhibitor, suberoylanilide hydroxamic acid (SAHA; a.k.a. vorinostat or Zolinza™) affects bone mass and remodeling in vivo. Male C57BL/6 mice received daily SAHA (100 mg/kg) or vehicle injections for three to four weeks. SAHA decreased trabecular bone volume fraction and trabecular number in the distal femur. Cortical bone at the femoral midshaft was not affected. SAHA reduced serum levels of P1NP, a bone formation marker, and also suppressed tibial mRNA levels of type I collagen, osteocalcin and osteopontin, but did not alter Runx2 or osterix transcripts. SAHA decreased histological measures of osteoblast number but interestingly increased indices of osteoblast activity including mineral apposition rate and bone formation rate. Neither serum (TRAcP 5b) nor histological markers of bone resorption were affected by SAHA. P1NP levels returned to baseline in animals which were allowed to recover for four weeks after four weeks of daily SAHA injections, but bone density remained low. In vitro, SAHA suppressed osteogenic colony formation, decreased osteoblastic gene expression, induced cell cycle arrest, and caused DNA damage in bone marrow-derived adherent cells. Collectively, these data demonstrate that bone loss following treatment with SAHA is primarily due to a reduction in osteoblast number. Moreover, these decreases in osteoblast number can be attributed to the deleterious effects of SAHA on immature osteoblasts, even while mature osteoblasts are resistant to the harmful effects and demonstrate increased activity in vivo, indicating that the response of osteoblasts to SAHA is dependent upon their differentiation state. These studies suggest that

  20. On-ground housing in “Mice Drawer System” (MDS) cage affects locomotor behaviour but not anxiety in male mice

    NASA Astrophysics Data System (ADS)

    Simone, Luciano; Bartolomucci, Alessandro; Palanza, Paola; Parmigiani, Stefano

    2008-03-01

    In the present study adult male mice were housed for 21 days in a housing modules of the Mice Drawer System (MDS). MDS is the facility that will support the research on board the International Space Station (ISS). Our investigation focused on: circadian rhythmicity of wide behavioural categories such as locomotor activity, food intake/drinking and resting; emotionality in the elevated plus maze (EPM); body weight. Housing in the MDS determined a strong up-regulation of activity and feeding behaviour and a concomitant decrease in inactivity. Importantly, housing in the MDS disrupted circadian rhythmicity in mice and also determined a decrease in body weight. Finally, when mice were tested in the EPM a clear hyperactivity (i.e. increased total transitions) was found, while no evidence for altered anxiety was detected. In conclusion, housing adult male mice in the MDS housing modules may affect their behaviour, circadian rhythmicity while having no effect on anxiety. It is suggested that to allow adaptation to the peculiar housing allowed by MDS a longer housing duration is needed.

  1. Parental stress affects the emotions and behaviour of children up to adolescence: a Greek prospective, longitudinal study.

    PubMed

    Bakoula, Chryssa; Kolaitis, Gerasimos; Veltsista, Alexandra; Gika, Artemis; Chrousos, George P

    2009-11-01

    Systematic research about the continuity of mental health problems from childhood to adolescence is limited, but necessary to design effective prevention and intervention strategies. We used a population-based representative sample of Greek adolescents, followed-up from birth to the age of 18 years, to assess early influences on and the persistence of mental health problems in youth. We examined the role of peripartum, early development and parental characteristics in predicting mental health problems in childhood and adolescence. Results suggest a strong relationship between behavioural problems in childhood and adolescence for both genders, while emotional problems were more likely to persist in boys. Age and sex-specific models revealed significant positive associations between higher scores on the behavioural and emotional problems scales and higher frequency of accidents in preschool years, physical punishment in early childhood, lack of parental interest in child's school and activities, and perceived maternal stress in all children. Perceived paternal stress was associated with higher scores on the Total and Internalizing problems scales in the total population. Our results suggest that early interventions are necessary as mental health problems strongly persist from childhood to late adolescence. The adverse effects of parental stress and poor care-giving practices on child's psychopathology need to be recognised and improved. PMID:19206015

  2. Do ambient electromagnetic fields affect behaviour? A demonstration of the relationship between geomagnetic storm activity and suicide.

    PubMed

    Berk, Michael; Dodd, Seetal; Henry, Margaret

    2006-02-01

    The relationship between ambient electromagnetic fields and human mood and behaviour is of great public health interest. The relationship between Ap indices of geomagnetic storm activity and national suicide statistics for Australia from 1968 to 2002 was studied. Ap index data was normalised so as to be globally uniform and gave a measure of storm activity for each day. A geomagnetic storm event was defined as a day in which the Ap index was equal to or exceeded 100 nT. Suicide data was a national tally of daily male and female death figures where suicide had been documented as the cause of death. A total of 51 845 males and 16 327 females were included. The average number of suicides was greatest in spring for males and females, and lowest in autumn for males and summer for females. Suicide amongst females increased significantly in autumn during concurrent periods of geomagnetic storm activity (P = .01). This pattern was not observed in males (P = .16). This suggests that perturbations in ambient electromagnetic field activity impact behaviour in a clinically meaningful manner. The study furthermore raises issues regarding other sources of stray electromagnetic fields and their effect on mental health. PMID:16304696

  3. The Phosphorylation and Distribution of Cortactin Downstream of Integrin α9β1 Affects Cancer Cell Behaviour.

    PubMed

    Høye, Anette M; Couchman, John R; Wewer, Ulla M; Yoneda, Atsuko

    2016-01-01

    Integrins, a family of heterodimeric adhesion receptors are implicated in cell migration, development and cancer progression. They can adopt conformations that reflect their activation states and thereby impact adhesion strength and migration. Integrins in an intermediate activation state may be optimal for migration and we have shown previously that fully activated integrin α9β1 corresponds with less migratory behaviour in melanoma cells. Here, we aimed to identify components associated with the activation status of α9β1. Using cancer cell lines with naturally occuring high levels of this integrin, activation by α9β1-specific ligands led to upregulation of fibronectin matrix assembly and tyrosine phosphorylation of cortactin on tyrosine 470 (Y470). Specifically, cortactin phosphorylated on Y470, but not Y421, redistributed together with α9β1 to focal adhesions where active β1 integrin also localises, upon integrin activation. This was commensurate with reduced migration. The localisation and phosphorylation of cortactin Y470 was regulated by Yes kinase and PTEN phosphatase. Cortactin levels influenced fibronectin matrix assembly and active β1 integrin on the cell surface, being inversely correlated with migratory behaviour. This study underlines the complex interplay between cortactin and α9β1 integrin that regulates cell-extracellular matrix interactions. PMID:27339664

  4. The Phosphorylation and Distribution of Cortactin Downstream of Integrin α9β1 Affects Cancer Cell Behaviour

    PubMed Central

    Høye, Anette M.; Couchman, John R.; Wewer, Ulla M.; Yoneda, Atsuko

    2016-01-01

    Integrins, a family of heterodimeric adhesion receptors are implicated in cell migration, development and cancer progression. They can adopt conformations that reflect their activation states and thereby impact adhesion strength and migration. Integrins in an intermediate activation state may be optimal for migration and we have shown previously that fully activated integrin α9β1 corresponds with less migratory behaviour in melanoma cells. Here, we aimed to identify components associated with the activation status of α9β1. Using cancer cell lines with naturally occuring high levels of this integrin, activation by α9β1-specific ligands led to upregulation of fibronectin matrix assembly and tyrosine phosphorylation of cortactin on tyrosine 470 (Y470). Specifically, cortactin phosphorylated on Y470, but not Y421, redistributed together with α9β1 to focal adhesions where active β1 integrin also localises, upon integrin activation. This was commensurate with reduced migration. The localisation and phosphorylation of cortactin Y470 was regulated by Yes kinase and PTEN phosphatase. Cortactin levels influenced fibronectin matrix assembly and active β1 integrin on the cell surface, being inversely correlated with migratory behaviour. This study underlines the complex interplay between cortactin and α9β1 integrin that regulates cell-extracellular matrix interactions. PMID:27339664

  5. How Growing Complexity of Consumer Choices and Drivers of Consumption Behaviour Affect Demand for Animal Source Foods.

    PubMed

    Perry, B D; Grace, D C

    2015-12-01

    Many societies are spoiled for choice when they purchase meat and other livestock products, and around the globe food choice has grown dramatically in the last two decades. What is more, besides the cost and obvious health concerns influencing commodity section, an increasing proportion of choices is made to contribute to the achievement of certain ideals, such as natural resource management, climate change mitigation, animal welfare concerns and personal lifestyle. At the same time, human health considerations are becoming more important for consumption choices as richer societies, and increasingly the urban poor in low- and middle-income countries, face an unprecedented epidemic of over-consumption and associated diet-related non-communicable diseases. Animal source foods are considered significant contributors to this trend. This paper reviews this complicated arena, and explores the range of considerations that influence consumers' preferences for meat and other animal source foods. This paper also argues that deeper drivers of consumption behaviour of many foods may act in opposition to the articulated preferences for choices around animal source food consumption. We review how the returns to different causes are being valued, how emerging metrics are helping to manage and influence consumption behaviours, and draw conclusions regarding options which influence food choice. PMID:26682899

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

    PubMed Central

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

    2014-01-01

    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 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 36 week-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 14 week-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

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

    PubMed

    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

  8. Uptake of postprandial lipoproteins into bone in vivo: impact on osteoblast function.

    PubMed

    Niemeier, Andreas; Niedzielska, Dagmara; Secer, Rukiye; Schilling, Arndt; Merkel, Martin; Enrich, Carlos; Rensen, Patrick C N; Heeren, Joerg

    2008-08-01

    Dietary lipids and lipophilic vitamins are transported by postprandial lipoproteins and are required for bone metabolism. Despite that, it remains unknown whether bone cells are involved in the uptake of circulating postprandial lipoproteins in vivo. The current study was performed to investigate a putative participation of bone in the systemic postprandial lipoprotein metabolism in mice, to identify potentially involved cell type populations and to analyze whether lipoprotein uptake affects bone function in vivo. As a model for the postprandial state, chylomicron remnants (CR) were injected intravenously into mice. Next to the liver and compared to other organs, bone appeared to be the second most important organ for the clearance of radiolabeled CR particles from the circulation in vivo. In addition, uptake of radiolabeled CR by primary murine osteoblasts and hepatocytes was quantified to be in a similar range in vitro. A complementary approach with fluorescently labeled CR and immunohistochemical staining for apoE proved that intact CR particles were taken up into bone and liver. Electron microscopy localization studies of bone sections revealed CR uptake into sinusoidal endothelial cells, macrophages and osteoblasts. The relative amount of radiolabeled CR uptake into femoral cortical bone, representing predominantly osteoblasts, and bone marrow, representing predominantly non-osteoblast cells, was within the same range. Most importantly, the injection of vitamin K1-enriched CR resulted in an increase of the degree of osteocalcin carboxylation in vivo while total osteocalcin concentrations remained unaffected, giving functional proof that osteoblasts process CR in vivo. In conclusion, here we demonstrate that bone is involved in the postprandial lipoprotein metabolism in mice. Osteoblasts participate in CR clearance from the circulation, which has a direct impact on the secretory function of osteoblasts. PMID:18538644

  9. Co-localisation of host plant resistance QTLs affecting the performance and feeding behaviour of the aphid Myzus persicae in the peach tree

    PubMed Central

    Sauge, M-H; Lambert, P; Pascal, T

    2012-01-01

    The architecture and action of quantitative trait loci (QTL) contributing to plant resistance mechanisms against aphids, the largest group of phloem-feeding insects, are not well understood. Comparative mapping of several components of resistance to the green peach aphid (Myzus persicae) was undertaken in Prunus davidiana, a wild species related to peach. An interspecific F1 population of Prunus persica var. Summergrand × P. davidiana clone P1908 was scored for resistance (aphid colony development and foliar damage) and 17 aphid feeding behaviour traits monitored by means of the electrical penetration graph technique. Seven resistance QTLs were detected, individually explaining 6.1–43.1% of the phenotypic variation. Consistency was shown over several trials. Nine QTLs affecting aphid feeding behaviour were identified. All resistance QTLs except one co-located with QTLs underlying aphid feeding behaviour. A P. davidiana resistance allele at the major QTL was associated with drastic reductions in phloem sap ingestion by aphids, suggesting a phloem-based resistance mechanism. Resistance was also positively correlated with aphid salivation into sieve elements, suggesting an insect response to restore the appropriate conditions for ingestion after phloem occlusion. No significant QTL was found for traits characterising aphid mouthpart activity in plant tissues other than phloem vessels. Two QTLs with effects on aphid feeding behaviour but without effect on resistance were identified. SSR markers linked to the main QTLs involved in resistance are of potential use in marker-assisted selection for aphid resistance. Linking our results with the recent sequencing of the peach genome may help clarify the physiological resistance mechanisms. PMID:21897441

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

    PubMed

    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

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

    PubMed Central

    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

  12. Hypoxia Modulates the Phenotype of Osteoblasts Isolated From Knee Osteoarthritis Patients, Leading to Undermineralized Bone Nodule Formation

    PubMed Central

    Chang, Joan; Jackson, Sonya G; Wardale, John; Jones, Simon W

    2014-01-01

    Objective To investigate the role of hypoxia in the pathology of osteoarthritic (OA) bone by exploring its effect on the phenotype of isolated primary osteoblasts from patients with knee OA. Methods OA bone samples were collected at the time of elective joint replacement surgery for knee or hip OA. Normal bone samples were collected postmortem from cadaver donors. Primary osteoblasts were isolated from knee OA bone chips and cultured under normoxic or hypoxic (2% O2) conditions. Alkaline phosphatase activity was quantified using an enzymatic assay, and osteopontin and prostaglandin E2 (PGE2) production was assayed by enzyme-linked immunosorbent assay. Total RNA was extracted from bone and osteoblasts, and gene expression was profiled by quantitative reverse transcription–polymerase chain reaction. Results Human OA bone tissue sections stained positively for carbonic anhydrase IX, a biomarker of hypoxia, and exhibited differential expression of genes that mediate the vasculature and blood coagulation as compared to those found in normal bone. Culture of primary osteoblasts isolated from knee OA bone under hypoxic conditions profoundly affected the osteoblast phenotype, including the expression of genes that mediate bone matrix, bone remodeling, and bone vasculature. Hypoxia also increased the expression of cyclooxygenase 2 and the production of PGE2 by OA osteoblasts. Osteoblast expression of type II collagen α1 chain, angiopoietin-like 4, and insulin-like growth factor binding protein 1 was shown to be mediated by hypoxia-inducible factor 1α. Chronic hypoxia reduced osteoblast- mineralized bone nodule formation. Conclusion These findings demonstrate that hypoxia can induce pathologic changes in osteoblast functionality consistent with an OA phenotype, providing evidence that hypoxia is a key driver of OA pathology. PMID:24574272

  13. Id4, a new candidate gene for senile osteoporosis, acts as a molecular switch promoting osteoblast differentiation.

    PubMed

    Tokuzawa, Yoshimi; Yagi, Ken; Yamashita, Yzumi; Nakachi, Yutaka; Nikaido, Itoshi; Bono, Hidemasa; Ninomiya, Yuichi; Kanesaki-Yatsuka, Yukiko; Akita, Masumi; Motegi, Hiromi; Wakana, Shigeharu; Noda, Tetsuo; Sablitzky, Fred; Arai, Shigeki; Kurokawa, Riki; Fukuda, Toru; Katagiri, Takenobu; Schönbach, Christian; Suda, Tatsuo; Mizuno, Yosuke; Okazaki, Yasushi

    2010-07-01

    Excessive accumulation of bone marrow adipocytes observed in senile osteoporosis or age-related osteopenia is caused by the unbalanced differentiation of MSCs into bone marrow adipocytes or osteoblasts. Several transcription factors are known to regulate the balance between adipocyte and osteoblast differentiation. However, the molecular mechanisms that regulate the balance between adipocyte and osteoblast differentiation in the bone marrow have yet to be elucidated. To identify candidate genes associated with senile osteoporosis, we performed genome-wide expression analyses of differentiating osteoblasts and adipocytes. Among transcription factors that were enriched in the early phase of differentiation, Id4 was identified as a key molecule affecting the differentiation of both cell types. Experiments using bone marrow-derived stromal cell line ST2 and Id4-deficient mice showed that lack of Id4 drastically reduces osteoblast differentiation and drives differentiation toward adipocytes. On the other hand knockdown of Id4 in adipogenic-induced ST2 cells increased the expression of Ppargamma2, a master regulator of adipocyte differentiation. Similar results were observed in bone marrow cells of femur and tibia of Id4-deficient mice. However the effect of Id4 on Ppargamma2 and adipocyte differentiation is unlikely to be of direct nature. The mechanism of Id4 promoting osteoblast differentiation is associated with the Id4-mediated release of Hes1 from Hes1-Hey2 complexes. Hes1 increases the stability and transcriptional activity of Runx2, a key molecule of osteoblast differentiation, which results in an enhanced osteoblast-specific gene expression. The new role of Id4 in promoting osteoblast differentiation renders it a target for preventing the onset of senile osteoporosis. PMID:20628571

  14. Repeated forced swim stress differentially affects formalin-evoked nociceptive behaviour and the endocannabinoid system in stress normo-responsive and stress hyper-responsive rat strains.

    PubMed

    Jennings, Elaine M; Okine, Bright N; Olango, Weredeselam M; Roche, Michelle; Finn, David P

    2016-01-01

    Repeated exposure to a homotypic stressor such as forced swimming enhances nociceptive responding in rats. However, the influence of genetic background on this stress-induced hyperalgesia is poorly understood. The aim of the present study was to compare the effects of repeated forced swim stress on nociceptive responding in Sprague-Dawley (SD) rats versus the Wistar Kyoto (WKY) rat strain, a genetic background that is susceptible to stress, negative affect and hyperalgesia. Given the well-documented role of the endocannabinoid system in stress and pain, we investigated associated alterations in endocannabinoid signalling in the dorsal horn of the spinal cord and amygdala. In SD rats, repeated forced swim stress for 10 days was associated with enhanced late phase formalin-evoked nociceptive behaviour, compared with naive, non-stressed SD controls. In contrast, WKY rats exposed to 10 days of swim stress displayed reduced late phase formalin-evoked nociceptive behaviour. Swim stress increased levels of monoacylglycerol lipase (MAGL) mRNA in the ipsilateral side of the dorsal spinal cord of SD rats, an effect not observed in WKY rats. In the amygdala, swim stress reduced anandamide (AEA) levels in the contralateral amygdala of SD rats, but not WKY rats. Additional within-strain differences in levels of CB1 receptor and fatty acid amide hydrolase (FAAH) mRNA and levels of 2-arachidonylglycerol (2-AG) were observed between the ipsilateral and contralateral sides of the dorsal horn and/or amygdala. These data indicate that the effects of repeated stress on inflammatory pain-related behaviour are different in two rat strains that differ with respect to stress responsivity and affective state and implicate the endocannabinoid system in the spinal cord and amygdala in these differences. PMID:25988529

  15. Primary Human Osteoblasts in Response to 25-Hydroxyvitamin D3, 1,25-Dihydroxyvitamin D3 and 24R,25-Dihydroxyvitamin D3

    PubMed Central

    van der Meijden, Karen; Lips, Paul; van Driel, Marjolein; Heijboer, Annemieke C.; Schulten, Engelbert A. J. M.; den Heijer, Martin; Bravenboer, Nathalie

    2014-01-01

    The most biologically active metabolite 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) has well known direct effects on osteoblast growth and differentiation in vitro. The precursor 25-hydroxyvitamin D3 (25(OH)D3) can affect osteoblast function via conversion to 1,25(OH)2D3, however, it is largely unknown whether 25(OH)D3 can affect primary osteoblast function on its own. Furthermore, 25(OH)D3 is not only converted to 1,25(OH)2D3, but also to 24R,25-dihydroxyvitamin D3 (24R,25(OH)2D3) which may have bioactivity as well. Therefore we used a primary human osteoblast model to examine whether 25(OH)D3 itself can affect osteoblast function using CYP27B1 silencing and to investigate whether 24R,25(OH)2D3 can affect osteoblast function. We showed that primary human osteoblasts responded to both 25(OH)D3 and 1,25(OH)2D3 by reducing their proliferation and enhancing their differentiation by the increase of alkaline phosphatase, osteocalcin and osteopontin expression. Osteoblasts expressed CYP27B1 and CYP24 and synthesized 1,25(OH)2D3 and 24R,25(OH)2D3 dose-dependently. Silencing of CYP27B1 resulted in a decline of 1,25(OH)2D3 synthesis, but we observed no significant differences in mRNA levels of differentiation markers in CYP27B1-silenced cells compared to control cells after treatment with 25(OH)D3. We demonstrated that 24R,25(OH)2D3 increased mRNA levels of alkaline phosphatase, osteocalcin and osteopontin. In addition, 24R,25(OH)2D3 strongly increased CYP24 mRNA. In conclusion, the vitamin D metabolites 25(OH)D3, 1,25(OH)2D3 and 24R,25(OH)2D3 can affect osteoblast differentiation directly or indirectly. We showed that primary human osteoblasts not only respond to 1,25(OH)2D3, but also to 24R,25(OH)2D3 by enhancing osteoblast differentiation. This suggests that 25(OH)D3 can affect osteoblast differentiation via conversion to the active metabolite 1,25(OH)2D3, but also via conversion to 24R,25(OH)2D3. Whether 25(OH)D3 has direct actions on osteoblast function needs further

  16. EGF Inhibits Wnt/β-Catenin-Induced Osteoblast Differentiation by Promoting β-Catenin Degradation.

    PubMed

    Boonanantanasarn, Kanitsak; Lee, Hye-Lim; Baek, Kyunghwa; Woo, Kyung Mi; Ryoo, Hyun-Mo; Baek, Jeong-Hwa; Kim, Gwan-Shik

    2015-12-01

    Bone morphogenetic protein (BMP) and canonical Wnts are representative developmental signals that enhance osteoblast differentiation and bone formation. Previously, we demonstrated that epidermal growth factor (EGF) inhibits BMP2-induced osteoblast differentiation by inducing Smurf1 expression. However, the regulatory role of EGF in Wnt/β-catenin-induced osteoblast differentiation has not been elucidated. In this study, we investigated the effect of EGF on Wnt/β-catenin signaling-induced osteoblast differentiation using the C2C12 cell line. EGF significantly suppressed the expression of osteoblast marker genes, which were induced by Wnt3a and a GSK-3β inhibitor. EGF increased the expression levels of Smurf1 mRNA and protein. Smurf1 knockdown rescued Wnt/β-catenin-induced osteogenic marker gene expression in the presence of EGF. EGF treatment or Smurf1 overexpression did not affect β-catenin mRNA expression levels, but reduced β-catenin protein levels and TOP-Flash activity. EGF and Smurf1 promoted β-catenin ubiquitination. Co-immunoprecipitation and GST pull-down assays showed that Smurf1 associates with β-catenin. These results suggest that EGF/Smurf1 inhibits Wnt/β-catenin-induced osteogenic differentiation and that Smurf1 downregulates Wnt/β-catenin signaling by enhancing proteasomal degradation of β-catenin. PMID:26015066

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

    PubMed

    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

  18. Advanced glycation end products suppress osteoblastic differentiation of stromal cells by activating endoplasmic reticulum stress.

    PubMed

    Tanaka, Ken-ichiro; Yamaguchi, Toru; Kaji, Hiroshi; Kanazawa, Ippei; Sugimoto, Toshitsugu

    2013-08-30

    Advanced glycation end products (AGEs) are involved in bone quality deterioration in diabetes mellitus. We previously showed that AGE2 or AGE3 inhibited osteoblastic differentiation and mineralization of mouse stromal ST2 cells, and also induced apoptosis and decreased cell growth. Although quality management for synthesized proteins in endoplasmic reticulum (ER) is crucial for the maturation of osteoblasts, the effects of AGEs on ER stress in osteoblast lineage are unknown. We thus examined roles of ER stress in AGE2- or AGE3-induced suppression of osteoblastogenesis of ST2 cells. An ER stress inducer, thapsigargin (TG), induced osteoblastic differentiation of ST2 cells by increasing the levels of Osterix, type 1 collagen (Col1), alkaline phosphatase (ALP) and osteocalcin (OCN) mRNA. AGE2 or AGE3 suppressed the levels of ER stress sensors such as IRE1α, ATF6 and OASIS, while they increased the levels of PERK and its downstream molecules, ATF4. A reduction in PERK level by siRNA did not affect the AGEs-induced suppression of the levels of Osterix, Col1 and OCN mRNA. In conclusion, AGEs inhibited the osteoblastic differentiation of stromal cells by suppressing ER stress sensors and accumulating abnormal proteins in the cells. This process might accelerate AGEs-induced suppression of bone formation found in diabetes mellitus. PMID:23933252

  19. Effects of a hydroxyapatite-based biomaterial on gene expression in osteoblast-like cells.

    PubMed

    Sibilla, P; Sereni, A; Aguiari, G; Banzi, M; Manzati, E; Mischiati, C; Trombelli, L; del Senno, L

    2006-04-01

    Biostite is a hydroxyapatite-derived biomaterial that is used in periodontal and bone reconstructive procedures due to its osteoconductive properties. Since the molecular effects of this biomaterial on osteoblasts are still unknown, we decided to assess whether it may specifically modulate osteoblast functions in vitro. We found that a brief exposure to Biostite significantly reduced the proliferation of MG-63 and SaOS-2 osteoblast-like cells to approximately 50% of the plateau value. Furthermore, gene array analysis of MG-63 cells showed that Biostite caused a differential expression of 37 genes which are involved in cell proliferation and interaction, and related to osteoblast differentiation and tissue regeneration. Results were confirmed by RT-PCR, Western blot, and by an increase in alkaline phosphatase (ALP) specific activity. Biostite also increased levels of polycystin-2, a mechano-sensitive Ca(2+) channel, a promising new marker of bone cell differentiation. Biostite, therefore, may directly affect osteoblasts by enhancing chondro/osteogenic gene expression and cytoskeleton-related signaling pathways, which may contribute to its clinical efficacy. PMID:16567558

  20. FoxO1 Protein Cooperates with ATF4 Protein in Osteoblasts to Control Glucose Homeostasis*

    PubMed Central

    Kode, Aruna; Mosialou, Ioanna; Silva, Barbara C.; Joshi, Sneha; Ferron, Mathieu; Rached, Marie Therese; Kousteni, Stavroula

    2012-01-01

    The Forkhead transcription factor FoxO1 inhibits through its expression in osteoblasts β-cell proliferation, insulin secretion, and sensitivity. At least part of the FoxO1 metabolic functions result from its ability to suppress the activity of osteocalcin, an osteoblast-derived hormone favoring glucose metabolism and energy expenditure. In searching for mechanisms mediating the metabolic actions of FoxO1, we focused on ATF4, because this transcription factor also affects glucose metabolism through its expression in osteoblasts. We show here that FoxO1 co-localizes with ATF4 in the osteoblast nucleus, and physically interacts with and promotes the transcriptional activity of ATF4. Genetic experiments demonstrate that FoxO1 and ATF4 cooperate to increase glucose levels and decrease glucose tolerance. These effects result from a synergistic effect of the two transcription factors to suppress the activity of osteocalcin through up-regulating expression of the phosphatase catalyzing osteocalcin inactivation. As a result, insulin production by β-cells and insulin signaling in the muscle, liver and white adipose tissue are compromised and fat weight increases by the FoxO1/ATF4 interaction. Taken together these observations demonstrate that FoxO1 and ATF4 cooperate in osteoblasts to regulate glucose homeostasis. PMID:22298775

  1. Severity and timing: How prenatal stress exposure affects glial developmental, emotional behavioural and plasma neurosteroid responses in guinea pig offspring.

    PubMed

    Bennett, Greer A; Palliser, Hannah K; Walker, David; Hirst, Jonathan

    2016-08-01

    Prenatal stress has been associated with a variety of developmental changes in offspring, notably those associated with brain development and subsequent risk for neuropathologies later in life. Recently, the importance of the timing and the severity of the stressor during pregnancy has been emphasized with neurosteroids including allopregnanolone implicated in the regulation of stress and also for endogenous neuroprotection in offspring. Prenatal stress was induced using strobe light exposure in pregnant guinea pigs (term 71days) in three defined stress exposure groups (Gestational Age (GA)35-65, GA50-65 and GA60-65). Stress was induced for 2h (9-11am) every 5days via strobe light exposure. A fetal cohort were euthanased at term with fetal brains and plasma collected. Anxiety-like behaviour was evaluated at 18 days of age in a separate cohort of offspring with brains and plasma collected at 21days of age. Markers for mature oligodendrocytes and reactive astrocytes were measured in the CA1 region of the hippocampus and the subcortical white matter. The neurosteroid allopregnanolone was measured by radioimmunoassay in offspring plasma. In the CA1 region of the hippocampus, fetuses from all stress groups showed reduced expression of mature oligodendrocytes and reactive astrocytes. By juvenility, all male stress exposure groups had recovered to levels of unaffected controls with the exception of the GA35-65 stress group. In juvenile females, mature oligodendrocyte marker expression was reduced in all stress groups and reactive astrocyte expression was reduced in the GA35-65 and GA60-65 stress groups by juvenility. Increased reactive astrocyte expression was also apparent in the subcortical white matter in both sexes both at term and at juvenility. Prenatally stressed offspring spent less time exploring in the object exploration test and also entered the inner zone of the open field less than controls at 18days of age. Circulating allopregnanolone concentrations were

  2. Exploring Students' Behavioural Patterns during Online Peer Assessment from the Affective, Cognitive, and Metacognitive Perspectives: A Progressive Sequential Analysis

    ERIC Educational Resources Information Center

    Cheng, Kun-Hung; Hou, Huei-Tse

    2015-01-01

    Previous research regarding peer assessment has investigated the relationships between peer feedback and learners' performance. However, few studies investigate in-depth learning processes during technology-assisted peer assessment activities, particularly from affective, cognitive, and metacognitive perspectives. This study conducts a series of…

  3. Osteoblasts of calvaria induce higher numbers of osteoclasts than osteoblasts from long bone.

    PubMed

    Wan, Qilong; Schoenmaker, Ton; Jansen, Ineke D C; Bian, Zhuan; de Vries, Teun J; Everts, Vincent

    2016-05-01

    Several studies have demonstrated the existence of functional differences between osteoclasts harbored in different bones. The mechanisms involved in the occurrence of such a heterogeneity are not yet understood. Since cells of the osteoblast lineage play a critical role in osteoclastogenesis, osteoclast heterogeneity may be due to osteoblasts that differ at the different bone sites. In the present study we evaluated possible differences in the capacity of calvaria and long bone osteoblasts to induce osteoclastogenesis. Osteoblasts were isolated from calvaria and long bone of mice and co-cultured with osteoclast precursors obtained from bone marrow of both types of bone, spleen and peripheral blood. Irrespective of the source of the precursors, a significantly higher number of TRACP-positive multinucleated cells were formed with calvaria osteoblasts. The expression of osteoclastogenesis related genes was analyzed by qPCR. OPG was significantly higher expressed by long bone osteoblasts. The RANKL/OPG ratio and TNF-α gene expression were significantly higher in calvaria osteoblast cultures. OPG added to the culture system inhibited osteoclastogenesis in both groups. Blocking TNF-α had no effect on osteoclastogenesis. Calvaria and long bone osteoblasts were pre-stimulated with VitD3 for 5days. Subsequently, osteoclast precursors were added to these cultures. After a co-culture of 6days, it was shown that VitD3 pre-stimulation of long bone osteoblasts strongly improved their capacity to induce osteoclast formation. This coincided with an increased ratio of RANKL/OPG. Taken together, the data demonstrated differences in the capacity of calvaria and long bone osteoblasts to induce osteoclastogenesis. This appeared to be due to differences in the expression of RANKL and OPG. VitD3 pre-stimulation improved the ability of long bone osteoblasts to induce osteoclast formation. Our findings demonstrate bone-site specific differences in osteoblast-mediated formation of

  4. Experiments with osteoblasts cultured under hypergravity conditions

    NASA Technical Reports Server (NTRS)

    Kacena, Melissa A.; Todd, Paul; Gerstenfeld, Louis C.; Landis, William J.

    2004-01-01

    To understand further the role of gravity in osteoblast attachment, osteoblasts were subjected to hypergravity conditions in vitro. Scanning electron microscopy of all confluent coverslips from FPA units show that the number of attached osteoblasts was similar among gravitational levels and growth durations (90 cells/microscopic field). Specifically, confluent 1.0 G control cultures contained an average of 91 +/- 8 cells/field, 3.3 G samples had 88 +/- 8 cells/field, and 4.0 G cultures averaged 90 +/- 7 cells/field. The sparsely plated cultures assessed by immunohistochemistry also had similar numbers of cells at each time point (l.0 G was similar to 3.3 and 4.0 G), but cell number changed from one time point to the next as those cells proliferated. Immunohistochemistry of centrifuged samples showed an increase in number (up to 160% increase) and thickness (up to 49% increase) of actin fibers, a decrease in intensity of fibronectin fluorescence (18-23% decrease) and an increase in number of vinculin bulbs (202-374% increase in number of vinculin bulbs/area). While hypergravity exposure did not alter the number of attached osteoblasts, it did result in altered actin, fibronectin, and vinculin elements, changing some aspects of osteoblast- substrate adhesion.

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

    SciTech Connect

    Hong, Dun; Chen, Hai-Xiao; Yu, Hai-Qiang; Liang, Yong; Wang, Carrie; Lian, Qing-Quan; Deng, Hai-Teng; Ge, Ren-Shan

    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.

  6. Skeletal Collagen Turnover by the Osteoblast

    NASA Technical Reports Server (NTRS)

    Partridge, Nicola C.

    1997-01-01

    Among the most overt negative changes experienced by man and experimental animals under conditions of weightlessness are the loss of skeletal mass and attendant hypercalciuria. These clearly result from some disruption in the balance between bone formation and bone resorption (i.e. remodelling) which appears to be due to a decrease in the functions of the osteoblast. In the studies funded by this project, the clonal osteoblastic cell line, UMR 106-01, has been used to investigate the regulation of collagenase and Tissue Inhibitors of MetalloProteases (TIMPs). This project has shed light on the comprehensive role of the osteoblast in the remodelling process, and, in so doing, provided some insight into how the process might be disrupted under conditions of microgravity.

  7. Nanostructured niobium oxide coatings influence osteoblast adhesion.

    PubMed

    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

  8. [The regulation of various organs by osteoblasts].

    PubMed

    Sato, Shingo; Takeda, Shu

    2016-05-01

    It has been recently demonstrated that osteocalcin, which is secreted from osteoblasts, plays a significant role in glucose metabolism, fat metabolism, mail fertility, and brain functions. It has been also revealed that fibroblast growth factor 23 (FGF23), which is secreted from osteocytes, has an important role in phosphate metabolism or calcium homeostasis. These findings suggest that bone is not only a target organ of hormones but also involved in regulating other organs as an endocrine organ. Bone forms regulatory network with various organs to maintain the whole body homeostasis. This review introduces the regulation of various organs by osteoblasts. PMID:27117618

  9. Triclosan Blocks Mmp 13 Expression in Hormone-Stimulated Osteoblasts

    PubMed Central

    Barnes, Virginia Monsul; Xu, Tao; Shimizu, Emi; Jefcoat, Steven; Vasilov, Anatoliy; Qin, Ling; Partridge, Nicola C.

    2014-01-01

    Background Matrix metalloproteinase-13 (Mmp-13) is an important enzyme for the modulation of bone turnover and gingival recession. Elevated levels of Mmp-13 are associated with alveolar bone resorption, periodontal ligament destruction, and gingival attachment loss, which are the clinical symptoms of periodontal disease. Continued evidence suggests periodontal disease contributes to oral tissue destruction and is linked to numerous systemic conditions. Triclosan is a long standing, proven antibacterial and anti-inflammatory agent found in the only FDA-approved dentifrice for the treatment of plaque and gingivitis. Methods This study examined the inhibitory effects of triclosan on lipopolysaccharide (LPS), parathyroid hormone (PTH) and prostaglandin E2 (PGE2) induced expression of Mmp-13 in UMR 106-01 cells, an osteoblastic osteosarcoma cell line. The cells were stimulated with PTH or PGE2 to induce Mmp-13 mRNA expression and Real Time RT-PCR was performed to determine gene expression levels. Western blot analysis assessed the presence or absence of protein degradation or inhibition of protein synthesis. Mmp-13 Promoter Reporter Assay was utilized to explore possible direct effects of triclosan on the Mmp-13 promoter. Results Triclosan significantly reduced PTH or PGE2 elevated expression of Mmp-13 in osteoblastic cells without affecting basal levels of the mRNA. Surprisingly, triclosan enhanced the expression of c-fos and amphiregulin mRNA. A promoter assay indicated triclosan directly inhibits the activation of the PTH-responsive minimal promoter of Mmp-13. Conclusion Our data appear to have identified a nuclear mechanism of action of triclosan which accounts for triclosan’s ability to inhibit PTH or PGE2 induced Mmp-13 expression in osteoblastic cells. PMID:23368947

  10. Alpha-adrenergic blocker mediated osteoblastic stem cell differentiation

    SciTech Connect

    Choi, Yoon Jung; Lee, Jue Yeon; Lee, Seung Jin; Chung, Chong-Pyoung; Park, Yoon Jeong

    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

  11. Polonium behaviour in reservoirs potentially affected by acid mine drainage (AMD) in the Iberian Pyrite Belt (SW of Spain).

    PubMed

    Blasco, M; Gázquez, M J; Pérez-Moreno, S M; Grande, J A; Valente, T; Santisteban, M; de la Torre, M L; Bolívar, J P

    2016-02-01

    The province of Huelva is one of the areas most affected by acid mine drainage (AMD) in the world, which can produce big enhancements and fractionations in the waters affected by AMD. There are very few studies on this issue, and none on polonium-210. Twenty-two water reservoirs were sampled, and the (210)Po was measured in both dissolution and particulate phases. The (210)Po concentrations in the waters were in the same order of magnitude to those ones for unperturbed systems, although the data published to particulate matter are very scarce. A mean value and standard uncertainty for (210)Po of 0.25 ± 0.03 mBq L(-1) in the dissolved matter, and 62 ± 9 mBq g(-1) in the particulate matter can be established as base line for the reservoirs of the Huelva area. The distribution coefficients (kd) range from 10(4) to 10(6) L kg(-1), in agreement to the found ones by other authors for the case of neutral waters, but being the lowest values for the more acidic reservoirs. It has been also found that (210)Po has a high tendency to be associated to the particulate matter for neutral-alkaline waters, however, under extreme acid conditions (pH < 3), increases the Po tendency to be associated to the dissolved phase. Therefore, the main conclusion obtained in this work is that AMD has no a significant influence on the total activity concentration of (210)Po in the waters of reservoirs, but the acidity has a clear influence on its distribution between both dissolved and the particulate phases. PMID:26650826

  12. Effect of therapeutic levels of doxycycline and minocycline in the proliferation and differentiation of human bone marrow osteoblastic cells.

    PubMed

    Gomes, Pedro Sousa; Fernandes, Maria Helena

    2007-03-01

    Semi-synthetic tetracyclines (TCs) have been reported to reduce pathological bone resorption through several mechanisms, although their effect over bone physiological metabolism is not yet fully understood. The present study aims at evaluate the behaviour of osteoblastic-induced human bone marrow cells regarding proliferation and functional activity, in the presence of representative therapeutic concentrations of doxycycline and minocycline. First passage human osteoblastic bone marrow cells were cultured for 35 days in conditions known to favor osteoblastic differentiation. Doxycycline (1-25 micro g/ml) or minocycline (1-50 micro g/ml) were added continuously, with the culture medium, twice a week with every medium change. Cultures were characterised at several time points for cell proliferation and function. Present data showed that 1 micro g/ml of both tetracyclines, level representative of that attained in plasma and crevicular fluid with the standard therapeutic dosage, increased significantly the proliferation of human bone marrow osteoblastic cells without altering their specific phenotype and functional activity. Long-term exposure to these TCs induced a significant increase in the number of active osteoblastic cells that yielded a proportional amount of a normal mineralised matrix, suggesting a potential application in therapeutic approaches aiming to increase bone formation. The presence of higher levels of these agents led to a dose-dependent deleterious effect over cell culture, delaying cell proliferation and differentiation. PMID:17141175

  13. Influence of increased mechanical loading by hypergravity on the microtubule cytoskeleton and prostaglandin E2 release in primary osteoblasts

    NASA Technical Reports Server (NTRS)

    Searby, Nancy D.; Steele, Charles R.; Globus, Ruth K.

    2005-01-01

    Cells respond to a wide range of mechanical stimuli such as fluid shear and strain, although the contribution of gravity to cell structure and function is not understood. We hypothesized that bone-forming osteoblasts are sensitive to increased mechanical loading by hypergravity. A centrifuge suitable for cell culture was developed and validated, and then primary cultures of fetal rat calvarial osteoblasts at various stages of differentiation were mechanically loaded using hypergravity. We measured microtubule network morphology as well as release of the paracrine factor prostaglandin E2 (PGE2). In immature osteoblasts, a stimulus of 10x gravity (10 g) for 3 h increased PGE2 2.5-fold and decreased microtubule network height 1.12-fold without affecting cell viability. Hypergravity (3 h) caused dose-dependent (5-50 g) increases in PGE2 (5.3-fold at 50 g) and decreases (1.26-fold at 50 g) in microtubule network height. PGE2 release depended on duration but not orientation of the hypergravity load. As osteoblasts differentiated, sensitivity to hypergravity declined. We conclude that primary osteoblasts demonstrate dose- and duration-dependent sensitivity to gravitational loading, which appears to be blunted in mature osteoblasts.

  14. Assessment of the role of flavonoids for inducing osteoblast differentiation in isolated mouse bone marrow derived mesenchymal stem cells.

    PubMed

    Srivastava, Swati; Bankar, Rohini; Roy, Partha

    2013-06-15

    Quercetin and rutin are common flavonoids in fruit and vegetables, and have been reported to affect bone development. However, the effect of flavonoids on osteoblast differentiation remains a matter of controversy. In the present study, mouse bone marrow mesenchymal stem cells (BMMSCs) were isolated and characterized for their use in osteoblast differentiation using two flavonoids, quercetin and rutin. BMMSCs were cultured in various concentrations of quercetin and rutin during the osteoblast differentiation period of 10 days. Both quercetin and rutin were found to up regulate the osteoblast differentiation in dose dependent manner, albeit to lesser extent in case of former than that of latter. Quercetin and rutin also increased alkaline phosphatase activity by about 150 and 240% and demonstrated mineralization up to 110 and 200% respectively as compared to control (which was considered as 100%). Further, both the flavonoids were also found to increase the expression of some of the prominent markers for differentiation of osteoblast like osteopontin, osterix, RunX2, osteoprotegerin and osteocalcin. The current data suggests that certain classes of flavonoids like rutin and quercetin can be used in the cure and management of osteodegenerative disorders due to their osteoblast specific differentiation activities. PMID:23570998

  15. Health Service Utilization for Mental, Behavioural and Emotional Problems among Conflict-Affected Population in Georgia: A Cross-Sectional Study

    PubMed Central

    Gotsadze, George; Patel, Vikram; McKee, Martin; Uchaneishvili, Maia; Rukhadze, Natia

    2015-01-01

    Background There is large gap in mental illness treatment globally and potentially especially so in war-affected populations. The study aim was to examine health care utilization patterns for mental, behavioural and emotional problems among the war-affected adult population in the Republic of Georgia. Methods A cross-sectional household survey was conducted among 3600 adults affected by 1990s and 2008 armed conflicts in Georgia. Service use was measured for the last 12 months for any mental, emotional or behavioural problems. TSQ, PHQ-9 and GAD-7 were used to measure current symptoms of PTSD, depression and anxiety. Descriptive and regression analyses were used. Results Respondents were predominantly female (65.0%), 35.8% were unemployed, and 56.0% covered by the government insurance scheme. From the total sample, 30.5% had symptoms of at least one current mental disorder. Among them, 39.0% sought care for mental problems, while 33.1% expressed facing barriers to accessing care and so did not seek care. General practitioners (29%) and neurologists (26%) were consulted by the majority of those with a current mental disorder who accessed services, while use of psychiatric services was far more limited. Pharmacotherapy was the predominant type of care (90%). Female gender (OR 1.50, 95% CI: 1.25, 1.80), middle-age (OR 1.83, 95% CI: 1.48, 2.26) and older-age (OR 1.62, 95% CI: 1.19, 2.21), possession of the state insurance coverage (OR 1.55, 95% CI: 1.30, 1.86), current PTSD symptoms (OR 1.56, 95% CI: 1.29, 1.90) and depression (OR 2.12, 95% CI: 1.70, 2.65) were associated with higher rates of health service utilization, while employed were less likely to use services (OR 0.71, 95% CI: 0.55, 0.89). Conclusions Reducing financial access barriers and increasing awareness and access to local care required to help reduce the burden of mental disorders among conflict-affected persons in Georgia. PMID:25853246

  16. Prolonged Survival of Transplanted Osteoblastic Cells Does Not Directly Accelerate the Healing of Calvarial Bone Defects.

    PubMed

    Kitami, Megumi; Kaku, Masaru; Rocabado, Juan Marcelo Rosales; Ida, Takako; Akiba, Nami; Uoshima, Katsumi

    2016-09-01

    Considering the increased interest in cell-based bone regeneration, it is necessary to reveal the fate of transplanted cells and their substantive roles in bone regeneration. The aim of this study was to analyze the fate of transplanted cells and the effect of osteogenic cell transplantation on calvarial bone defect healing. An anti-apoptotic protein, heat shock protein (HSP) 27, was overexpressed in osteoblasts. Then, the treated osteoblasts were transplanted to calvarial bone defect and their fate was analyzed to evaluate the significance of transplanted cell survival. Transient overexpression of Hsp27 rescued MC3T3-E1 osteoblastic cells from H2 O2 -induced apoptosis without affecting osteoblastic differentiation in culture. Transplantation of Hsp27-overexpressing cells, encapsulated in collagen gel, showed higher proliferative activity, and fewer apoptotic cells in comparison with control cells. After 4-week of transplantation, both control cell- and Hsp27 overexpressed cell-transplanted groups showed significantly higher new bone formation in comparison with cell-free gel-transplantation group. Interestingly, the prolonged survival of transplanted osteoblastic cells by Hsp27 did not provide additional effect on bone healing. The transplanted cells in collagen gel survived for up to 4-week but did not differentiate into bone-forming osteoblasts. In conclusion, cell-containing collagen gel accelerated calvarial bone defect healing in comparison with cell-free collagen gel. However, prolonged survival of transplanted cells by Hsp27 overexpression did not provide additional effect. These results strongly indicate that cell transplantation-based bone regeneration cannot be explained only by the increment of osteogenic cells. Further studies are needed to elucidate the practical roles of transplanted cells that will potentiate successful bone regeneration. J. Cell. Physiol. 231: 1974-1982, 2016. © 2016 Wiley Periodicals, Inc. PMID:26754153

  17. Overexpression of BCLXL in Osteoblasts Inhibits Osteoblast Apoptosis and Increases Bone Volume and Strength.

    PubMed

    Moriishi, Takeshi; Fukuyama, Ryo; Miyazaki, Toshihiro; Furuichi, Tatsuya; Ito, Masako; Komori, Toshihisa

    2016-07-01

    The Bcl2 family proteins, Bcl2 and BclXL, suppress apoptosis by preventing the release of caspase activators from mitochondria through the inhibition of Bax subfamily proteins. We reported that BCL2 overexpression in osteoblasts using the 2.3 kb Col1a1 promoter increased osteoblast proliferation, failed to reduce osteoblast apoptosis, inhibited osteoblast maturation, and reduced the number of osteocyte processes, leading to massive osteocyte death. We generated BCLXL (BCL2L1) transgenic mice using the same promoter to investigate BCLXL functions in bone development and maintenance. Bone mineral density in the trabecular bone of femurs was increased, whereas that in the cortical bone was similar to that in wild-type mice. Osteocyte process formation was unaffected and bone structures were similar to those in wild-type mice. A micro-CT analysis showed that trabecular bone volume in femurs and vertebrae and the cortical thickness of femurs were increased. A dynamic bone histomorphometric analysis revealed that the mineralizing surface was larger in trabecular bone, and the bone-formation rate was increased in cortical bone. Serum osteocalcin but not TRAP5b was increased, BrdU-positive osteoblastic cell numbers were increased, TUNEL-positive osteoblastic cell numbers were reduced, and osteoblast marker gene expression was enhanced in BCLXL transgenic mice. The three-point bending test indicated that femurs were stronger in BCLXL transgenic mice than in wild-type mice. The frequency of TUNEL-positive primary osteoblasts was lower in BCLXL transgenic mice than in wild-type mice during cultivation, and osteoblast differentiation was enhanced but depended on cell density, indicating that enhanced differentiation was mainly owing to reduced apoptosis. Increased trabecular and cortical bone volumes were maintained during aging in male and female mice. These results indicate that BCLXL overexpression in osteoblasts increased the trabecular and cortical bone volumes with

  18. Osteoblastic response to pectin nanocoating on titanium surfaces.

    PubMed

    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. Effects of microgravity on osteoblast growth activation

    NASA Technical Reports Server (NTRS)

    Hughes-Fulford, M.; Lewis, M. L.

    1996-01-01

    Space flight is an environmental condition where astronauts can lose up to 19% of weight-bearing bone during long duration missions. We used the MC3T3-E1 osteoblast to investigate bone cell growth in microgravity (10(-6) to 10(-9)g). Osteoblasts were launched on the STS-56 shuttle flight in a quiescent state with 0.5% fetal calf serum (FCS) medium and growth activation was initiated by adding fresh medium with 10% FCS during microgravity exposure. Four days after serum activation, the cells were fixed before return to normal Earth gravity. Ground controls were treated in parallel with the flight samples in identical equipment. On landing, cell number, cell cytoskeleton, glucose utilization, and prostaglandin synthesis in flight (n = 4) and ground controls (n = 4) were examined. The flown osteoblasts grew slowly in microgravity with total cell number significantly reduced (55 +/- 6 vs 141 +/- 8 cells per microscopic field). The cytoskeleton of the flight osteoblasts had a reduced number of stress fibers and a unique abnormal morphology. Nuclei in the ground controls were large and round with punctate Hoechst staining of the DNA nucleosomes. The flight nuclei were 30% smaller than the controls (P < 0.0001) and oblong in shape, with fewer punctate areas. Due to their reduced numbers, the cells activated in microgravity used significantly less glucose than ground controls (80.2 +/- 0.7 vs 50.3 +/- 3.7 mg of glucose/dl remaining in the medium) and had reduced prostaglandin E2 (PGE2) synthesis when compared to controls (57.3 +/- 17 vs 138.3 +/- 41 pmol/ml). Cell viability was normal since, on a per-cell basis, glucose use and prostaglandin synthesis were comparable for flight and ground samples. Taken together, these data suggest that growth activation in microgravity results in reduced growth, causing reduced glucose utilization and reduced prostaglandin synthesis, with significantly altered actin cytoskeleton in osteoblasts.

  20. Effects of farnesyl pyrophosphate accumulation on calvarial osteoblast differentiation.

    PubMed

    Weivoda, Megan M; Hohl, Raymond J

    2011-08-01

    Statins, drugs commonly used to lower serum cholesterol, have been shown to stimulate osteoblast differentiation and bone formation. Statins inhibit 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A reductase (HMGCR), the first step of the isoprenoid biosynthetic pathway, leading to the depletion of the isoprenoids farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). The effects of statins on bone have previously been attributed to the depletion of GGPP, because the addition of exogenous GGPP prevented statin-stimulated osteoblast differentiation in vitro. However, in a recent report, we demonstrated that the specific depletion of GGPP did not stimulate but, in fact, inhibited osteoblast differentiation. This led us to hypothesize that isoprenoids upstream of GGPP play a role in the regulation of osteoblast differentiation. We demonstrate here that the expression of HMGCR and FPP synthase decreased during primary calvarial osteoblast differentiation, correlating with decreased FPP and GGPP levels during differentiation. Zaragozic acid (ZGA) inhibits the isoprenoid biosynthetic pathway enzyme squalene synthase, leading to an accumulation of the squalene synthase substrate FPP. ZGA treatment of calvarial osteoblasts led to a significant increase in intracellular FPP and resulted in inhibition of osteoblast differentiation as measured by osteoblastic gene expression, alkaline phosphatase activity, and matrix mineralization. Simultaneous HMGCR inhibition prevented the accumulation of FPP and restored osteoblast differentiation. In contrast, specifically inhibiting GGPPS to lower the ZGA-induced increase in GGPP did not restore osteoblast differentiation. The specificity of HMGCR inhibition to restore osteoblast differentiation of ZGA-treated cultures through the reduction in isoprenoid accumulation was confirmed with the addition of exogenous mevalonate. Similar to ZGA treatment, exogenous FPP inhibited the mineralization of primary calvarial osteoblasts

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

    PubMed

    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

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

    PubMed Central

    Eleniste, Pierre P.; 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 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

  3. Modulation of osteoblast attachment and growth in vitro by inertial forces

    NASA Astrophysics Data System (ADS)

    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.

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

    NASA Technical Reports Server (NTRS)

    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

  5. Development and characterization of a mouse floxed Bmp2 osteoblast cell line that retains osteoblast genotype and phenotype

    PubMed Central

    Wu, Li-an; Feng, Junsheng; Wang, Lynn; Mu, Yan-dong; Baker, Andrew; Donly, Kevin J.; Harris, Stephen E.; MacDougall, Mary; Chen, Shuo

    2011-01-01

    Bone morphogenetic protein 2 (Bmp2) is essential for osteoblast differentiation and osteogenesis. Generation of floxed Bmp2 osteoblast cell lines is a valuable tool for studying the effects of Bmp2 on osteoblast differentiation and its signaling pathways during skeletal metabolism. Due to relatively limited sources of primary osteoblasts, we have developed cell lines that serve as good surrogate models for the study of osteoblast cell differentiation and bone mineralization. In this study, we established and characterized immortalized mouse floxed Bmp2 osteoblast cell lines. Primary mouse floxed Bmp2 osteoblasts were transfected with pSV3-neo and clonally selected. These transfected cells were verified by PCR and immunohistochemistry. To determine the genotype and phenotype of the immortalized cells, cell morphology, proliferation, differentiation and mineralization were analyzed. Also, expression of osteoblast-related gene markers including Runx2, Osx, ATF4, Dlx3, bone sialoprotein, dentin matrix protein 1, osteonectin, osteocalcin and osteopontin were examined by quantitative RT-PCR and immunohistochemistry. These results showed that immortalized floxed Bmp2 osteoblasts had a higher proliferation rate but preserved their genotypic and phenotypic characteristics similar to the primary cells. Thus, we, for the first time, describe the development of immortalized mouse floxed Bmp2 osteoblast cell lines and present a useful model to study osteoblast biology mediated by BMP2 and its downstream signaling transduction pathways. PMID:21271257

  6. Resveratrol Increases Osteoblast Differentiation In Vitro Independently of Inflammation.

    PubMed

    Ornstrup, Marie Juul; Harsløf, Torben; Sørensen, Lotte; Stenkjær, Liselotte; Langdahl, Bente Lomholt; Pedersen, Steen Bønløkke

    2016-08-01

    Low-grade inflammation negatively affects bone. Resveratrol is a natural compound proven to possess both anti-inflammatory and bone protective properties. However, it is uncertain if the bone effects are mediated though anti-inflammatory effects. Firstly, we investigated if resveratrol affects proliferation and differentiation of human bone marrow-derived mesenchymal stem cells. Secondly, we investigated if inflammation negatively affects proliferation and differentiation, and if resveratrol counteracts this through anti-inflammatory effects. Mesenchymal stem cells were obtained from bone marrow aspiration in 13 healthy individuals and cultured towards the osteoblast cell lineage. The cells were stimulated with resveratrol, lipopolysaccharide (LPS), LPS + resveratrol, or vehicle (control) for 21 days. Compared to control, resveratrol decreased cell number by 35 % (p < 0.05) and induced differentiation (a 3-fold increase in alkaline phosphatase (p < 0.002), while P1NP and OPG showed similar trends). LPS induced inflammation with a 44-fold increase in interleukin-6 (p < 0.05) and an extremely prominent increase in interleukin-8 production (p < 0.05) relative to control. In addition, LPS increased cell count (p < 0.05) and decreased differentiation (a reduction in P1NP production (p < 0.02)). Co-stimulation with LPS + resveratrol did not reduce interleukin-6 or interleukin-8, but nonetheless, cell count was reduced (p < 0.05) and alkaline phosphatase, P1NP, and OPG increased (p < 0.05 for all). Thus, resveratrol stimulates osteoblast differentiation independently of inflammation. PMID:27000750

  7. Beta Adrenergic Receptor Stimulation Suppresses Cell Migration in Association with Cell Cycle Transition in Osteoblasts-Live Imaging Analyses Based on FUCCI System.

    PubMed

    Katsumura, Sakie; Ezura, Yoichi; Izu, Yayoi; Shirakawa, Jumpei; Miyawaki, Atsushi; Harada, Kiyoshi; Noda, Masaki

    2016-02-01

    Osteoporosis affects over 20 million patients in the United States. Among those, disuse osteoporosis is serious as it is induced by bed-ridden conditions in patients suffering from aging-associated diseases including cardiovascular, neurological, and malignant neoplastic diseases. Although the phenomenon that loss of mechanical stress such as bed-ridden condition reduces bone mass is clear, molecular bases for the disuse osteoporosis are still incompletely understood. In disuse osteoporosis model, bone loss is interfered by inhibitors of sympathetic tone and adrenergic receptors that suppress bone formation. However, how beta adrenergic stimulation affects osteoblastic migration and associated proliferation is not known. Here we introduced a live imaging system, fluorescent ubiquitination-based cell cycle indicator (FUCCI), in osteoblast biology and examined isoproterenol regulation of cell cycle transition and cell migration in osteoblasts. Isoproterenol treatment suppresses the levels of first entry peak of quiescent osteoblastic cells into cell cycle phase by shifting from G1 /G0 to S/G2 /M and also suppresses the levels of second major peak population that enters into S/G2 /M. The isoproterenol regulation of osteoblastic cell cycle transition is associated with isoproterenol suppression on the velocity of migration. This isoproterenol regulation of migration velocity is cell cycle phase specific as it suppresses migration velocity of osteoblasts in G1 phase but not in G1 /S nor in G2 /M phase. Finally, these observations on isoproterenol regulation of osteoblastic migration and cell cycle transition are opposite to the PTH actions in osteoblasts. In summary, we discovered that sympathetic tone regulates osteoblastic migration in association with cell cycle transition by using FUCCI system. PMID:26192605

  8. Loss of Osteoblast Runx3 Produces Severe Congenital Osteopenia

    PubMed Central

    Bauer, Omri; Sharir, Amnon; Kimura, Ayako; Hantisteanu, Shay; Takeda, Shu

    2015-01-01

    Congenital osteopenia is a bone demineralization condition that is associated with elevated fracture risk in human infants. Here we show that Runx3, like Runx2, is expressed in precommitted embryonic osteoblasts and that Runx3-deficient mice develop severe congenital osteopenia. Runx3-deficient osteoblast-specific (Runx3fl/fl/Col1α1-cre), but not chondrocyte-specific (Runx3fl/fl/Col1α2-cre), mice are osteopenic. This demonstrates that an osteoblastic cell-autonomous function of Runx3 is required for proper osteogenesis. Bone histomorphometry revealed that decreased osteoblast numbers and reduced mineral deposition capacity in Runx3-deficient mice cause this bone formation deficiency. Neonatal bone and cultured primary osteoblast analyses revealed a Runx3-deficiency-associated decrease in the number of active osteoblasts resulting from diminished proliferation and not from enhanced osteoblast apoptosis. These findings are supported by Runx3-null culture transcriptome analyses showing significant decreases in the levels of osteoblastic markers and increases in the levels of Notch signaling components. Thus, while Runx2 is mandatory for the osteoblastic lineage commitment, Runx3 is nonredundantly required for the proliferation of these precommitted cells, to generate adequate numbers of active osteoblasts. Human RUNX3 resides on chromosome 1p36, a region that is associated with osteoporosis. Therefore, RUNX3 might also be involved in human bone mineralization. PMID:25605327

  9. Vitamin D and gene networks in human osteoblasts

    PubMed Central

    van de Peppel, Jeroen; van Leeuwen, Johannes P. T. M.

    2014-01-01

    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. PMID:24782782

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

    PubMed

    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

  11. Ethyl-2, 5-dihydroxybenzoate displays dual activity by promoting osteoblast differentiation and inhibiting osteoclast differentiation.

    PubMed

    Kwon, Byeong-Ju; Lee, Mi Hee; Koo, Min-Ah; Kim, Min Sung; Seon, Gyeung Mi; Han, Jae-Jin; Park, Jong-Chul

    2016-03-11

    The interplay between bone-forming osteoblasts and bone-resorbing osteoclasts is essential for balanced bone remodeling. In this study, we evaluate the ability of ethyl-2, 5-dihyrdoxybenzoate (E-2, 5-DHB) to affect both osteoblast and osteoclast differentiation for bone regeneration. Osteogenic differentiation of human mesenchymal stem cells (hMSCs) was quantified by measuring alkaline phosphatase (ALP) activity and calcium deposition. To evaluate osteoclast differentiation, we investigated the effect of E-2, 5-DHB on RANKL-activated osteoclastogenesis in RAW 264.7 cells. E-2, 5-DHB enhanced ALP activity and inhibited RAW 264.7 cell osteoclastogenesis in vitro. To assess the in vivo activity of E-2, 5-DHB, hMSCs were delivered subcutaneosuly alone or in combination with E-2, 5-DHB in an alginate gel into the backs of nude-mice. Histological and immunohistochemical evaluation showed significantly higher calcium deposition in the E-2, 5-DHB group. Osteocalcin (OCN) was highly expressed in cells implanted in the gels containing E-2, 5-DHB. Our results suggest that E-2, 5-DHB can effectively enhance osteoblast differentiation and inhibit osteoclast differentiation both in vitro and in vivo. Understanding the dual function of E-2, 5-DHB on osteoblast and osteoclast differentiation will aid in future development of E-2, 5-DHB as a material for bone tissue engineering. PMID:26869515

  12. Pre-adsorbed type-I collagen structure-dependent changes in osteoblastic phenotype

    SciTech Connect

    Hanagata, Nobutaka . E-mail: HANAGATA.Nobutaka@nims.go.jp; Takemura, Taro; Monkawa, Akira; Ikoma, Toshiyuki; Tanaka, Junzo

    2006-06-16

    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 {alpha}{sub 2}- and {alpha}{sub 5}-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.

  13. Increased Gs Signaling in Osteoblasts Reduces Bone Marrow and Whole-Body Adiposity in Male Mice.

    PubMed

    Cain, Corey J; Valencia, Joel T; Ho, Samantha; Jordan, Kate; Mattingly, Aaron; Morales, Blanca M; Hsiao, Edward C

    2016-04-01

    Bone is increasingly recognized as an endocrine organ that can regulate systemic hormones and metabolism through secreted factors. Although bone loss and increased adiposity appear to be linked clinically, whether conditions of increased bone formation can also change systemic metabolism remains unclear. In this study, we examined how increased osteogenesis affects metabolism by using an engineered G protein-coupled receptor, Rs1, to activate Gs signaling in osteoblastic cells in ColI(2.3)(+)/Rs1(+) transgenic mice. We previously showed that these mice have dramatically increased bone formation resembling fibrous dysplasia of the bone. We found that total body fat was significantly reduced starting at 3 weeks of age. Furthermore, ColI(2.3)(+)/Rs1(+) mice showed reduced O2 consumption and respiratory quotient measures without effects on food intake and energy expenditure. The mice had significantly decreased serum triacylglycerides, leptin, and adiponectin. Resting glucose and insulin levels were unchanged; however, glucose and insulin tolerance tests revealed increased sensitivity to insulin. The mice showed resistance to fat accumulation from a high-fat diet. Furthermore, ColI(2.3)(+)/Rs1(+) mouse bones had dramatically reduced mature adipocyte differentiation, increased Wingless/Int-1 (Wnt) signaling, and higher osteoblastic glucose utilization than controls. These findings suggest that osteoblasts can influence both local and peripheral adiposity in conditions of increased bone formation and suggest a role for osteoblasts in the regulation of whole-body adiposity and metabolic homeostasis. PMID:26901092

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

    PubMed

    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

  15. How attempts to meet others' unrealistic expectations affect health: health-promoting behaviours as a mediator between perfectionism and physical health.

    PubMed

    Harrison, Fleur; Craddock, Alan E

    2016-01-01

    The traits of perfectionism have been associated with health and longevity. Theoretically and empirically, health behaviours are considered a primary mechanism through which such associations of personality and health occur. However, scant evidence to date indicates behaviours did not mediate between perfectionism and health as anticipated. The aim of the current research was therefore to rigorously examine whether health behaviours mediated associations of perfectionism and physical health-related quality of life (HRQL). A sample of 263 students completed questionnaires measuring subtypes of perfectionism, HRQL, self-efficacy and health-promoting behaviours. Hierarchical regression analyses investigated predictors of physical HRQL and health-promoting behaviours. Non-parametric bootstrapping techniques assessed whether health-promoting behaviours mediated significant associations between perfectionism and physical HRQL. Socially prescribed perfectionism (SPP) significantly predicted poorer physical HRQL, and this association was mediated by health-promoting behaviours, a unique finding. Self-oriented perfectionism did not significantly predict physical HRQL, but was linked with more numerous health-promoting behaviours. In conclusion, results suggest that individuals higher in SPP, who are overly concerned with evaluation by others and with meeting perceived unrealistically high standards of performance, performed fewer health-promoting behaviours, and this mediated the association between SPP and poorer physical HRQL. More broadly, perfectionism predicted physical HRQL and engagement or lack thereof in health-promoting behaviours and should be considered as part of health promotion strategies. PMID:26167729

  16. 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.

    PubMed

    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

  17. BK Knockout by TALEN-Mediated Gene Targeting in Osteoblasts: KCNMA1 Determines the Proliferation and Differentiation of Osteoblasts

    PubMed Central

    Hei, Hongya; Gao, Jianjun; Dong, Jibin; Tao, Jie; Tian, Lulu; Pan, Wanma; Wang, Hongyu; Zhang, Xuemei

    2016-01-01

    Large conductance calcium-activated potassium (BK) channels participate in many important physiological functions in excitable tissues such as neurons, cardiac and smooth muscles, whereas the knowledge of BK channels in bone tissues and osteoblasts remains elusive. To investigate the role of BK channels in osteoblasts, we used transcription activator-like effector nuclease (TALEN) to establish a BK knockout cell line on rat ROS17/2.8 osteoblast, and detected the proliferation and mineralization of the BK-knockout cells. Our study found that the BK-knockout cells significantly decreased the ability of proliferation and mineralization as osteoblasts, compared to the wild type cells. The overall expression of osteoblast differentiation marker genes in the BK-knockout cells was significantly lower than that in wild type osteoblast cells. The BK-knockout osteoblast cell line in our study displays a phenotype decrease in osteoblast function which can mimic the pathological state of osteoblast and thus provide a working cell line as a tool for study of osteoblast function and bone related diseases. PMID:27329042

  18. Effects of microgravity on osteoblast growth

    NASA Technical Reports Server (NTRS)

    Hughes-Fulford, M.; Tjandrawinata, R.; Fitzgerald, J.; Gasuad, K.; Gilbertson, V.

    1998-01-01

    Studies from space flights over the past two decades have demonstrated that basic physiological changes occur in humans during space flight. These changes include cephalic fluid shifts, loss of fluid and electrolytes, loss of muscle mass, space motion sickness, anemia, reduced immune response, and loss of calcium and mineralized bone. The cause of most of these manifestations is not known and until recently, the general approach was to investigate general systemic changes, not basic cellular responses to microgravity. Recently analyzed data from the 1973-1974 Skylabs disclose that there is a rise in the systemic hormone, cortisol, which may play a role in bone loss in flight. In two flights where bone growth was measured (Skylabs 3 and 4), the crew members had a significant loss of calcium accompanied by a rise in 24 hour urinary cortisol during the entire flight period. In ground-based work on osteoblasts, we have demonstrated that equivalent amounts of glucocorticoids can inhibit osteoblast cell growth. In addition, this laboratory has recently studied gene growth and activation of mouse osteoblasts (MC3T3-E1) during spaceflight. Osteoblast cells were grown on glass coverslips, loaded in the Biorack plunger boxes 18 hours before launch and activated 19 hours after launch in the Biorack incubator under microgravity conditions. The osteoblasts were launched in a serum deprived state, activated and collected in microgravity. Samples were collected at 29 hours after sera activation (0-g, n=4; 1-g, n=4). The osteoblasts were examined for changes in gene expression and cell morphology. Approximately one day after growth activation, remarkable differences were observed in gene expression in 0-g and 1-g flight samples. The 0-g activated cells had increased c-fos mRNA when compared to flight 1-g controls. The message of immediate early growth gene, cox-2 was decreased in the microgravity activated cells when compared to ground or 1-g flight controls. Cox-1 was not

  19. Human osteoblast response to PTFE surfaces.

    PubMed

    Walsh, W R; Olmedo, M; Kim, H D; Zou, L; Weiss, A P

    1994-01-01

    Recently, expanded polytetrafluoroethylene (ePTFE, Gortex) vascular grafts have been rolled and used for interpositional arthroplasties of the carpus in the wrist. Little data, however, are available on the response of human osteoblasts to ePTFE. In-vitro cell culture is a useful method to determine initial cell-biomaterial interactions. The present study explores the morphological and mitogenic response of human bone cells cultured on vascular grade ePTFE grafts. The present findings suggest that neither the inner nor the outer surface of ePTFE, in its present form, support osteoblast growth. PTFE may be a suitable material to act as a space filler for carpal bone interpositional arthroplasties. PMID:10150168

  20. Reduction in 50-kHz call-numbers and suppression of tickling-associated positive affective behaviour after lesioning of the lateral hypothalamic parvafox nucleus in rats.

    PubMed

    Roccaro-Waldmeyer, Diana M; Babalian, Alexandre; Müller, Annelies; Celio, Marco R

    2016-02-01

    The parvafox nucleus is located ventrolaterally in the lateral hypothalamic area (LHA). Its core and shell are composed of neurons expressing the calcium-binding protein parvalbumin (PV) and the transcription factor Foxb1, respectively. Given the known functions of the LHA and that the parvafox nucleus receives afferents from the lateral orbitofrontal cortex and projects to the periaqueductal gray matter, a functional role of this entity in the expression of positive emotions has been postulated. The purpose of the present study was to ascertain whether the deletion of neurons in the parvafox nucleus influenced the tickling-induced 50-kHz calls, which are thought to reflect positive affective states, in rats. To this end, tickling of the animals (heterospecific play) was combined with intracerebral injections of the excitotoxin kainic acid into the parvafox nucleus. The most pronounced surgery-associated reduction in 50-kHz call-numbers was observed in the group of rats in which, on the basis of PV-immunoreactive-cell counts in the parvafox nucleus, bilateral lesions had been successfully produced. Two other parameters that were implemented to quantify positive affective behaviour, namely, an approach towards and a following of the hand of the tickling experimenter, were likewise most markedly suppressed in the group of rats with bilaterally successful lesions. Furthermore, positive correlations were found between each of the investigated parameters. Our data afford evidence that the parvafox nucleus plays a role in the production of 50-kHz calls in rats, and, more generally, in the expression of positive emotions. PMID:26554726

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

    PubMed Central

    Wannenes, Francesca; Papa, Vincenza; Greco, Emanuela A.; Fornari, Rachele; Marocco, Chiara; Di Luigi, Luigi; Donini, Lorenzo M.; Lenzi, Andrea

    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 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. PMID:24963291

  2. Bone regenerates via dedifferentiation of osteoblasts in the zebrafish fin.

    PubMed

    Knopf, Franziska; Hammond, Christina; Chekuru, Avinash; Kurth, Thomas; Hans, Stefan; Weber, Christopher W; Mahatma, Gina; Fisher, Shannon; Brand, Michael; Schulte-Merker, Stefan; Weidinger, Gilbert

    2011-05-17

    While mammals have a limited capacity to repair bone defects, zebrafish can completely regenerate amputated bony structures of their fins. Fin regeneration is dependent on formation of a blastema, a progenitor cell pool accumulating at the amputation plane. It is unclear which cells the blastema is derived from, whether it forms by dedifferentiation of mature cells, and whether blastema cells are multipotent. We show that mature osteoblasts dedifferentiate and form part of the blastema. Osteoblasts downregulate expression of intermediate and late bone differentiation markers and induce genes expressed by bone progenitors. Dedifferentiated osteoblasts proliferate in a FGF-dependent manner and migrate to form part of the blastema. Genetic fate mapping shows that osteoblasts only give rise to osteoblasts in the regenerate, indicating that dedifferentiation is not associated with the attainment of multipotency. Thus, bone can regenerate from mature osteoblasts via dedifferentiation, a finding with potential implications for human bone repair. PMID:21571227

  3. Staphylococcus aureus vs. Osteoblast: Relationship and Consequences in Osteomyelitis.

    PubMed

    Josse, Jérôme; Velard, Frédéric; Gangloff, Sophie C

    2015-01-01

    Bone cells, namely osteoblasts and osteoclasts work in concert and are responsible for bone extracellular matrix formation and resorption. This homeostasis is, in part, altered during infections by Staphylococcus aureus through the induction of various responses from the osteoblasts. This includes the over-production of chemokines, cytokines and growth factors, thus suggesting a role for these cells in both innate and adaptive immunity. S. aureus decreases the activity and viability of osteoblasts, by induction of apoptosis-dependent and independent mechanisms. The tight relationship between osteoclasts and osteoblasts is also modulated by S. aureus infection. The present review provides a survey of the relevant literature discussing the important aspects of S. aureus and osteoblast interaction as well as the ability for antimicrobial peptides to kill intra-osteoblastic S. aureus, hence emphasizing the necessity for new anti-infectious therapeutics. PMID:26636047

  4. Staphylococcus aureus vs. Osteoblast: Relationship and Consequences in Osteomyelitis

    PubMed Central

    Josse, Jérôme; Velard, Frédéric; Gangloff, Sophie C.

    2015-01-01

    Bone cells, namely osteoblasts and osteoclasts work in concert and are responsible for bone extracellular matrix formation and resorption. This homeostasis is, in part, altered during infections by Staphylococcus aureus through the induction of various responses from the osteoblasts. This includes the over-production of chemokines, cytokines and growth factors, thus suggesting a role for these cells in both innate and adaptive immunity. S. aureus decreases the activity and viability of osteoblasts, by induction of apoptosis-dependent and independent mechanisms. The tight relationship between osteoclasts and osteoblasts is also modulated by S. aureus infection. The present review provides a survey of the relevant literature discussing the important aspects of S. aureus and osteoblast interaction as well as the ability for antimicrobial peptides to kill intra-osteoblastic S. aureus, hence emphasizing the necessity for new anti-infectious therapeutics. PMID:26636047

  5. Osteoblast differentiation is functionally associated with decreased AMP kinase activity.

    PubMed

    Kasai, Takayuki; Bandow, Kenjiro; Suzuki, Hiraku; Chiba, Norika; Kakimoto, Kyoko; Ohnishi, Tomokazu; Kawamoto, Shin-ichiro; Nagaoka, Eiichi; Matsuguchi, Tetsuya

    2009-12-01

    Osteoblasts, originating from mesenchymal stem cells, play a pivotal role in bone formation and mineralization. Several transcription factors including runt-related transcription factor 2 (Runx2) have been reported to be essential for osteoblast differentiation, whereas the cytoplasmic signal transduction pathways controlling the differentiation process have not been fully elucidated. AMP-activated protein kinase (AMPK) is a serine-threonine kinase generally regarded as a key regulator of cellular energy homeostasis, polarity, and division. Recent lines of evidence have indicated that the activity of the catalytic alpha subunit of AMPK is regulated through its phosphorylation by upstream AMPK kinases (AMPKKs) including LKB1. Here, we explored the role of AMPK in osteoblast differentiation using in vitro culture models. Phosphorylation of AMPKalpha was significantly decreased during osteoblastic differentiation in both primary osteoblasts and MC3T3-E1, a mouse osteoblastic cell line. Conversely, the terminal differentiation of primary osteoblasts and MC3T3-E1 cells, represented by matrix mineralization, was significantly inhibited by glucose restriction and stimulation with metformin, both of which are known activators of AMPK. Matrix mineralization of MC3T3-E1 cells was also inhibited by the forced expression of a constitutively active form of AMPKalpha. Metformin significantly inhibited gene expression of Runx2 along with osteoblast differentiation markers including osteocalcin (Ocn), bone sialo protein (Bsp), and osteopontin (Opn). Thus, our present data indicate that differentiation of osteoblasts is functionally associated with decreased AMPK activity. PMID:19725053

  6. Serotonin regulates osteoblast proliferation and function in vitro

    PubMed Central

    Dai, S.Q.; Yu, L.P.; Shi, X.; Wu, H.; Shao, P.; Yin, G.Y.; Wei, Y.Z.

    2014-01-01

    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-HT1A, 5-HT1B, 5-HT1D, 5-HT2A, 5-HT2B, and 5-HT2C) were found to exist in rat osteoblasts. Of these, 5-HT2A and 5-HT1B 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. PMID:25098615

  7. Enhanced and suppressed mineralization by acetoacetate and β-hydroxybutyrate in osteoblast cultures.

    PubMed

    Saito, Akihiro; Yoshimura, Kentaro; Miyamoto, Yoichi; Kaneko, Kotaro; Chikazu, Daichi; Yamamoto, Matsuo; Kamijo, Ryutaro

    2016-04-29

    It is known that diabetes aggravates alveolar bone loss associated with periodontitis. While insulin depletion increases the blood concentration of ketone bodies, i.e., acetoacetate and β-hydroxybutyrate, their roles in bone metabolism have not been much studied until today. We investigated the effects of acetoacetate and β-hydroxybutyrate on mineralization of extracellular matrix in cultures of mouse osteoblastic MC3T3-E1 cells and primary mouse osteoblasts in the presence and absence of bone morphogenetic protein-2. Acetoacetate potentiated alkaline phosphatase activity in MC3T3-E1 cells in a concentration-dependent manner, ranging from physiological to pathological concentrations (0.05-5 mmol/L). In contrast, β-hydroxybutyrate lowered it in the same experimental settings. Mineralization in cultures of these cells was also up-regulated by acetoacetate and down-regulated by β-hydroxybutyrate. Similar results were obtained in cultures of mouse primary osteoblasts. Neither alkaline phosphatase mRNA nor its protein expression in MC3T3-E1 cells was affected by acetoacetate or β-hydroxybutyrate, indicating that these ketone bodies control the enzyme activity of alkaline phosphatase in osteoblasts and hence their mineralization bi-directionally. Finally, either gene silencing of monocarboxylate transporter-1, a major transmembrate transporter for ketone bodies, nullified the effects of ketone bodies on alkaline phosphatase activity in MC3T3-E1 cells. Collectively, we found that ketone bodies bidirectionally modulates osteoblast functions, which suggests that ketone bodies are important endogenous factors that regulate bone metabolism in both physiological and pathological situations. PMID:27018251

  8. Sex dependent regulation of osteoblast response to implant surface properties by systemic hormones

    PubMed Central

    2010-01-01

    Background Osseointegration depends on the implant surface, bone quality and the local and systemic host environment, which can differ in male and female patients. This study was undertaken in order to determine if male and female cells respond differently to titanium surfaces that have micron-scale roughness and if interactions of calciotropic hormones [1α,25(OH)2D3 and 17β-oestradiol (E2)] and microstructured surfaces on osteoblasts are sex dependent. Methods Osteoblasts from 6-week old Sprague-Dawley rats were cultured on tissue culture polystyrene (TCPS) or on titanium (Ti) disks with two different surface topographies, a smooth pretreated (PT) surface and a coarse grit-blasted/acid-etched (SLA) surface, and treated with 1α,25(OH)2D3, E2, or E2 conjugated to bovine serum albumin (E2-BSA). Results Male and female cells responded similarly to Ti microstructure with respect to cell number and levels of osteocalcin, transforming growth factor-β1, osteoprotegerin and prostaglandin E2 in their conditioned media, exhibiting a more differentiated phenotype on SLA than on PT or TCPS. E2 and E2-BSA increased differentiation and local factor production, an effect that was microstructure dependent and found only in female osteoblasts. 1α,25(OH)2D3 increased osteoblast differentiation and local factor production in female and male cells, but the effect was more robust in male cells. Conclusions Male and female rat osteoblasts respond similarly to surface microstructure but exhibit sexual dimorphism in substrate-dependent responses to systemic hormones. Oestrogen affected only female cells while 1α,25(OH)2D3 had a greater effect on male cells. These results suggest that successful osseointegration in males and females may depend on the implant surface design and correct levels of calciotropic hormones. PMID:21208469

  9. Modelling the Factors that Affect Individuals' Utilisation of Online Learning Systems: An Empirical Study Combining the Task Technology Fit Model with the Theory of Planned Behaviour

    ERIC Educational Resources Information Center

    Yu, Tai-Kuei; Yu, Tai-Yi

    2010-01-01

    Understanding learners' behaviour, perceptions and influence in terms of learner performance is crucial to predict the use of electronic learning systems. By integrating the task-technology fit (TTF) model and the theory of planned behaviour (TPB), this paper investigates the online learning utilisation of Taiwanese students. This paper provides a…

  10. Osteoblast adhesion to orthopaedic implant alloys: effects of cell adhesion molecules and diamond-like carbon coating.

    PubMed

    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 < 0.05) by 24 hours. Coating the alloys with diamond-like carbon did not alter osteoblast adhesion, whereas fibronectin pretreatment increased cell binding 2.6-fold (p < 0.05). In contrast, fibrinogen, vitronectin, and laminin did not 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

  11. Histone Deacetylase Inhibition Promotes Osteoblast Maturation by Altering the Histone H4 Epigenome and Reduces Akt Phosphorylation*

    PubMed Central

    Dudakovic, Amel; Evans, Jared M.; Li, Ying; Middha, Sumit; McGee-Lawrence, Meghan E.; van Wijnen, Andre J.; Westendorf, Jennifer J.

    2013-01-01

    Bone has remarkable regenerative capacity, but this ability diminishes during aging. Histone deacetylase inhibitors (HDIs) promote terminal osteoblast differentiation and extracellular matrix production in culture. The epigenetic events altered by HDIs in osteoblasts may hold clues for the development of new anabolic treatments for osteoporosis and other conditions of low bone mass. To assess how HDIs affect the epigenome of committed osteoblasts, MC3T3 cells were treated with suberoylanilide hydroxamic acid (SAHA) and subjected to microarray gene expression profiling and high-throughput ChIP-Seq analysis. As expected, SAHA induced differentiation and matrix calcification of osteoblasts in vitro. ChIP-Seq analysis revealed that SAHA increased histone H4 acetylation genome-wide and in differentially regulated genes, except for the 500 bp upstream of transcriptional start sites. Pathway analysis indicated that SAHA increased the expression of insulin signaling modulators, including Slc9a3r1. SAHA decreased phosphorylation of insulin receptor β, Akt, and the Akt substrate FoxO1, resulting in FoxO1 stabilization. Thus, SAHA induces genome-wide H4 acetylation and modulates the insulin/Akt/FoxO1 signaling axis, whereas it promotes terminal osteoblast differentiation in vitro. PMID:23940046

  12. Osteoblast adhesion to orthopaedic implant alloys: Effects of cell adhesion molecules and diamond-like carbon coating

    SciTech Connect

    Kornu, R.; Kelly, M.A.; Smith, R.L.; Maloney, W.J.

    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 < 0.05) by 24 hours. Coating the alloys with diamond-like carbon did not alter osteoblast adhesion, whereas fibronectin pretreatment increased cell binding 2.6-fold (p < 0.05). In contrast, fibrinogen, vitronectin, and laminin did not 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. 40 refs., 4 figs.

  13. Socio-economic and behavioural factors affecting the prevalence of Ascaris infection in a low-country tea plantation in Sri Lanka.

    PubMed

    Gunawardena, G S A; Karunaweera, N D; Ismail, M M

    2004-09-01

    The identification of the factors that affect the prevalences of geohelminthiases should help to maximize the effectiveness of programmes for the control of these diseases. In the present study, the relationships between the prevalence and intensity of human infection with Ascaris and the availability of sanitary facilities, socio-economic status and personal health habits have been explored in Sri Lanka. The 176 subjects, who lived on a low-country tea plantation, were aged 2-50 years (median = 13 years) and were investigated between the July and December of 2000. When the prevalence and intensity of Ascaris infection were determined, using Kato-Katz smears, 50.0% of the subjects were found to be secreting the eggs of the parasite. Almost all (96.6%) of the subjects lived in terraces of one-room houses built by the plantation owners, and only 30.7% had access to a latrine. Most (90.3%) obtained their drinking water from common taps, and 48.8% boiled their drinking water. The subjects who only drank water that had been boiled and those who washed their hands before meals were relatively unlikely to be infected (P < 0.05 for each). In congested living conditions with poor sanitary facilities, the level of faecal contamination of the environment is invariably high. Even under these conditions, however, good hygiene and the boiling of all drinking water can reduce the risks of Ascaris infection. In the study setting and in similar environments, regular anthelmintic therapy, improvements in housing conditions and sanitary facilities, and health education, to promote risk-reducing patterns of behaviour, would all be beneficial. PMID:15324467

  14. Osteoblast response to biomimetically altered titanium surfaces.

    PubMed

    Nebe, J Barbara; Müller, Lenka; Lüthen, Frank; Ewald, Andrea; Bergemann, Claudia; Conforto, Egle; Müller, Frank A

    2008-11-01

    Bioinert titanium (Ti) materials are generally encapsulated by fibrous tissue after implantation into the living body. To improve the bone-bonding ability of Ti implants, we activated commercially pure titanium (cpTi) by a simple chemical pre-treatment in HCl and NaOH. Subsequently, we exposed the treated samples to simulated body fluid (SBF) for 2 (TiCT) and 14 days (TiHCA), respectively, to mimic the early stages of bone bonding and to investigate the in vitro response of osteoblasts on thus altered biomimetic surfaces. Sample surfaces were characterized by scanning electron microscopy, energy-dispersive X-ray analysis, cross-sectional transmission electron microscopy analyses, Fourier transform infrared and Raman spectroscopy. It was shown that the efflorescence consisting of sodium titanate that is present on pre-treated cpTi surfaces transformed to calcium titanate after 2 days in SBF. After 14 days in SBF a homogeneous biomimetic apatite layer precipitated. Human osteoblasts (MG-63) revealed a well spread morphology on both functionalized Ti surfaces. On TiCT, the gene expression of the differentiation proteins alkaline phosphatase (ALP) and bone sialo protein was increased after 2 days. On both TiCT and TiHCA, the collagen I and ALP expression on the protein level was enhanced at 7 and 14 days. The TiCT and the TiHCA surfaces reveal the tendency to increase the differentiated cell function of MG-63 osteoblasts. Thus, chemical pre-treatment of titanium seems to be a promising method to generate osteoconductive surfaces. PMID:18595788

  15. Proliferation, differentiation and apoptosis in connexin43-null osteoblasts

    NASA Technical Reports Server (NTRS)

    Furlan, F.; Lecanda, F.; Screen, J.; Civitelli, R.

    2001-01-01

    Osteoblasts are highly coupled by gap junctions formed primarily by connexin43 (Cx43). We have shown that interference with Cx43 expression or function disrupts transcriptional regulation of osteoblast genes, and that deletion of Cx43 in the mouse causes skeletal malformations, delayed mineralization, and osteoblast dysfunction. Here, we studied the mechanisms by which genetic deficiency of Cx43 alters osteoblast development. While cell proliferation rates were similar in osteoblastic cells derived from calvaria of Cx43-null and wild type mice, camptothecin-induced apoptosis was 3-fold higher in mutant compared to wild type osteoblasts. When grown in mineralizing medium, Cx43-null cells were able to produce mineralized matrix but it took one week longer to reach the same mineralization levels as in normal cells. Likewise, expression of alkaline phosphatase activity per cell--a marker of osteoblast differentiation--was maximal only 2 weeks later in Cx43-null relative to wild-type cells. These observations suggest that Cx43 is important for a normal and timely development of the osteoblastic phenotype. Delayed differentiation and increase programmed cell death may explain the skeletal phenotype of Cx43-null mice.

  16. Developmental Regulation of the Collagenase-3 Promoter in Osteoblasts

    NASA Technical Reports Server (NTRS)

    Partridge, N. C.; Yang, Y.; DAlonzo, R. C.; Winchester, S. K.

    1999-01-01

    Previously, we have shown that collagenase-3 MRNA is developmentally expressed in normal, differentiating rat osteoblasts. In vivo, the gene is expressed in a tissue-specific fashion in hypertrophic chondrocytes and osteoblasts and developmentally regulated. Our studies aim at determining the promoter elements and proteins binding to the promoter responsible for tissue and developmental regulation of collagenase-3.

  17. Signaling and transcriptional regulation in osteoblast commitment and differentiation

    PubMed Central

    Huang, Wei; Yang, Shuying; Shao, Jianzhong; Li, Yi-Ping

    2013-01-01

    The major event that triggers osteogenesis is the transition of mesenchymal stem cells into bone forming, differentiating osteoblast cells. Osteoblast differentiation is the primary component of bone formation, exemplified by the synthesis, deposition and mineralization of extracellular matrix. Although not well understood, osteoblast differentiation from mesenchymal stem cells is a well-orchestrated process. Recent advances in molecular and genetic studies using gene targeting in mouse enable a better understanding of the multiple factors and signaling networks that control the differentiation process at a molecular level. Osteoblast commitment and differentiation are controlled by complex activities involving signal transduction and transcriptional regulation of gene expression. We review Wnt signaling pathway and Runx2 regulation network, which are critical for osteoblast differentiation. Many other factors and signaling pathways have been implicated in regulation of osteoblast differentiation in a network manner, such as the factors Osterix, ATF4, and SATB2 and the TGF-beta, Hedgehog, FGF, ephrin, and sympathetic signaling pathways. This review summarizes the recent advances in the studies of signaling transduction pathways and transcriptional regulation of osteoblast cell lineage commitment and differentiation. The knowledge of osteoblast commitment and differentiation should be applied towards the development of new diagnostic and therapeutic alternatives for human bone diseases. PMID:17485283

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

    SciTech Connect

    Nifuji, Akira; Ideno, Hisashi; Ohyama, Yoshio; Takanabe, Rieko; Araki, Ryoko; Abe, Masumi; Noda, Masaki; Shibuya, Hiroshi

    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.

  19. Birth Origin Differentially Affects Depressive-Like Behaviours: Are Captive-Born Cynomolgus Monkeys More Vulnerable to Depression than Their Wild-Born Counterparts?

    PubMed Central

    Camus, Sandrine MJ.; Rochais, Céline; Blois-Heulin, Catherine; Li, Qin; Hausberger, Martine; Bezard, Erwan

    2013-01-01

    Background Adverse early-life experience might lead to the expression of abnormal behaviours in animals and the predisposition to psychiatric disorder (e.g. major depressive disorder) in Humans. Common breeding processes employ weaning and housing conditions different from what happens in the wild. Methods The present study, therefore, investigated whether birth origin impacts the possible existence of spontaneous atypical/abnormal behaviours displayed by 40 captive-born and 40 wild-born socially-housed cynomolgus macaques in farming conditions using an unbiased ethological scan-sampling analysis followed by multifactorial correspondence and hierarchical clustering analyses. Results We identified 10 distinct profiles (groups A to J) that significantly differed on several behaviours, body postures, body orientations, distances between individuals and locations in the cage. Data suggest that 4 captive-born and 1 wild-born animals (groups G and J) present depressive-like symptoms, unnatural early life events thereby increasing the risk of developing pathological symptoms. General differences were also highlighted between the captive- and wild-born populations, implying the expression of differential coping mechanisms in response to the same captive environment. Conclusions Birth origin thus impacts the development of atypical ethologically-defined behavioural profiles, reminiscent of certain depressive-like symptoms. The use of unbiased behavioural observations might allow the identification of animal models of human mental/behavioural disorders and their most appropriate control groups. PMID:23861787

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

    PubMed

    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

  1. Effect of fibronectin adsorption on osteoblastic cellular responses to hydroxyapatite and alumina.

    PubMed

    Kawashita, Masakazu; Hasegawa, Maki; Kudo, Tada-Aki; Kanetaka, Hiroyasu; Miyazaki, Toshiki; Hashimoto, Masami

    2016-12-01

    Initial cellular responses following implantation are important for inducing osteoconduction. We investigated cell adhesion, spreading, proliferation and differentiation of mouse MC3T3-E1 osteoblastic cells on untreated or fibronectin (Fn)-coated discs of hydroxyapatite (HAp) or alpha-type alumina (α-Al2O3). Fn coating significantly enhanced adhesion and spreading of MC3T3-E1 cells on HAp, but did not affect MC3T3-E1 cell proliferation and differentiation on HAp or α-Al2O3. Fn-coated HAp likely does not stimulate pre-osteoblast cells to initiate the process of osteoconduction; however, Fn adsorption might affect the response of inflammatory cells to the implanted material or, in conjunction with other serum proteins, stimulate pre-osteoblast cell proliferation and differentiation. Further studies on the effect of serum proteins in cell culture and the efficacy of Fn-coated HAp and α-Al2O3in vivo are warranted. PMID:27612826

  2. Physical vapour deposition of zirconia on titanium: fabrication, characterization and interaction with human osteoblast cells.

    PubMed

    Kaluđerović, Milena R; Mändl, Stephan; Kohlweyer, Hannes; Graf, Hans-Ludwig

    2015-12-01

    The physical vapor deposition of zirconia was used to prepare two new titanium-based surfaces M1 and M2 with a different layer thickness. These novel surfaces were characterized for chemistry, topography and morphology by surface and solid state techniques. Primary osteoblast cells were used for in vitro studies. DAPI assay was applied for cell proliferation, while for bone sialoprotein (BSP), osteonectin and transforming growth factor-β (TGF-β) expression immunohistochemical analyses were employed. Materials M1 and M2 affected cell proliferation accordingly to their surface roughness with their impact on cell number being between the impact of two rough (Ticer, SS) and two smooth surfaces (Ti cp and Cercon). Different influence of the investigated materials on the osteoblastic production of BSP (all materials similar impact), ON (Cercon-higher; SS-lower for others) and TGF-β (Cercon different) was found. PMID:26507200

  3. Differential responses of osteoblasts and macrophages upon Staphylococcus aureus infection

    PubMed Central

    2014-01-01

    Background Staphylococcus aureus (S. aureus) is one of the primary causes of bone infections which are often chronic and difficult to eradicate. Bacteria like S. aureus may survive upon internalization in cells and may be responsible for chronic and recurrent infections. In this study, we compared the responses of a phagocytic cell (i.e. macrophage) to a non-phagocytic cell (i.e. osteoblast) upon S. aureus internalization. Results We found that upon internalization, S. aureus could survive for up to 5 and 7 days within macrophages and osteoblasts, respectively. Significantly more S. aureus was internalized in macrophages compared to osteoblasts and a significantly higher (100 fold) level of live intracellular S. aureus was detected in macrophages compared to osteoblasts. However, the percentage of S. aureus survival after infection was significantly lower in macrophages compared to osteoblasts at post-infection days 1–6. Interestingly, macrophages had relatively lower viability in shorter infection time periods (i.e. 0.5-4 h; significant at 2 h) but higher viability in longer infection time periods (i.e. 6–8 h; significant at 8 h) compared to osteoblasts. In addition, S. aureus infection led to significant changes in reactive oxygen species production in both macrophages and osteoblasts. Moreover, infected osteoblasts had significantly lower alkaline phosphatase activity at post-infection day 7 and infected macrophages had higher phagocytosis activity compared to non-infected cells. Conclusions S. aureus was found to internalize and survive within osteoblasts and macrophages and led to differential responses between osteoblasts and macrophages. These findings may assist in evaluation of the pathogenesis of chronic and recurrent infections which may be related to the intracellular persistence of bacteria within host cells. PMID:25059520

  4. Hydrocarbon Deposition Attenuates Osteoblast Activity on Titanium

    PubMed Central

    Hayashi, R.; Ueno, T.; Migita, S.; Tsutsumi, Y.; Doi, H.; Ogawa, T.; Hanawa, T.; Wakabayashi, N.

    2014-01-01

    Although the reported percentage of bone-implant contact is far lower than 100%, the cause of such low levels of bone formation has rarely been investigated. This study tested the negative biological effect of hydrocarbon deposition onto titanium surfaces, which has been reported to be inevitable. Osteogenic MC3T3-E1 cells were cultured on titanium disks on which the carbon concentration was experimentally regulated to achieve carbon/titanium (C/Ti) ratios of 0.3, 0.7, and 1.0. Initial cellular activities such as cell attachment and cell spreading were concentration-dependently suppressed by the amount of carbon on the titanium surface. The osteoblastic functions of alkaline phosphatase activity and calcium mineralization were also reduced by more than 40% on the C/Ti (1.0) surface. These results indicate that osteoblast activity is influenced by the degree of hydrocarbon contamination on titanium implants and suggest that hydrocarbon decomposition before implant placement may increase the biocompatibility of titanium. PMID:24868012

  5. Effect of dual delivery of antibiotics (vancomycin and cefazolin) and BMP-7 from chitosan microparticles on Staphylococcus epidermidis and pre-osteoblasts in vitro.

    PubMed

    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

  6. Fabrication and in vivo evaluation of an osteoblast-conditioned nano-hydroxyapatite/gelatin composite scaffold for bone tissue regeneration.

    PubMed

    Samadikuchaksaraei, Ali; Gholipourmalekabadi, Mazaher; Erfani Ezadyar, Elham; Azami, Mahmoud; Mozafari, Masoud; Johari, Behrooz; Kargozar, Saeid; Jameie, Seyed Behnamedin; Korourian, Alireza; Seifalian, Alexander M

    2016-08-01

    In this study, the effects of osteoblast-conditioning on mechanical behavior, biocompatibility, biodegradation and osteoinductive properties of a nano-hydroxyapatite/gelatin (HA/GEL) nanocomposite scaffold was investigated. The scaffold was fabricated using the layer solvent casting combined with the freeze-drying and lamination techniques. The scaffolds were conditioned by culture of osteoblasts on their surface and their elimination by a repeated freeze-thawing process. The potential of the osteoblast-conditioned HA/GEL (HA/GEL/OC) scaffold to support cell adhesion and growth and its cytotoxicity was assessed in vitro using rat mesenchymal stem cells. For in vivo studies, the HA/GEL/OC nanocomposite was implanted in the critical size bone defect created on rat calvarium and studied after 7, 30 and 90 days. The results showed that mechanical and in vitro biological properties of the scaffold were not affected by the process of conditioning. However, in vivo studies demonstrated that osteoblast-conditioning enhanced biocompatibility and osteoinductivity and of the nanocomposite scaffold. The osteoblast conditioning also accelerated collagen content during the bone healing. In the experimental group that received the HA/GEL/OC and MSCs, the newly formed bone occupied almost the entire defect (93.4 ± 3.3%) within 3 months. In conclusion, this study indicates that osteoblast-conditioning is a viable strategy for the development of bone tissue engineering scaffolds. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2001-2010, 2016. PMID:27027855

  7. Impaired cell cycle regulation of osteoblast-related transcription factor Runx2/Cbfa1 in osteosarcoma cells

    PubMed Central

    San Martin, Inga; Varela, Nelson; Gaete, Marcia; Villegas, Karina; Osorio, Mariana; Tapia, Julio C.; Antonelli, Marcelo; Mancilla, Edna; Lian, Jane B.; Stein, Janet L.; Stein, Gary S; van Wijnen, Andre J.; Galindo, Mario

    2011-01-01

    In mammals, bone differentiation requires the functional expression of the Runx2/Cbfβ heterodimeric complex. Our previous results indicate that Runx2 is also a suppressor of pre-osteoblast proliferation by affecting cell cycle progression at G1. Runx2 levels are cell cycle regulated, oscillating from a maximum during early G1 to a minimum during late G1, S and mitosis phases in proliferating pre-osteoblasts Nevertheless, there is no information concerning Cbfβ gene expression during the cell cycle nor on Runx2 cell cycle expression in bone cancer cells. We analyzed Runx2 and Cbfβ gene expression during cell cycle progression in the pre-osteoblast MC3T3 and osteosarcoma ROS and SaOS cell lines. The expected reduction of Runx2 protein level was observed in MC3T3 cells arrested in late G1 or M phase using mimosine or nocodazole, respectively. However, this reduction was not observed in the cell cycle arrested osteosarcoma cells. Cbfβ protein levels were not regulated during the cell cycle in pre-osteoblasts and osteosarcoma cells. Using cells synchronized in late G1 and mitosis we found that Runx2 levels, but not Cbfβ levels, were cell cycle regulated in MC3T3 osteoblasts. Interestingly, both factors showed a constitutively elevated expression throughout the cell cycle in osteosarcoma cells. Proteasome inhibition by MG132 prevented cell cycle-dependent downregulation of Runx2 protein levels in osteoblasts, but not in osteosarcoma. We propose that Runx2 is involved in tumoral osteosarcoma progression. Altogether, deregulated Runx2 expression throughout the cell cycle seems to constitute a central mechanism in the pathogenesis of osteosarcoma. PMID:19739101

  8. Nanotopography Directs Mesenchymal Stem Cells to Osteoblast Lineage through Regulation of microRNA-SMAD-BMP-2 Circuit

    PubMed Central

    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

  9. Inorganic pyrophosphatase induces type I collagen in osteoblasts

    PubMed Central

    Polewski, Monika D.; Johnson, Kristen A.; Foster, Melissa; Millán, José Luis; Terkeltaub, Robert

    2009-01-01

    Introduction The physiologic selectivity of calcification in bone tissue reflects selective co-expression by osteoblasts of fibrillar collagen I and of tissue nonspecific alkaline phosphatase (TNAP), which hydrolyzes the calcification inhibitor pyrophosphate (PPi) and generates phosphate (Pi). Humans and mice deficient in the PPi-generating ecto-enzyme NPP1 demonstrate soft tissue calcification, occurring at sites of extracellular matrix expansion. Significantly, the function in osteoblasts of cytosolic inorganic pyrophosphatase (abbreviated iPPiase), which generates Pi via PPi hydrolysis with neutral pH optimum, remains unknown. We assessed iPPiase in Enpp1−/− and wild type (WT) mouse osteoblasts and we tested the hypothesis that iPPiase regulates collagen I expression. Methods We treated mouse calvarial osteoblasts with ascorbate and β-glycerol phosphate to promote calcification, and we assessed cytosolic Pi and PPi levels, sodium-dependent Pi uptake, Pit-1 Pi co-transporter expression, and iPPiase and TNAP activity and expression. We also assessed the function of transfected Ppa1 in osteoblasts. Results Inorganic pyrophosphatase but not TNAP was elevated in Enpp1−/− calvariae in situ. Cultured primary Enpp1−/− calvarial osteoblasts demonstrated increased calcification despite flat TNAP activity rather than physiologic TNAP up-regulation seen in WT osteoblasts. Despite decreased cytosolic PPi in early culture, Enpp1−/− osteoblasts maintained cytosolic Pi levels comparable to WT osteoblasts, in association with increased iPPiase, enhanced sodium-dependent Pi uptake and expression of Pit-1, and markedly increased collagen I synthesis. Suppression of collagen synthesis in Enpp1−/− osteoblasts using 3,4-dehydroproline markedly suppressed calcification. Last, transfection of Ppa1 in WT osteoblasts increased cytosolic Pi and decreased cytosolic but not extracellular PPi, and induced both collagen I synthesis and calcification. Conclusions Increased

  10. Thyrostimulin Regulates Osteoblastic Bone Formation During Early Skeletal Development.

    PubMed

    Bassett, J H Duncan; van der Spek, Anne; Logan, John G; Gogakos, Apostolos; Bagchi-Chakraborty, Jayashree; Murphy, Elaine; van Zeijl, Clementine; Down, Jenny; Croucher, Peter I; Boyde, Alan; Boelen, Anita; Williams, Graham R

    2015-09-01

    The ancestral glycoprotein hormone thyrostimulin is a heterodimer of unique glycoprotein hormone subunit alpha (GPA)2 and glycoprotein hormone subunit beta (GPB)5 subunits with high affinity for the TSH receptor. Transgenic overexpression of GPB5 in mice results in cranial abnormalities, but the role of thyrostimulin in bone remains unknown. We hypothesized that thyrostimulin exerts paracrine actions in bone and determined: 1) GPA2 and GPB5 expression in osteoblasts and osteoclasts, 2) the skeletal consequences of thyrostimulin deficiency in GPB5 knockout (KO) mice, and 3) osteoblast and osteoclast responses to thyrostimulin treatment. Gpa2 and Gpb5 expression was identified in the newborn skeleton but declined rapidly thereafter. GPA2 and GPB5 mRNAs were also expressed in primary osteoblasts and osteoclasts at varying concentrations. Juvenile thyrostimulin-deficient mice had increased bone volume and mineralization as a result of increased osteoblastic bone formation. However, thyrostimulin failed to induce a canonical cAMP response or activate the noncanonical Akt, ERK, or mitogen-activated protein kinase (P38) signaling pathways in primary calvarial or bone marrow stromal cell-derived osteoblasts. Furthermore, thyrostimulin did not directly inhibit osteoblast proliferation, differentiation or mineralization in vitro. These studies identify thyrostimulin as a negative but indirect regulator of osteoblastic bone formation during skeletal development. PMID:26018249

  11. The scent of stress: environmental challenge in the peripartum environment of mice affects emotional behaviours of the adult offspring in a sex-specific manner.

    PubMed

    Lerch, S; Dormann, C; Brandwein, C; Gass, P; Chourbaji, S

    2016-06-01

    Early adverse experiences are known to influence the risk of developing psychiatric disorders later. To shed further light on the development of laboratory mice, we systematically examined the influence of a prenatal or postnatal olfactory stressor, namely unfamiliar male mouse faeces, presented to pregnant or nursing mouse dams. Maternal and offspring behaviours were then examined. Maternal behaviours relative to controls revealed changes in nest building by the pregnant dams exposed to the unfamiliar faeces. There were no differences among groups on pup retrieval or exploration by the dams. Behavioural phenotyping of male and female offspring as adults included measures of exploration, anxiety, social and depressive-like behaviours. Additionally, serum corticosterone was assessed as a marker of physiological stress response. Group differences were dependent on the sex of the adult offspring. Males raised by dams that were stressed during pregnancy presented elevated emotionality as indicated by increased numbers of faecal boluses in the open field paradigm. Consistent with the effects of prenatal stress on the males only the prenatally stressed females had higher body weights than their respective controls. Indeed, males in both experimental groups had higher circulating corticosterone levels. By contrast, female offspring of dams exposed to the olfactory stressor after parturition were more anxious in the O-maze as indicated by increased latencies in entering the exposed areas of the maze. These findings emphasize the necessity for researchers to consider the pre- and postnatal environments, even of mice with almost identical genetic backgrounds, in designing experiments and interpreting their data. PMID:26408077

  12. Defective Multilayer Carbon Nanotubes Increase Alkaline Phosphatase Activity and Bone-Like Nodules in Osteoblast Cultures.

    PubMed

    Zancanela, Daniela Cervelle; Simaã, Ana Maria Sper; Matsubara, Elaine Yoshiko; Rosolen, José Maurício; Ciancaglini, Pietro

    2016-02-01

    Carbon nanotubes (CNT) is one of the most studied biomaterials, and issues about its cytotoxicity remain. The objective of our study was to investigate the in vitro influence of defective CNT on culture growth and on the formation of mineralized matrix nodules by primary osteoblastic cells grown in plastic or titanium (Ti) surfaces. Cellular viability, alkaline phosphatase activity and formation of mineral nodules were evaluated, besides the CNT characterization tests. The CNT studies showed better cell viability for osteoblasts incubated at stationary phase of culture in the presence of Ti (about 70%), but for the other phases, the cells suffered a significant reduction in viability. A peak of maximum alkaline phosphatase activity in the intermediate stage of growth (14 days of culture), which is characteristic for osteoblasts, was not affected, regardless of the presence of Ti or combination of CNT and Ti. Mineralized matrix nodules grew much more when the cells were incubated with CNT in the last 2 phases than when incubated in the first week, mainly when the cultures were grown on Ti discs. This study provides information for the application of CNT associated or not with Ti in processes of mineralization biostimulation. PMID:27433601

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

    PubMed Central

    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

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

    PubMed

    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

  15. Dihydroorotate dehydrogenase depletion hampers mitochondrial function and osteogenic differentiation in osteoblasts.

    PubMed

    Fang, JingXian; Yamaza, Haruyoshi; Uchiumi, Takeshi; Hoshino, Yoshihiro; Masuda, Keiji; Hirofuji, Yuta; Wagener, Frank A D T G; Kang, Dongchon; Nonaka, Kazuaki

    2016-06-01

    Mutation of the dihydroorotate dehydrogenase (DHODH) gene is responsible for Miller syndrome, which is characterized by craniofacial malformations with limb abnormalities. We previously demonstrated that DHODH was involved in forming a mitochondrial supercomplex and that mutated DHODH led to protein instability, loss of enzyme activity, and increased levels of reactive oxygen species in HeLa cells. To explore the etiology of Miller syndrome in more detail, we investigated the effects of DHODH inhibition in the cells involved in skeletal structure. Dihydroorotate dehydrogenase in MC3T3-E1 cells derived from mouse calvaria osteoblast precursor cells was knocked down by specific small interfering RNAs (siRNAs), and cell proliferation, ATP production, and expression of bone-related genes were investigated in these cells. After depletion of DHODH using specific siRNAs, inhibition of cell proliferation and cell cycle arrest occurred in MC3T3-E1 cells. In addition, ATP production was reduced in whole cells, especially in mitochondria. Furthermore, the levels of runt-related transcription factor 2 (Runx2) and osteocalcin (Ocn) mRNAs were lower in DHODH siRNA-treated cells compared with controls. These data suggest that depletion of DHODH affects the differentiation and maturation of osteoblasts. This study shows that mitochondrial dysfunction by DHODH depletion in osteoblasts can be directly linked to the abnormal bone formation in Miller syndrome. PMID:27086500

  16. Osteoblast Adhesion of Breast Cancer Cells with Scanning Acoustic Microscopy

    NASA Astrophysics Data System (ADS)

    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

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

    PubMed

    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

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

    PubMed

    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

  19. Bone formation by three-dimensional stromal osteoblast culture in biodegradable polymer scaffolds

    NASA Technical Reports Server (NTRS)

    Ishaug, S. L.; Crane, G. M.; Miller, M. J.; Yasko, A. W.; Yaszemski, M. J.; Mikos, A. G.; McIntire, L. V. (Principal Investigator)

    1997-01-01

    Bone formation was investigated in vitro by culturing stromal osteoblasts in three-dimensional (3-D), biodegradable poly(DL-lactic-co-glycolic acid) foams. Three polymer foam pore sizes, ranging from 150-300, 300-500, and 500-710 microns, and two different cell seeding densities, 6.83 x 10(5) cells/cm2 and 22.1 x 10(5) cells/cm2, were examined over a 56-day culture period. The polymer foams supported the proliferation of seeded osteoblasts as well as their differentiated function, as demonstrated by high alkaline phosphatase activity and deposition of a mineralized matrix by the cells. Cell number, alkaline phosphatase activity, and mineral deposition increased significantly over time for all the polymer foams. Osteoblast foam constructs created by seeding 6.83 x 10(5) cells/cm2 on foams with 300-500 microns pores resulted in a cell density of 4.63 x 10(5) cells/cm2 after 1 day in culture; they had alkaline phosphatase activities of 4.28 x 10(-7) and 2.91 x 10(-6) mumol/cell/min on Days 7 and 28, respectively; and they had a cell density that increased to 18.7 x 10(5) cells/cm2 by Day 56. For the same constructs, the mineralized matrix reached a maximum penetration depth of 240 microns from the top surface of the foam and a value of 0.083 mm for mineralized tissue volume per unit of cross sectional area. Seeding density was an important parameter for the constructs, but pore size over the range tested did not affect cell proliferation or function. This study suggests the feasibility of using poly(alpha-hydroxy ester) foams as scaffolding materials for the transplantation of autogenous osteoblasts to regenerate bone tissue.

  20. Cathepsin S controls adipocytic and osteoblastic differentiation, bone turnover, and bone microarchitecture.

    PubMed

    Rauner, M; Föger-Samwald, U; Kurz, M F; Brünner-Kubath, C; Schamall, D; Kapfenberger, A; Varga, P; Kudlacek, S; Wutzl, A; Höger, H; Zysset, P K; Shi, G P; Hofbauer, L C; Sipos, W; Pietschmann, P

    2014-07-01

    Cathepsin S is a cysteine protease that controls adipocyte differentiation and has been implicated in vascular and metabolic complications of obesity. Considering the inverse relation of osteoblasts and adipocytes and their mutual precursor cell, we hypothesized that cathepsin S may also affect osteoblast differentiation and bone remodeling. Thus, the fat and bone phenotypes of young (3 months old) and aged (12 or 18 months old) cathepsin S knock-out (KO) and wild-type (WT) mice were determined. Cathepsin S KO mice had a normal body weight at both ages investigated, even though the amount of subscapular and gonadal fat pads was reduced by 20%. Further, cathepsin S deficiency impaired adipocyte formation (-38%, p<0.001), which was accompanied by a lower expression of adipocyte-related genes and a reduction in serum leptin, IL-6 and CCL2 (p<0.001). Micro-CT analysis revealed an unchanged trabecular bone volume fraction and density, while tissue mineral density was significantly lower in cathepsin S KO mice at both ages. Aged KO mice further had a lower cortical bone mass (-2.3%, p<0.05). At the microarchitectural level, cathepsin S KO mice had thinner trabeculae (-8.3%), but a better connected trabecular network (+24%). Serum levels of the bone formation marker type 1 procollagen amino-terminal-propeptide and osteocalcin were both 2-3-fold higher in cathepsin S KO mice as was the mineralized surface. Consistently, osteogenic differentiation was increased 2-fold along with an increased expression of osteoblast-specific genes. Interestingly, serum levels of C-terminal telopeptide of type I collagen were also higher (+43%) in cathepsin S KO mice as were histological osteoclast parameters and ex vivo osteoclast differentiation. Thus, cathepsin S deficiency alters the balance between adipocyte and osteoblast differentiation, increases bone turnover, and changes bone microarchitecture. Therefore, bone and fat metabolisms should be monitored when using cathepsin S

  1. The effects of BIG-3 on osteoblast differentiation are not dependent upon endogenously produced BMPs

    SciTech Connect

    Gori, Francesca; Demay, Marie B. . E-mail: demay@helix.mgh.harvard.edu

    2005-03-10

    BMPs play an important role in both intramembranous and endochondral ossification. BIG-3, BMP-2-induced gene 3 kb, encodes a WD-40 repeat protein that accelerates the program of osteoblastic differentiation in vitro. To examine the potential interactions between BIG-3 and the BMP-2 pathway during osteoblastic differentiation, MC3T3-E1 cells stably transfected with BIG-3 (MC3T3E1-BIG-3), or with the empty vector (MC3T3E1-EV), were treated with noggin. Noggin treatment of pooled MC3T3E1-EV clones inhibited the differentiation-dependent increase in AP activity observed in the untreated MC3T3E1-EV clones but did not affect the increase in AP activity in the MC3T3E1-BIG-3 clones. Noggin treatment decreased the expression of Runx2 and type I collagen mRNAs and impaired mineralized matrix formation in MC3T3E1-EV clones but not in MC3T3E1-BIG-3 clones. To determine whether the actions of BIG-3 on osteoblast differentiation converged upon the BMP pathway or involved an alternate signaling pathway, Smad1 phosphorylation was examined. Basal phosphorylation of Smad1 was not altered in the MC3T3E1-BIG-3 clones. However, these clones did not exhibit the noggin-dependent decrease in phosphoSmad1 observed in the MC3T3E1-EV clones, nor did it decrease nuclear localization of phosphoSmad1. These observations suggest that BIG-3 accelerates osteoblast differentiation in MC3T3-E1 cells by inducing phosphorylation and nuclear translocation of Smad1 independently of endogenously produced BMPs.

  2. Suppression of osteoblast differentiation during weightlessness

    NASA Technical Reports Server (NTRS)

    Roberts, W. E.; Mozsary, P. G.; Morey, E. R.

    1982-01-01

    It is pointed out that associated with weightlessness is a marked depression or arrest of bone formation. Although the mechanism of this effect is unknown, it probably involves a failure of osteogenic induction. The present study's objective is to determine if weightlessness alters osteoblast differentiation, as evidenced by a change in relative distribution of large to small nuclei in rat moral periodontal ligament of the maxilla. In conjunction with the U.S./USSR Biological Satellite Program, male Wistar rats were flown aboard a modified Soviet Vostok spacecraft (Cosmos 1129). The results of the study are discussed. Morphometric investigations suggest that depleted numbers of preosteoblasts may be an important factor in the inhibition of bone formation during weightlessness.

  3. Osteoblast adhesion to clodronate-hydroxyapatite composite

    NASA Astrophysics Data System (ADS)

    Luo, E.; Liu, X.; Wei, S. C.; Cai, X. X.; Hu, J.

    2008-11-01

    The present study combined clodronate, one of bisphosphonates, to the surface of HA scaffold by chelation. The cell culture test and gene expression test were performed and evaluated the effect of clodronate modifying the HA on co-culturing osteoblast with HA scaffold in vitro. Characteristic peaks in XPS and FT-IR spectra indicated clodronate being immobilized on the surface of HA. The cell culture test indicated that the cells actively proliferated on the scaffolds. The results of RT-PCR showed there was no significant difference between two groups in expression of RANKL gene, while the expression of OPG gene showed higher expression in Clodronate-HA group. Clodronate had no obvious effect on the cytocompatibility of HA, and Clodronate-HA might be used as bone scaffold with potential ability to improve osteogenesis.

  4. Role of Integrin in Mechanical Loading of Osteoblasts

    NASA Technical Reports Server (NTRS)

    Globus, Ruth; Demsky, Caroline

    2000-01-01

    Mechanical forces generated by gravity, weightbearing, and muscle contraction play a key role in the genesis and maintenance of skeletal structure. The molecular mechanisms that mediate changes in osteoblast activity in response to altered patterns of skeletal loading are not known, and a better understanding of these processes may be essential for developing effective treatment strategies to prevent disuse osteoporosis. We have elucidated specific integrin/ECM (extracellular matrix) interactions that are required for osteoblast differentiation and survival and have developed a useful loading system to further explore the molecular basis of mechano-sensitivity of osteoblasts. The long term goal of our collaborative research is to understand how the ECM and cell adhesion proteins and integrins interaction to mediate the response of osteoblasts and their progenitors to mechanical loading. We suggest that integrin/ECM interactions are crucial for basic cellular processes, including differentiation and survival, as well as to participate in detecting and mediating cellular responses to mechanical stimuli.

  5. Vegfa regulates perichondrial vascularity and osteoblast differentiation in bone development

    PubMed Central

    Duan, Xuchen; Murata, Yurie; Liu, Yanqiu; Nicolae, Claudia; Olsen, Bjorn R.; Berendsen, Agnes D.

    2015-01-01

    ABSTRACT Vascular endothelial growth factor A (Vegfa) has important roles in endochondral bone formation. Osteoblast precursors, endothelial cells and osteoclasts migrate from perichondrium into primary ossification centers of cartilage templates of future bones in response to Vegfa secreted by (pre)hypertrophic chondrocytes. Perichondrial osteolineage cells also produce Vegfa, but its function is not well understood. By deleting Vegfa in osteolineage cells in vivo, we demonstrate that progenitor-derived Vegfa is required for blood vessel recruitment in perichondrium and the differentiation of osteoblast precursors in mice. Conditional deletion of Vegfa receptors indicates that Vegfa-dependent effects on osteoblast differentiation are mediated by Vegf receptor 2 (Vegfr2). In addition, Vegfa/Vegfr2 signaling stimulates the expression and activity of Indian hedgehog, increases the expression of β-catenin and inhibits Notch2. Our findings identify Vegfa as a regulator of perichondrial vascularity and osteoblast differentiation at early stages of bone development. PMID:25977369

  6. Vertically, interconnected carbon nanowalls as biocompatible scaffolds for osteoblast cells

    NASA Astrophysics Data System (ADS)

    Ion, Raluca; Vizireanu, Sorin; Luculescu, Catalin; Cimpean, Anisoara; Dinescu, Gheorghe

    2016-07-01

    The response of MC3T3-E1 pre-osteoblasts to vertically aligned, interconnected carbon nanowalls prepared by plasma enhanced chemical vapor deposition on silicon substrate has been evaluated in terms of cell adhesion, viability and cell proliferation. The behavior of osteoblasts seeded on carbon nanowalls was analyzed in parallel and compared with the behavior of the cells maintained in contact with tissue culture polystyrene (TCPS). The results demonstrate that osteoblasts adhere and remain viable in the long term on carbon nanowalls. Moreover, on the investigated scaffold cell proliferation was significantly promoted, although to a lower extent than on TCPS. Overall, the successful culture of osteoblasts on carbon nanowalls coated substrate confirms the biocompatibility of this scaffold, which could have potential applications in the development of orthopedic biomaterials.

  7. Could Dromedary Camels Develop Stereotypy? The First Description of Stereotypical Behaviour in Housed Male Dromedary Camels and How It Is Affected by Different Management Systems

    PubMed Central

    Padalino, Barbara; Aubé, Lydiane; Fatnassi, Meriem; Monaco, Davide; Khorchani, Touhami; Hammadi, Mohamed; Lacalandra, Giovanni Michele

    2014-01-01

    Dromedary camel husbandry has recently been evolving towards a semi-intensive system, due to the changes in use of the animal and the settlement of nomadic populations. Captivity could restrict its social activities, limiting the expression of various behavioural needs and causing the manifestation of stereotypy. The aims of this trial were, firstly, to identify and describe some stereotypical behaviours in captive male dromedary camels used for artificial insemination and, secondly, to study the effects on them of the following husbandry management systems: i) housing in single boxes for 24 hours (H24), ii) housing in single boxes for 23 hours with one hour free in the paddock (H23), and iii) housing in single boxes for 22 hours 30 min with 1 h of paddock time and 30 min exposure to a female camel herd (ExF). Every day, the camels were filmed in their single box in the morning for 30 minutes to record their behavioural activities and a focal animal sampling ethogram was filled in. In this study, male camels showed both oral and locomotor stereotypy most frequently when the bulls were reared in H24. Overall, this preliminary study is a starting point in the identification of stereotypies in male camels, reporting the positive effects of spending one hour outdoor and of social interaction with females. PMID:24586522

  8. Could dromedary camels develop stereotypy? The first description of stereotypical behaviour in housed male dromedary camels and how it is affected by different management systems.

    PubMed

    Padalino, Barbara; Aubé, Lydiane; Fatnassi, Meriem; Monaco, Davide; Khorchani, Touhami; Hammadi, Mohamed; Lacalandra, Giovanni Michele

    2014-01-01

    Dromedary camel husbandry has recently been evolving towards a semi-intensive system, due to the changes in use of the animal and the settlement of nomadic populations. Captivity could restrict its social activities, limiting the expression of various behavioural needs and causing the manifestation of stereotypy. The aims of this trial were, firstly, to identify and describe some stereotypical behaviours in captive male dromedary camels used for artificial insemination and, secondly, to study the effects on them of the following husbandry management systems: i) housing in single boxes for 24 hours (H24), ii) housing in single boxes for 23 hours with one hour free in the paddock (H23), and iii) housing in single boxes for 22 hours 30 min with 1 h of paddock time and 30 min exposure to a female camel herd (ExF). Every day, the camels were filmed in their single box in the morning for 30 minutes to record their behavioural activities and a focal animal sampling ethogram was filled in. In this study, male camels showed both oral and locomotor stereotypy most frequently when the bulls were reared in H24. Overall, this preliminary study is a starting point in the identification of stereotypies in male camels, reporting the positive effects of spending one hour outdoor and of social interaction with females. PMID:24586522

  9. Do umbilical outpouchings affect the behaviour or clinical condition of pigs during 6 h housing in a pre-transport pick-up facility?

    PubMed

    Schild, Sarah-Lina Aa; Rousing, Tine; Jensen, Henrik E; Barington, Kristiane; Herskin, Mette S

    2015-08-01

    This study focused on behavioural and clinical effects of umbilical outpouchings (UOs) in pigs. Matched pairs of pigs with UOs (diameter 12 cm; range 4-20; diagnosed p.m. as hernia or non-hernia) and controls (N=28) were compared during a 6-h stay in a pick-up facility. Overall, skin lesion scores were increased after the 6-h stay. Behaviour of the UO-pigs differed from the controls (a shorter latency to lie down (P<0.05) and decreased aggression (P<0.05)). Pigs with umbilical hernia showed e.g. increased sitting (P<0.05) and decreased lying (P<0.05) compared to pigs with non-hernia UOs. No effects of the size of the OUs were found. These results are among the first to establish knowledge about UO-pigs and suggest that a stay in a pick-up facility can be challenging for pig welfare. The behavioural findings suggest that UO-pigs, and especially pigs with hernia, may be less fit for mixing and housing in barren environments. PMID:26267102

  10. Effect of static magnetic fields on osteoblasts and fibroblasts in vitro.

    PubMed

    McDonald, F

    1993-01-01

    In vitro assays were made of the effect of a static magnetic field of a neodymium magnet on cellular behavior. The cell turnover rate was examined by the incorporation of radioactive thymidine, and anabolic processes were measured by the incorporation of radioactive proline. Cell cultures of fibroblast- and osteoblast-like cells of the neonatal rat calvarium were assayed to determine uptakes of radioactive thymidine and proline; these assays were performed in conjunction with examination of an explant of the rat calvarium. The cells were assayed after exposure to a field for 1-, 3-, 5-, 7-, and 10-day periods. Cells were exposed to north and south poles with a pole-face flux density of 0.61 T; control cultures were exposed to an unmagnetised piece of neodymium. After sham exposure or exposure to the magnetic field, 50 microCuries/ml of culture media of isotope were added to the culture medium. The cultures were returned to an incubator for 6 h. Then, following centrifugation, the supernatant was assayed for radioactivity in a scintillation counter after addition of 3 ml of scintillation fluid. A statistically significant magnetic stimulation of turnover rate and synthesis of fibroblasts was found, but stimulation of osteoblasts did not occur. Conversely, the explants, which represent the osteoblasts and fibroblasts in an organised system, showed a statistically significant inhibition in uptake of the radioactive label. The data indicate both variability and diversity of cellular behaviour, and they accentuate the need for caution in the interpretation of effects of static magnetic fields. PMID:8323569

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

    PubMed

    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

  12. In situ quantitative evaluation of osteoblastic collagen synthesis under cyclic strain by using second-harmonic-generation microscope

    NASA Astrophysics Data System (ADS)

    Matsubara, Oki; Hase, Eiji; Minamikawa, Takeo; Yasui, Takeshi; Sato, Katsuya

    2016-03-01

    Osteoblast-produced collagen matrix in bone is influenced by the mechanical stimulus from their surroundings. However, it has been still unclear how mechanical stimulus affects collagen production by osteoblasts. Therefore, it is strongly required to investigate the characteristics of osteoblastic bone regenerative tissue engineering. Recently, second-harmonic-generation (SHG) microscope has attracted attention for in situ visualization of collagen fiber because of less invasiveness, unstaining and no fixation, as well as high spatial resolution and 3D imaging. Using SHG microscopy, one can track the temporal dynamics of collagen fiber during the cultured period of the sample. We applied cyclic stretch strain to osteoblasts (MC3T3-E1) by using originally developed cell stretching device. The stimulation time was set to 5min or 3hours with same strain 5% and same frequency 0.5Hz. Cells were seeded onto the PDMS (polydimethylsiloxane) rubber chamber at a density of 50,000 cells/cm2 and cultured in α-MEM with 10% FBS, 1% P/S, 1% Ascorbic acid, 0.2% hydrocortisone and 2% β-Glycerophosphate. SHG imaging was carried out every 7 days. As a result, we confirmed from SHG image that the collagen production was enhanced by the cyclic stretch strain, stretch stimulation time and stretch application term.

  13. Polygonatum sibiricum polysaccharide inhibits osteoporosis by promoting osteoblast formation and blocking osteoclastogenesis through Wnt/β-catenin signalling pathway

    PubMed Central

    Du, Li; Nong, Meng-Ni; Zhao, Jin-Min; Peng, Xiao-Ming; Zong, Shao-Hui; Zeng, Gao-Feng

    2016-01-01

    Bone homeostasis is maintained by a balance between bone formation by osteoblasts and bone resorption by osteoclasts. Osteoporosis occurs when osteoclast activity surpasses osteoblast activity. Our previous studies showed the plant-derived natural polysaccharide (Polygonatum sibiricum polysaccharide or PSP) had significant anti-ovariectomy (OVX)-induced osteoporosis effects in vivo, but the mechanisms of PSP’s anti-osteoporosis effect remains unclear. In this study, we assessed PSP’s effect on the generation of osteoblast and osteoclast in vitro. This study showed that PSP promoted the osteogenic differentiation of mouse bone marrow stromal cells (BMSCs) without affecting BMPs signaling pathway. This effect was due to the increased nuclear accumulation of β-catenin, resulting in a higher expression of osteoblast-related genes. Furthermore, the study showed PSP could inhibit the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis and exert prophylatic protection against LPS-induced osteolysis in vivo. This effect was also related to the increased nuclear accumulation of β-catenin, resulting in the decreased expression of osteoclast-related genes. In conclusion, our results showed that PSP effectively promoted the osteogenic differentiation of mouse BMSCs and suppressed osteoclastogenesis; therefore, it could be used to treat osteoporosis. PMID:27554324

  14. Overexpression of the Transcriptional Factor Runx2 in Osteoblasts Abolishes the Anabolic Effect of Parathyroid Hormone in Vivo

    PubMed Central

    Merciris, Didier; Marty, Caroline; Collet, Corinne; de Vernejoul, Marie-Christine; Geoffroy, Valerie

    2007-01-01

    There is convincing evidence that Runx2 could be a regulator of the anabolic action of parathyroid hormone (PTH) in bone. We therefore decided to determine how Runx2 overexpression in osteoblasts affects the anabolic response to PTH. Transgenic osteoporotic female mice overexpressing Runx2 (TG) and their wild-type littermates (WT) were treated with PTH (100 μg/kg/day, 7 days a week) or with the vehicle for 6 weeks. Unexpectedly, Runx2 overexpression blunted the increase in the mineral density and volume of bone induced by intermittent PTH in WT mice. Our findings also indicate that PTH failed to increase bone formation in TG mice overexpressing Runx2. This abolition of the effect of PTH by Runx2 overexpression was attributable to a decrease in the differentiation of osteoblastic cells both in vivo and in vitro. Finally, we showed that less cAMP was induced by PTH and that there were fewer PTH binding sites in TG than WT osteoblasts. In conclusion, our findings demonstrate that in vivo a high level of Runx2 abolishes the anabolic effect of PTH, probably via a decrease in the sensitivity of TG osteoblasts to PTH, and that the level of expression of Runx2 is critical if PTH is to produce its anabolic effect on bone in vivo. PMID:17456773

  15. Osteoblast-released Matrix Vesicles, Regulation of Activity and Composition by Sulfated and Non-sulfated Glycosaminoglycans.

    PubMed

    Schmidt, Johannes R; Kliemt, Stefanie; Preissler, Carolin; Moeller, Stephanie; von Bergen, Martin; Hempel, Ute; Kalkhof, Stefan

    2016-02-01

    Our aging population has to deal with the increasing threat of age-related diseases that impair bone healing. One promising therapeutic approach involves the coating of implants with modified glycosaminoglycans (GAGs) that mimic the native bone environment and actively facilitate skeletogenesis. In previous studies, we reported that coatings containing GAGs, such as hyaluronic acid (HA) and its synthetically sulfated derivative (sHA1) as well as the naturally low-sulfated GAG chondroitin sulfate (CS1), reduce the activity of bone-resorbing osteoclasts, but they also induce functions of the bone-forming cells, the osteoblasts. However, it remained open whether GAGs influence the osteoblasts alone or whether they also directly affect the formation, composition, activity, and distribution of osteoblast-released matrix vesicles (MV), which are supposed to be the active machinery for bone formation. Here, we studied the molecular effects of sHA1, HA, and CS1 on MV activity and on the distribution of marker proteins. Furthermore, we used comparative proteomic methods to study the relative protein compositions of isolated MVs and MV-releasing osteoblasts. The MV proteome is much more strongly regulated by GAGs than the cellular proteome. GAGs, especially sHA1, were found to severely impact vesicle-extracellular matrix interaction and matrix vesicle activity, leading to stronger extracellular matrix formation and mineralization. This study shows that the regulation of MV activity is one important mode of action of GAGs and provides information on underlying molecular mechanisms. PMID:26598647

  16. Systems Genetic Analysis of Osteoblast-Lineage Cells

    PubMed Central

    Calabrese, Gina; Bennett, Brian J.; Orozco, Luz; Kang, Hyun M.; Eskin, Eleazar; Dombret, Carlos; De Backer, Olivier; Lusis, Aldons J.; Farber, Charles R.

    2012-01-01

    The osteoblast-lineage consists of cells at various stages of maturation that are essential for skeletal development, growth, and maintenance. Over the past decade, many of the signaling cascades that regulate this lineage have been elucidated; however, little is known of the networks that coordinate, modulate, and transmit these signals. Here, we identify a gene network specific to the osteoblast-lineage through the reconstruction of a bone co-expression network using microarray profiles collected on 96 Hybrid Mouse Diversity Panel (HMDP) inbred strains. Of the 21 modules that comprised the bone network, module 9 (M9) contained genes that were highly correlated with prototypical osteoblast maker genes and were more highly expressed in osteoblasts relative to other bone cells. In addition, the M9 contained many of the key genes that define the osteoblast-lineage, which together suggested that it was specific to this lineage. To use the M9 to identify novel osteoblast genes and highlight its biological relevance, we knocked-down the expression of its two most connected “hub” genes, Maged1 and Pard6g. Their perturbation altered both osteoblast proliferation and differentiation. Furthermore, we demonstrated the mice deficient in Maged1 had decreased bone mineral density (BMD). It was also discovered that a local expression quantitative trait locus (eQTL) regulating the Wnt signaling antagonist Sfrp1 was a key driver of the M9. We also show that the M9 is associated with BMD in the HMDP and is enriched for genes implicated in the regulation of human BMD through genome-wide association studies. In conclusion, we have identified a physiologically relevant gene network and used it to discover novel genes and regulatory mechanisms involved in the function of osteoblast-lineage cells. Our results highlight the power of harnessing natural genetic variation to generate co-expression networks that can be used to gain insight into the function of specific cell-types. PMID

  17. Time-lapse Raman imaging of osteoblast differentiation

    NASA Astrophysics Data System (ADS)

    Hashimoto, Aya; Yamaguchi, Yoshinori; Chiu, Liang-Da; Morimoto, Chiaki; Fujita, Katsumasa; Takedachi, Masahide; Kawata, Satoshi; Murakami, Shinya; Tamiya, Eiichi

    2015-07-01

    Osteoblastic mineralization occurs during the early stages of bone formation. During this mineralization, hydroxyapatite (HA), a major component of bone, is synthesized, generating hard tissue. Many of the mechanisms driving biomineralization remain unclear because the traditional biochemical assays used to investigate them are destructive techniques incompatible with viable cells. To determine the temporal changes in mineralization-related biomolecules at mineralization spots, we performed time-lapse Raman imaging of mouse osteoblasts at a subcellular resolution throughout the mineralization process. Raman imaging enabled us to analyze the dynamics of the related biomolecules at mineralization spots throughout the entire process of mineralization. Here, we stimulated KUSA-A1 cells to differentiate into osteoblasts and conducted time-lapse Raman imaging on them every 4 hours for 24 hours, beginning 5 days after the stimulation. The HA and cytochrome c Raman bands were used as markers for osteoblastic mineralization and apoptosis. From the Raman images successfully acquired throughout the mineralization process, we found that β-carotene acts as a biomarker that indicates the initiation of osteoblastic mineralization. A fluctuation of cytochrome c concentration, which indicates cell apoptosis, was also observed during mineralization. We expect time-lapse Raman imaging to help us to further elucidate osteoblastic mineralization mechanisms that have previously been unobservable.

  18. Time-lapse Raman imaging of osteoblast differentiation

    PubMed Central

    Hashimoto, Aya; Yamaguchi, Yoshinori; Chiu, Liang-da; Morimoto, Chiaki; Fujita, Katsumasa; Takedachi, Masahide; Kawata, Satoshi; Murakami, Shinya; Tamiya, Eiichi

    2015-01-01

    Osteoblastic mineralization occurs during the early stages of bone formation. During this mineralization, hydroxyapatite (HA), a major component of bone, is synthesized, generating hard tissue. Many of the mechanisms driving biomineralization remain unclear because the traditional biochemical assays used to investigate them are destructive techniques incompatible with viable cells. To determine the temporal changes in mineralization-related biomolecules at mineralization spots, we performed time-lapse Raman imaging of mouse osteoblasts at a subcellular resolution throughout the mineralization process. Raman imaging enabled us to analyze the dynamics of the related biomolecules at mineralization spots throughout the entire process of mineralization. Here, we stimulated KUSA-A1 cells to differentiate into osteoblasts and conducted time-lapse Raman imaging on them every 4 hours for 24 hours, beginning 5 days after the stimulation. The HA and cytochrome c Raman bands were used as markers for osteoblastic mineralization and apoptosis. From the Raman images successfully acquired throughout the mineralization process, we found that β-carotene acts as a biomarker that indicates the initiation of osteoblastic mineralization. A fluctuation of cytochrome c concentration, which indicates cell apoptosis, was also observed during mineralization. We expect time-lapse Raman imaging to help us to further elucidate osteoblastic mineralization mechanisms that have previously been unobservable. PMID:26211729

  19. Time-lapse Raman imaging of osteoblast differentiation.

    PubMed

    Hashimoto, Aya; Yamaguchi, Yoshinori; Chiu, Liang-da; Morimoto, Chiaki; Fujita, Katsumasa; Takedachi, Masahide; Kawata, Satoshi; Murakami, Shinya; Tamiya, Eiichi

    2015-01-01

    Osteoblastic mineralization occurs during the early stages of bone formation. During this mineralization, hydroxyapatite (HA), a major component of bone, is synthesized, generating hard tissue. Many of the mechanisms driving biomineralization remain unclear because the traditional biochemical assays used to investigate them are destructive techniques incompatible with viable cells. To determine the temporal changes in mineralization-related biomolecules at mineralization spots, we performed time-lapse Raman imaging of mouse osteoblasts at a subcellular resolution throughout the mineralization process. Raman imaging enabled us to analyze the dynamics of the related biomolecules at mineralization spots throughout the entire process of mineralization. Here, we stimulated KUSA-A1 cells to differentiate into osteoblasts and conducted time-lapse Raman imaging on them every 4 hours for 24 hours, beginning 5 days after the stimulation. The HA and cytochrome c Raman bands were used as markers for osteoblastic mineralization and apoptosis. From the Raman images successfully acquired throughout the mineralization process, we found that β-carotene acts as a biomarker that indicates the initiation of osteoblastic mineralization. A fluctuation of cytochrome c concentration, which indicates cell apoptosis, was also observed during mineralization. We expect time-lapse Raman imaging to help us to further elucidate osteoblastic mineralization mechanisms that have previously been unobservable. PMID:26211729

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

    PubMed

    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 (p<0.05). More importantly, we identified the preferential alignment of osteoblasts in the direction of the microcracks 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

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

    PubMed Central

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

    2014-01-01

    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 (p<0.05). More importantly, we identified the preferential alignment of osteoblasts in the direction of the microcracks 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 microscope 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

  2. Candida glabrata survives and replicates in human osteoblasts.

    PubMed

    Muñoz-Duarte, Ana Rosa; Castrejón-Jiménez, Nayeli Shantal; Baltierra-Uribe, Shantal Lizbeth; Pérez-Rangel, Sofia Judith; Carapia-Minero, Natalee; Castañeda-Sánchez, Jorge Ismael; Luna-Herrera, Julieta; López-Santiago, Rubén; Rodríguez-Tovar, Aída Verónica; García-Pérez, Blanca Estela

    2016-06-01

    Candida glabrata is an opportunistic pathogen that is considered the second most common cause of candidiasis after Candida albicans Many characteristics of its mechanisms of pathogenicity remain unknown. Recent studies have focused on determining the events that underlie interactions between C. glabrata and immune cells, but the relationship between this yeast and osteoblasts has not been studied in detail. The aim of this study was to determine the mechanisms of interaction between human osteoblasts and C. glabrata, and to identify the roles played by some of the molecules that are produced by these cells in response to infection. We show that C. glabrata adheres to and is internalized by human osteoblasts. Adhesion is independent of opsonization, and internalization depends on the rearrangement of the actin cytoskeleton. We show that C. glabrata survives and replicates in osteoblasts and that this intracellular behavior is related to the level of production of nitric oxide and reactive oxygen species. Opsonized C. glabrata stimulates the production of IL-6, IL-8 and MCP-1 cytokines. Adhesion and internalization of the pathogen and the innate immune response of osteoblasts require viable C. glabrata These results suggest that C. glabrata modulates immunological mechanisms in osteoblasts to survive inside the cell. PMID:27073253

  3. Spleen tyrosine kinase suppresses osteoblastic differentiation through MAPK and PKCα.

    PubMed

    Yoshida, Kiyoshi; Higuchi, Chikahisa; Nakura, Akio; Yoshikawa, Hideki

    2011-08-12

    Spleen tyrosine kinase (Syk) is a non-receptor protein kinase present in abundance in a wide range of hematopoietic cells. Syk reportedly plays a crucial role in immune signaling in B cells and cells bearing Fcγ-activation receptors. The role of syk in osteoblastic differentiation has not been well elucidated. We report herein the role of syk in osteoblastic differentiation. We investigated the effects of two syk inhibitors on osteoblastic differentiation in mouse preosteoblastic MC3T3-E1 cells and bone marrow stromal ST2 cells. Expression of syk was detected in these two cell lines. Two syk inhibitors stimulated mRNA expression of osteoblastic markers (ALP, Runx2, Osterix). Mineralization of extracellular matrix was also promoted by treatment with syk inhibitors. Knockdown of Syk caused increased mRNA expression of osteoblastic markers. In addition, syk inhibitor and knockdown of Syk suppressed phosphorylation of mitogen-activated protein kinase (MAPK) and protein kinase Cα (PKCα). Our results indicate that syk might regulate osteoblastic differentiation through MAPK and PKCα. PMID:21782794

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

    PubMed

    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

  5. Evolution of the osteoblast: skeletogenesis in gar and zebrafish

    PubMed Central

    2012-01-01

    Background Although the vertebrate skeleton arose in the sea 500 million years ago, our understanding of the molecular fingerprints of chondrocytes and osteoblasts may be biased because it is informed mainly by research on land animals. In fact, the molecular fingerprint of teleost osteoblasts differs in key ways from that of tetrapods, but we do not know the origin of these novel gene functions. They either arose as neofunctionalization events after the teleost genome duplication (TGD), or they represent preserved ancestral functions that pre-date the TGD. Here, we provide evolutionary perspective to the molecular fingerprints of skeletal cells and assess the role of genome duplication in generating novel gene functions. We compared the molecular fingerprints of skeletogenic cells in two ray-finned fish: zebrafish (Danio rerio)--a teleost--and the spotted gar (Lepisosteus oculatus)--a "living fossil" representative of a lineage that diverged from the teleost lineage prior to the TGD (i.e., the teleost sister group). We analyzed developing embryos for expression of the structural collagen genes col1a2, col2a1, col10a1, and col11a2 in well-formed cartilage and bone, and studied expression of skeletal regulators, including the transcription factor genes sox9 and runx2, during mesenchymal condensation. Results Results provided no evidence for the evolution of novel functions among gene duplicates in zebrafish compared to the gar outgroup, but our findings shed light on the evolution of the osteoblast. Zebrafish and gar chondrocytes both expressed col10a1 as they matured, but both species' osteoblasts also expressed col10a1, which tetrapod osteoblasts do not express. This novel finding, along with sox9 and col2a1 expression in developing osteoblasts of both zebrafish and gar, demonstrates that osteoblasts of both a teleost and a basally diverging ray-fin fish express components of the supposed chondrocyte molecular fingerprint. Conclusions Our surprising finding that

  6. Raspberry ketone promotes the differentiation of C3H10T1/2 stem cells into osteoblasts.

    PubMed

    Takata, Tomoyo; Morimoto, Chie

    2014-03-01

    The decrease in the bone mass associated with osteoporosis caused by ovariectomy, aging, and other conditions is accompanied by an increase in bone marrow adipose tissue. The balance between osteoblasts and adipocytes is influenced by a reciprocal relationship. The development of modalities to promote local/systemic bone formation by inhibiting bone marrow adipose tissue is important in the treatment of fractures or metabolic bone diseases such as osteoporosis. In this study, we examined whether raspberry ketone [4-(4-hydroxyphenyl)butan-2-one; RK], which is one of the major aromatic compounds of red raspberry and exhibits anti-obesity action, could promote osteoblast differentiation in C3H10T1/2 stem cells. Confluent C3H10T1/2 stem cells were treated for 6 days with 10-100 μg/mL of RK in culture medium containing 10 nM all-trans-retinoic acid (ATRA) or 300 ng/mL recombinant human bone morphogenetic protein (rhBMP)-2 protein as an osteoblast-differentiating agent. RK in the presence of ATRA increased alkaline phosphatase (ALP) activity in a dose-dependent manner. RK in the presence of rhBMP-2 also increased ALP activity. RK in the presence of ATRA also increased the levels of mRNAs of osteocalcin, α1(I) collagen, and TGF-βs (TGF-β1, TGF-β2, and TGF-β3) compared with ATRA only. RK promoted the differentiation of C3H10T1/2 stem cells into osteoblasts. However, RK did not affect the inhibition of early-stage adipocyte differentiation. Our results suggest that RK enhances the differentiation of C3H10T1/2 stem cells into osteoblasts, and it may promote bone formation by an action unrelated to adipocyte differentiation. PMID:24404978

  7. Palmitic Acid Reduces Circulating Bone Formation Markers in Obese Animals and Impairs Osteoblast Activity via C16-Ceramide Accumulation.

    PubMed

    Alsahli, Ahmad; Kiefhaber, Kathryn; Gold, Tziporah; Muluke, Munira; Jiang, Hongfeng; Cremers, Serge; Schulze-Späte, Ulrike

    2016-05-01

    Obesity and impaired lipid metabolism increase circulating and local fatty acid (FA) levels. Our previous studies showed that a high high-saturated -fat diet induced greater bone loss in mice than a high high-unsaturated-fat diet due to increased osteoclast numbers and activity. The impact of elevated FA levels on osteoblasts is not yet clear. We induced obesity in 4 week old male mice using a palmitic acid (PA)- or oleic acid (OA)-enriched high fat high-fat diet (HFD) (20 % of calories from FA), and compared them to mice on a normal (R) caloric diet (10 % of calories from FA). We collected serum to determine FA and bone metabolism marker levels. Primary osteoblasts were isolated; cultured in PA, OA, or control (C) medium; and assessed for mineralization activity, gene expression, and ceramide levels. Obese animals in the PA and OA groups had significantly lower serum levels of bone formation markers P1NP and OC compared to normal weight animals (*p < 0.001), with the lowest marker levels in animals on an PA-enriched HFD (*p < 0.001). Accordingly, elevated levels of PA significantly reduced osteoblast mineralization activity in vitro (*p < 0.05). Elevated PA intake significantly increased C16 ceramide accumulation. This accumulation was preventable through inhibition of SPT2 (serine palmitoyl transferase 2) using myriocin. Elevated levels of PA reduce osteoblast function in vitro and bone formation markers in vivo. Our findings suggest that saturated PA can compromise bone health by affecting osteoblasts, and identify a potential mechanism through which obesity promotes bone loss. PMID:26758875

  8. Runx2 Controls Bone Resorption through the Down-Regulation of the Wnt Pathway in Osteoblasts.

    PubMed

    Haxaire, Coline; Haÿ, Eric; Geoffroy, Valérie

    2016-06-01

    The transcription factor Runx2 and the Wnt/β-catenin pathway are major regulators of bone formation. Our aim was to assess the interactions between the Wnt/β-catenin pathway and Runx2 that contribute to bone resorption. Our results indicate that the activity of the canonical Wnt/β-catenin pathway depends on Runx2. Runx2 overexpression inhibited β-catenin levels and activity in vitro and in vivo. Inhibition of Gsk3b using lithium chloride in Runx2-overexpressing osteoporotic female mice rescued the Wnt/β-catenin signaling in vivo and completely restored trabecular bone volume by increasing bone formation and decreasing bone resorption. The activation of Wnt/β-catenin signaling by lithium chloride treatment reduced the number and activity of bone marrow-derived osteoclast-like cells in vitro, suggesting that the restoration of trabecular bone in vivo was due to decreased bone resorption, consistent with the reduced receptor activator of NF-κB ligand/osteoprotegerin ratio in Runx2-overexpressing osteoblasts. Lithium chloride also increased osteoblast differentiation and activity in vitro in agreement with the increase in mineral apposition rate and osteocalcin expression detected in vivo. Our results indicate that the activity of the canonical Wnt/β-catenin pathway in osteoblast is modulated by Runx2. To conclude, our in vivo and in vitro results highlight the role of Runx2 as a negative regulator of Wnt/β-catenin pathway activity in osteoblasts and indicate that the abnormal Wnt/β-catenin activity seen in Runx2 transgenic mice affects both osteoblast and osteoclast differentiation and activity. PMID:27083516

  9. A novel microRNA targeting HDAC5 regulates osteoblast differentiation in mice and contributes to primary osteoporosis in humans.

    PubMed

    Li, Hui; Xie, Hui; Liu, Wei; Hu, Rong; Huang, Bi; Tan, Yan-Fei; Xu, Kang; Sheng, Zhi-Feng; Zhou, Hou-De; Wu, Xian-Ping; Luo, Xiang-Hang

    2009-12-01

    MicroRNAs (miRNAs) interfere with translation of specific target mRNAs and are thought to thereby regulate many cellular processes. Recent studies have suggested that miRNAs might play a role in osteoblast differentiation and bone formation. Here, we identify a new miRNA (miR-2861) in primary mouse osteoblasts that promotes osteoblast differentiation by repressing histone deacetylase 5 (HDAC5) expression at the post-transcriptional level. miR-2861 was found to be transcribed in ST2 stromal cells during bone morphogenetic protein 2-induced (BMP2-induced) osteogenesis, and overexpression of miR-2861 enhanced BMP2-induced osteoblastogenesis, whereas inhibition of miR-2861 expression attenuated it. HDAC5, an enhancer of runt-related transcription factor 2 (Runx2) degradation, was confirmed to be a target of miR-2861. In vivo silencing of miR-2861 in mice reduced Runx2 protein expression, inhibited bone formation, and decreased bone mass. Importantly, miR-2861 was found to be conserved in humans, and a homozygous mutation in pre-miR-2861 that blocked expression of miR-2861 was shown to cause primary osteoporosis in 2 related adolescents. Consistent with the mouse data, HDAC5 levels were increased and Runx2 levels decreased in bone samples from the 2 affected individuals. Thus, our studies show that miR-2861 plays an important physiological role in osteoblast differentiation and contributes to osteoporosis via its effect on osteoblasts. PMID:19920351

  10. Soy protein isolates prevent loss of bone quantity associated with obesity in rats through regulation of insulin signaling in osteoblasts.

    PubMed

    Chen, Jin-Ran; Zhang, Jian; Lazarenko, Oxana P; Cao, Jay J; Blackburn, Michael L; Badger, Thomas M; Ronis, Martin J J

    2013-09-01

    In both rodents and humans, excessive consumption of a typical Western diet high in saturated fats and cholesterol is known to result in disruption of energy metabolism and development of obesity and insulin resistance. However, how these high-fat, energy-dense diets affect bone development, morphology, and modeling is poorly understood. Here we show that male weanling rats fed a high-fat (HF) diet containing 45% fat and 0.5% cholesterol made with casein (HF-Cas) for 6 wk displayed a significant increase in bone marrow adiposity and insulin resistance. Substitution of casein with soy protein isolate (SPI) in the HF diet (HF-SPI) prevented these effects. Maintenance of bone quantity in the SPI-fed rats was associated with increased undercarboxylated osteocalcin secretion and altered JNK/IRS1/Akt insulin signaling in osteoblasts. The HF-Cas group had significantly greater serum nonesterified free fatty acid (NEFA) concentrations than controls, whereas the HF-SPI prevented this increase. In vitro treatment of osteoblasts or mesenchymal stromal ST2 cells with NEFAs significantly decreased insulin signaling. An isoflavone mixture similar to that found in serum of HF-SPI rats significantly increased in vitro osteoblast proliferation and blocked significantly reduced NEFA-induced insulin resistance. Finally, insulin/IGF1 was able to increase both osteoblast activity and differentiation in a set of in vitro studies. These results suggest that high-fat feeding may disrupt bone development and modeling; high concentrations of NEFAs and insulin resistance occurring with high fat intake are mediators of reduced osteoblast activity and differentiation; diets high in soy protein may help prevent high dietary fat-induced bone impairments; and the molecular mechanisms underlying the SPI-protective effects involve isoflavone-induced normalization of insulin signaling in bone. PMID:23776073

  11. Transglutaminase activity arising from Factor XIIIA is required for stabilization and conversion of plasma fibronectin into matrix in osteoblast cultures.

    PubMed

    Cui, Cui; Wang, Shuai; Myneni, Vamsee D; Hitomi, Kiyotaka; Kaartinen, Mari T

    2014-02-01

    Circulating plasma fibronectin (pFN), produced by hepatocytes, is a major component of the noncollagenous bone matrix where it was recently shown in vivo in mice to control the biomechanical quality as well as the mineral-to-matrix ratio in bone. FN fibrillogenesis is a process generally requiring FN binding to cellular integrins, and cellular tension to elongate and assemble the molecule. Whether soluble pFN undergoes cell-mediated assembly in bone is not fully established. FN is a well-known substrate for transglutaminases (TGs), which are protein-crosslinking enzymes capable of stabilizing macromolecular structures. The role of this modification regarding the function of FN in bone matrix has remained unknown. Osteoblasts express two TGs-transglutaminase 2 and Factor XIIIA-and we have shown that Factor XIIIA is the main TG active during osteoblast differentiation. In the present study, conducted using MC3T3-E1 osteoblast cultures and bone marrow stromal cells, we demonstrate that pFN requires a TG-mediated crosslinking step to form osteoblast matrix in vitro. This modification step is specific for pFN; cellular FN (EDA-FN) does not serve as a TG substrate. Inhibition of pFN assembly using a TG inhibitor, or depletion of pFN from cell culture serum, dramatically decreased total FN matrix assembly in the osteoblast cultures and affected both the quantity and quality of the type I collagen matrix, and decreased lysyl oxidase and alkaline phosphatase levels, resulting in decreased mineralization. Experiments with isozyme-specific substrate peptides showed that FXIIIA is responsible for the crosslinking of pFN. Addition of exogenous preactivated FXIIIA to osteoblast cultures promoted pFN assembly from the media into matrix. Exogenous TG2 had no effect. Analysis of pFN and EDA-FN fibrils by immunofluorescence microscopy demonstrated that they form distinct matrix network, albeit with minor overlap, suggesting different functions for the two FN forms. Further analysis

  12. Nacre extract restores the mineralization capacity of subchondral osteoarthritis osteoblasts.

    PubMed

    Brion, A; Zhang, G; Dossot, M; Moby, V; Dumas, D; Hupont, S; Piet, M H; Bianchi, A; Mainard, D; Galois, L; Gillet, P; Rousseau, M

    2015-12-01

    Osteoarthritis (OA) is the most common cause of joint chronic pain and involves the entire joints. Subchondral osteoarthritic osteoblasts present a mineralization defect and, to date, only a few molecules (Vitamin D3 and Bone Morphogenetic Protein2) could improve the mineralization potential of this cell type. In this context, we have tested for the first time the effect of nacre extract on the mineralization capacity of osteoblasts from OA patients. Nacre extract is known to contain osteogenic molecules which have demonstrated their activities notably on the MC3T3 pre-osteoblastic cell line. For this goal, molecules were extracted from nacre (ESM, Ethanol Soluble Matrix) and tested on osteoblasts of the subchondral bone from OA patients undergoing total knee replacement and on MC3T3 cells for comparison. We chose to investigate the mineralization with Alizarin Red staining and with the study of extracellular matrix (ECM) structure and composition. In a complementary way the structure of the ECM secreted during the mineralization phase was investigated using second harmonic generation (SHG). Nacre extract was able to induce the early presence (after 7 days) of precipitated calcium in cells. Raman spectroscopy and electron microscopy showed the presence of nanograins of an early crystalline form of calcium phosphate in OA osteoblasts ECM and hydroxyapatite in MC3T3 ECM. SHG collagen fibers signal was present in both cell types but lower for OA osteoblasts. In conclusion, nacre extract was able to rapidly restore the mineralization capacity of osteoarthritis osteoblasts, therefore confirming the potential of nacre as a source of osteogenic compounds. PMID:26496825

  13. Osteoblast hydraulic conductivity is regulated by calcitonin and parathyroid hormone

    NASA Technical Reports Server (NTRS)

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

    1996-01-01

    It is our hypothesis that osteoblasts play a major role in regulating bone (re)modeling by regulating interstitial fluid (ISF) flow through individual bone compartments. We hypothesize that osteoblasts of the blood-bone membrane lining the bone surfaces are capable of regulating transosseous fluid flow. This regulatory function of the osteoblasts was tested in vitro by culturing a layer of rat calvarial osteoblasts on porous membranes. Such a layer of osteoblasts subjected to 7.3 mm Hg of hydrostatic pressure posed a significant resistance to fluid flow across the cell layer similar in magnitude to the resistance posed by endothelial monolayers in vitro. The hydraulic conductivity, the volumetric fluid flux per unit pressure drop, of the osteoblast layer was altered in response to certain hormones. Hydraulic conductivity decreased approximately 40% in response to 33 nM parathyroid hormone, while it exhibited biphasic behavior in response to calcitonin: increased 40% in response to 100 nM calcitonin and decreased 40% in response to 1000 nM calcitonin. Further, activation of adenylate cyclase by forskolin dramatically increased the hydraulic conductivity, while elevation of intracellular calcium, [Ca2+]i, by the calcium ionophore A23187 initially decreased the hydraulic conductivity at 5 minutes before increasing conductivity by 30 minutes. These results suggest that cyclic adenosine monophosphate (cAMP) and [Ca2+]i may mediate changes in the osteoblast hydraulic conductivity. The increase in hydraulic conductivity in response to 100 nM calcitonin and the decrease in response to PTH suggest that the stimulatory and inhibitory effects on bone formation of calcitonin and parathyroid hormone, respectively, may be due in part to alterations in bone fluid flow.

  14. Effect of recombinant human bone morphogenetic protein-2 on bisphosphonate-treated osteoblasts

    PubMed Central

    Kwon, Taek-Kyun; Song, Jae-Min; Kim, In-Ryoung; Park, Bong-Soo; Kim, Chul-Hoon; Cheong, In-Kyo

    2014-01-01

    Objectives Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a side effect of bisphophonate therapy that has been reported in recent years. Osteoclastic inactivity by bisphosphonate is the known cause of BRONJ. Bone morphogenetic protein-2 (BMP-2) plays an important role in the development of bone. Recombinant human BMP-2 (rhBMP-2) is potentially useful as an activation factor for bone repair. We hypothesized that rhBMP-2 would enhance the osteoclast-osteoblast interaction related to bone remodeling. Materials and Methods Human fetal osteoblast cells (hFOB 1.19) were treated with 100 µM alendronate, and 100 ng/mL rhBMP-2 was added. Cells were incubated for a further 48 hours, and cell viability was measured using an MTT assay. Expression of the three cytokines from osteoblasts, receptor activator of nuclear factor-κB ligand (RANKL), osteoprotegerin (OPG), and macrophage colony-stimulating factor (M-CSF), were analyzed by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Results Cell viability was decreased to 82.75%±1.00% by alendronate and then increased to 110.43%±1.35% after treatment with rhBMP-2 (P<0.05, respectively). OPG, RANKL, and M-CSF expression were all decreased by alendronate treatment. RANKL and M-CSF expression were increased, but OPG was not significantly affected by rhBMP-2. Conclusion rhBMP2 does not affect OPG gene expression in hFOB, but it may increase RANKL and M-CSF gene expression. PMID:25551094

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

    PubMed

    Nishide, Yoriko; Tousen, Yuko; Tadaishi, Miki; Inada, Masaki; Miyaura, Chisato; Kruger, Marlena C; Ishimi, Yoshiko

    2015-11-01

    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. PMID:26516892

  16. Cbfa1-independent decrease in osteoblast proliferation, osteopenia, and persistent embryonic eye vascularization in mice deficient in Lrp5, a Wnt coreceptor

    PubMed Central

    Kato, Masaki; Patel, Millan S.; Levasseur, Regis; Lobov, Ivan; Chang, Benny H.-J.; Glass, Donald A.; Hartmann, Christine; Li, Lan; Hwang, Tae-Ho; Brayton, Cory F.; Lang, Richard A.; Karsenty, Gerard; Chan, Lawrence

    2002-01-01

    The low-density lipoprotein receptor–related protein (Lrp)-5 functions as a Wnt coreceptor. Here we show that mice with a targeted disruption of Lrp5 develop a low bone mass phenotype. In vivo and in vitro analyses indicate that this phenotype becomes evident postnatally, and demonstrate that it is secondary to decreased osteoblast proliferation and function in a Cbfa1-independent manner. Lrp5 is expressed in osteoblasts and is required for optimal Wnt signaling in osteoblasts. In addition, Lrp5-deficient mice display persistent embryonic eye vascularization due to a failure of macrophage-induced endothelial cell apoptosis. These results implicate Wnt proteins in the postnatal control of vascular regression and bone formation, two functions affected in many diseases. Moreover, these features recapitulate human osteoporosis-pseudoglioma syndrome, caused by LRP5 inactivation. PMID:11956231

  17. Quantitative assessment of the response of osteoblast- and macrophage-like cells to particles of Ni-free Fe-base alloys.

    PubMed

    Ciapetti, G; González-Carrasco, J L; Savarino, L; Montealegre, M A; Pagani, S; Baldini, N

    2005-03-01

    In the present study, the effect of mechanically alloyed particles of new FeAlCr alloys developed for potential applications as surgical implants has been tested on osteoblast- and macrophage-like cells and compared to particles of the Ti6Al4V alloy, for which there is a good clinical experience. After microstructural characterisation of the particles, cells were cultured with particles for 24-48 h using three different concentrations of particles, and the response of cells was quantified by assessment of viability, proliferation, and morphology. Mineralisation by osteoblasts was verified after 21 days. The amount of aluminium and chromium ions in the culture medium of macrophages was measured by graphite furnace atomic absorption and phagocytosis of particles assessed by light microscopy. Viability and proliferation of osteoblast- and macrophage-like cells were substantially unaffected by the presence of particles of the new alloys, which were phagocytosed according to their size. Aluminium and chromium ions were released in the culture medium, but no direct correlation with the cell behaviour was found. In vitro mineralisation was achieved by osteoblasts in due time. The new alloys are well tolerated in in vitro systems, and, due to their chemical and mechanical characteristics, they are under development for surgical implants. PMID:15353196

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

    NASA Technical Reports Server (NTRS)

    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.

  19. Repercussions of NSAIDS drugs on bone tissue: the osteoblast.

    PubMed

    García-Martínez, O; De Luna-Bertos, E; Ramos-Torrecillas, J; Manzano-Moreno, F J; Ruiz, C

    2015-02-15

    Non-steroidal anti-inflammatory drugs (NSAIDs) can act by modulating the behavior of osteoblasts, including their proliferation, differentiation, adhesion, and migration, but not all NSAIDs have these effects. Our objective was to update the information on this issue in a review of the literature in order to offer guidance on the prescription of the appropriate NSAID(s) to patients requiring bone tissue repair. To review current knowledge of this issue by searching for all relevant publications since 2001 in the MEDLINE, EMBASE and Cochrane Library databases, we used the following descriptors: bone tissue, osteoblast, NSAIDs, Anti-inflammatory drugs. Published studies show that most NSAIDs have an adverse effect on osteoblast growth by cell cycle arrest and apoptosis induction. The effect on differentiation varies according to the drug, dose, and treatment time. Osteoblast adhesion is increased and migration decreased by some NSAIDs, such as indomethacin and diclofenac. The antigenic profile or phagocytic function can also be modulated by NSAIDs. In general, NSAIDs have an adverse effect on bone tissue and given the routine administration of NSAIDs to individuals requiring bone repair, in which the osteoblast has an essential role, this effect on bone should be borne in mind. PMID:25625244

  20. Nanobioengineered electrospun composite nanofibers and osteoblasts for bone regeneration.

    PubMed

    Venugopal, Jayarama Reddy; Low, Sharon; Choon, Aw Tar; Kumar, A Bharath; Ramakrishna, Seeram

    2008-05-01

    Bone defects represent a medical and socioeconomic challenge. Engineering bioartificial bone tissues may help to solve problems related to donor site morbidity and size limitations. Nanofibrous scaffolds were electrospun into a blend of synthetic biodegradable polycaprolactone (PCL) with hydroxyapatite (HA) and natural polymer gelatin (Gel) at a ratio of 1:1:2 (PCL/HA/Gel) compared to PCL (9%), PCL/HA (1:1), and PCL/Gel (1:2) nanofibers. These fiber diameters were around 411 +/- 158 to 856 +/- 157 nm, and the pore size and porosity around 5-35 microm and 76-93%, respectively. The interconnecting porous structure of the nanofibrous scaffolds provides large surface area for cell attachment and sufficient space for nutrient transportation. The tensile property of composite nanofibrous scaffold (PCL/HA/Gel) was highly flexible and allows penetrating osteoblasts inside the scaffolds for bone tissue regeneration. Fourier transform infrared analysis showed that the composite nanofiber contains an amino group, a phosphate group, and carboxyl groups for inducing proliferation and mineralization of osteoblasts for in vitro bone formation. The cell proliferation (88%), alkaline phosphatase activity (77%), and mineralization (66%) of osteoblasts were significantly (P < 0.001) increased in composite nanofibrous scaffold compared to PCL nanofibrous scaffolds. Field emission scanning electron microscopic images showed that the composite nanofibers supported the proliferation and mineralization of osteoblast cells. These results show that the fabrication of electrospun PCL/HA/Gel composite nanofibrous scaffolds has potential for the proliferation and mineralization of osteoblasts for bone regeneration. PMID:18471168

  1. Effects of dissolved calcium and phosphorous on osteoblast responses.

    PubMed

    Ma, S; Yang, Y; Carnes, D L; Kim, K; Park, S; Oh, S H; Ong, J L

    2005-01-01

    The dissolution behavior of hydroxyapatite (HA) and its effect on the initial cellular response is of both fundamental and clinical importance. In this study, plasma-sprayed HA coatings were characterized by X-ray diffraction and Fourier transform infrared spectroscopy (FTIR). Calcium (Ca) and inorganic phosphorous (Pi) ions released from plasma-sprayed HA coatings within 3 weeks were measured by flame atomic absorption and colorimetrically molybdenum blue complex, respectively. To investigate the effect of dissolution of HA coatings on osteoblast response, additional Ca and Pi were added into the cell culture media to simulate the dissolution concentrations. Human embryonic palatal mesenchyme cells, an osteoblast precursor cell line, were used to evaluate the biological responses to enhanced Ca and Pi media over 2 weeks. Osteoblast differentiation and mineralization were measured by alkaline phosphatase-specific assay and 1,25 (OH)2 vitamin D3 stimulated osteocalcin production. The coatings exhibited an HA-type structure. FTIR indicated the possible presence of carbonates on the coatings. A dissolution study indicated a continual increase in Ca and Pi over time. In the cell culture study, enhanced osteoblast differentiation occurred in the presence of additional Ca concentration in the cell culture media. However, additional Pi concentration in the cell culture media was suggested to slow down osteoblast differentiation and mineralization. PMID:15871524

  2. Regulation of CYP27B1 mRNA Expression in Primary Human Osteoblasts.

    PubMed

    van der Meijden, K; van Essen, H W; Bloemers, F W; Schulten, E A J M; Lips, P; Bravenboer, N

    2016-08-01

    The enzyme 1α-hydroxylase (gene CYP27B1) catalyzes the synthesis of 1,25(OH)2D in both renal and bone cells. While renal 1α-hydroxylase is tightly regulated by hormones and 1,25(OH)2D itself, the regulation of 1α-hydroxylase in bone cells is poorly understood. The aim of this study was to investigate in a primary human osteoblast culture whether parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), calcitonin, calcium, phosphate, or MEPE affect mRNA levels of CYP27B1. Our results show that primary human osteoblasts in the presence of high calcium concentrations increase their CYP27B1 mRNA levels by 1.3-fold. CYP27B1 mRNA levels were not affected by PTH1-34, rhFGF23, calcitonin, phosphate, and rhMEPE. Our results suggest that the regulation of bone 1α-hydroxylase is different from renal 1α-hydroxylase. High calcium concentrations in bone may result in an increased local synthesis of 1,25(OH)2D leading to an enhanced matrix mineralization. In this way, the local synthesis of 1,25(OH)2D may contribute to the stimulatory effect of calcium on matrix mineralization. PMID:27016371

  3. Exposure to mobile phone electromagnetic field radiation, ringtone and vibration affects anxiety-like behaviour and oxidative stress biomarkers in albino wistar rats.

    PubMed

    Shehu, Abubakar; Mohammed, Aliyu; Magaji, Rabiu Abdussalam; Muhammad, Mustapha Shehu

    2016-04-01

    Research on the effects of Mobile phone radio frequency emissions on biological systems has been focused on noise and vibrations as auditory stressors. This study investigated the potential effects of exposure to mobile phone electromagnetic field radiation, ringtone and vibration on anxiety-like behaviour and oxidative stress biomarkers in albino wistar rats. Twenty five male wistar rats were randomly divided into five groups of 5 animals each: group I: exposed to mobile phone in switched off mode (control), group II: exposed to mobile phone in silent mode, group III: exposed to mobile phone in vibration mode, group IV: exposed to mobile phone in ringtone mode, group V: exposed to mobile phone in vibration and ringtone mode. The animals in group II to V were exposed to 10 min call (30 missed calls for 20 s each) per day for 4 weeks. Neurobehavioural studies for assessing anxiety were carried out 24 h after the last exposure and the animals were sacrificed. Brain samples were collected for biochemical evaluation immediately. Results obtained showed a significant decrease (P < 0.05) in open arm duration in all the experimental groups when compared to the control. A significant decrease (P < 0.05) was also observed in catalase activity in group IV and V when compared to the control. In conclusion, the results of the present study indicates that 4 weeks exposure to electromagnetic radiation, vibration, ringtone or both produced a significant effect on anxiety-like behavior and oxidative stress in young wistar rats. PMID:26546224

  4. Osteoblastic differentiation of monkey embryonic stem cells in vitro.

    PubMed

    Yamashita, Akihiro; Takada, Tatsuyuki; Narita, Junko; Yamamoto, Gaku; Torii, Ryuzo

    2005-01-01

    Monkey embryonic stem (ES) cell is a useful tool for preclinical studies of regenerative medicine. In this paper, we investigated whether monkey ES cells can be differentiated into osteoblasts in vitro using factors known to promote osteogenesis. We prepared embryoid bodies (EB) in the presence of retinoic acid (RA) and subsequently differentiated in the medium containing either dexamethasone (DEX) or bone morphogenetic protein (BMP)-2 in addition to osteogenic supplements (OS), specifically ascorbic acid and beta-glycerophosphate. RA treatment during EB formation induced osteoblastic marker genes, such as collagen type 1, osteopontin, and Cbfa1. For the expression of osteocalcin, however, cultivation with medium containing either DEX or BMP-2 in addition to OS was required. These results showed that osteoblasts could be derived from monkey ES cells in vitro and BMP-2 + OS was effective to induce calcification. PMID:16390259

  5. Role of syndecan-2 in osteoblast biology and pathology

    PubMed Central

    Mansouri, Rafik; Haÿ, Eric; Marie, Pierre J; Modrowski, Dominique

    2015-01-01

    Syndecans 1–4 are a family of transmembrane proteins composed of a core protein and glycosaminoglycan chains. Although the four syndecans have common functions, they appear to be connected to different signaling pathways, and their expression occurs in a cell- and development-specific pattern. In contrast to other syndecans, syndecan-2 expression increases during osteoblast differentiation. Mechanistically, syndecan-2 exerts multiple functions in cells of the osteoblast lineage as it serves as a co-receptor for fibroblast growth factors and Wnt proteins and controls cell adhesion, proliferation, differentiation and apoptosis. Recent studies indicate that syndecan-2 also contributes to osteosarcoma cell response to cytotoxic agents through interactions with Wnt/β-catenin signaling. Here we summarize our current understanding of the role of syndecan-2 in the control of osteoblast biology and pathology and discuss how syndecan-2 acts as a modulator of the bone cell microenvironment. PMID:25848534

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

    PubMed

    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

  7. Bone sialoprotein II synthesized by cultured osteoblasts contains tyrosine sulfate

    SciTech Connect

    Ecarot-Charrier, B.; Bouchard, F.; Delloye, C. )

    1989-11-25

    Isolated mouse osteoblasts that retain their osteogenic activity in culture were incubated with (35S) sulfate. Two radiolabeled proteins, in addition to proteoglycans, were extracted from the calcified matrix of osteoblast cultures. All the sulfate label in both proteins was in the form of tyrosine sulfate as assessed by amino acid analysis and thin layer chromatography following alkaline hydrolysis. The elution behavior on DEAE-Sephacel of the major sulfated protein and the apparent Mr on sodium dodecyl sulfate gels were characteristic of bone sialoprotein II extracted from rat. This protein was shown to cross-react with an antiserum raised against bovine bone sialoprotein II, indicating that bone sialoprotein II synthesized by cultured mouse osteoblasts is a tyrosine-sulfated protein. The minor sulfated protein was tentatively identified as bone sialoprotein I or osteopontin based on its elution properties on DEAE-Sephacel and anomalous behavior on sodium dodecyl sulfate gels similar to those reported for rat bone sialoprotein I.

  8. Calcium signals and calcium channels in osteoblastic cells

    NASA Technical Reports Server (NTRS)

    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.

  9. Depression of osteoblastic activity in immobilized limbs of suckling rats.

    PubMed

    Weinreb, M; Rodan, G A; Thompson, D D

    1991-07-01

    Recently we characterized the immobilization-related osteopenia in adult rats and showed that it is caused by increased bone resorption and decreased bone formation (Weinreb et al. 1989 Bone 10:187). To assess the effect of age on disuse osteopenia, this study investigated the effects of immobilization on bone turnover in very young, suckling rats. The 15-day-old rats underwent unilateral hind limb immobilization by sciatic neurectomy; the contralateral limb was left intact and served as control. Experimental or sham-operated animals were killed after 0, 2, 4, or 12 days postsurgery. Dry, fat-free weight and ash weight were determined in both femora, and both tibiae were subjected to static and dynamic histomorphometry. Immobilization caused a progressive deficit in bone mass in the immobilized limb compared to the contralateral intact limb but did not affect femoral longitudinal growth. The total mineral content in the immobilized femora was 13.6% less than that in the intact limb by day 12. Concomitantly, tibial cancellous bone area and perimeter declined in the immobilized limb by 37.3 and 32.2%, respectively. This reduction in bone mass in the tibiae of immobilized limbs was associated with increased bone resorption, expressed as osteoclast perimeter, number of osteoclasts per mm surface, and number of osteoclasts per mm2 tissue area. Bone formation was reduced as a result of impaired osteoblast activity as evidenced by (1) decreased endocortical and trabecular mineral apposition rate; (2) reduced trabecular mineral formation rate; (3) decreased percentage of ash of the femoral dry weight; and (4) increased volume of unmineralized osteoid in the tibial metaphysis.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1950676

  10. Enhanced osteoblast proliferation and collagen gene expression by estradiol

    SciTech Connect

    Ernest, M.; Schmid, Ch.; Froesch, E.R. )

    1988-04-01

    Estrogens play a crucial role in the development of postmenopausal osteoporosis. However, the mechanism by which estrogens exert their effects on bone is unknown. To examine possible direct effects of 17{beta}-estradiol on bone-forming cells, the authors used pure rat osteoblast-like cells in vitro as a model. Osteoblast-like cells prepared from calvaria of newborn rats were cultured serum-free in methylcellulose-containing medium for 21 days. Osteoblast-like cells proliferate selectively into clonally derived cell clusters of spherical morphorlogy. 17{beta}-Estradiol at concentrations of 0.1 nM and 1 nM enhanced osteoblast-like cell proliferation by 41% and 68% above vehicle-treated controls. The biologically inactive stereoisomer 17{alpha}-estradiol (same concentrations) had no effect. Moreover, the antiestrogen tamoxifen abolished the stimulation of osteoblast-like cell proliferation by 17{beta}-estradiol. After 21 days of culture, RNA was prepared and analyzed in a dot-hybridization assay for the abundance of pro{alpha}1(I) collagen mRNA. Steady-state mRNA levels were increased in cultures treated with 17{beta}-estradiol in a dose-dependent manner with maximal stimulation at 1 nM and 10 nM. At the same concentrations, the percentage of synthesized protein (labeled by ({sup 3}H)proline pulse) that was digestible by collagenase was increased, indicating that 17{beta}-estradiol acts as pretranslational levels to enhance synthesis of bone collagen. These data show that the osteoblast is a direct target for 17{beta}-estradiol.

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

    SciTech Connect

    Kimira, Yoshifumi; Ogura, Kana; Taniuchi, Yuri; Kataoka, Aya; Inoue, Naoki; Sugihara, Fumihito; Nakatani, Sachie; Shimizu, Jun; Wada, Masahiro; Mano, Hiroshi

    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.

  12. Full-scale experimental and numerical study about structural behaviour of a thin-walled cold-formed steel building affected by ground settlements due to land subsidence

    NASA Astrophysics Data System (ADS)

    Ortiz, J. A.; Hernández, L. A.; Hernández, M.; Pacheco, J.; Zermeño, M. E.; Salinas, R.

    2015-11-01

    Land subsidence due to ground water withdrawal is a problem in many places around the world (Poland, 1984). This causes differential ground settlements that affect masonry structures, because these structural materials do not exhibit an adequate performance beyond a certain level of angular distortion. This work presents the experimental and numerical results about a study regarding the performance of a full-scale thin-walled cold-formed steel building affected by ground differential settlements due to land subsidence. The experimental stage consisted in the construction of a test-building to be subjected to differential settlements in laboratory. The numerical stage consisted in performing a numerical non-linear static pull-down analysis simulating the differential ground settlements of the test-building. The results show that the structural performance of the tested building was very suitable in terms of ductility.

  13. Age-related changes in human oestrogen receptor alpha function and levels in osteoblasts.

    PubMed Central

    Ankrom, M A; Patterson, J A; d'Avis, P Y; Vetter, U K; Blackman, M R; Sponseller, P D; Tayback, M; Robey, P G; Shapiro, J R; Fedarko, N S

    1998-01-01

    Oestrogen receptors (ERs) are present in human osteoblasts and mediate anti-resorptive effects on bone. Human osteoblast-like cells derived from different aged healthy female donors not on hormone replacement therapy were utilized under well-defined conditions in vitro to investigate ER function and levels. Treatment with 0.1 nM oestradiol-17beta of cell strains derived from eight young women (less than 50 years of age) increased hydroxyproline levels significantly [an average (2.2+/-0.1 S.E.M.)-fold increase], whereas cells derived from nine older women (more than 50 years of age) were not significantly affected. Similarly, cell strains, derived from younger women, transfected with a consensus oestrogen-responsive element linked to chloramphenicol acetyltransferase exhibited a greater response to oestrogen than strains derived from older women. When basal ERalpha levels were measured by enzyme immunoassay and normalized on a per cell basis, osteoblast-like strains derived from younger women (n=24) had a mean value of 2.54+/-0.16 fmol of ERalpha per 10(6) cells. In contrast, strains derived from older women (n=20) had a mean value of 5.44+/-0.48 fmol of ERalpha per 10(6) cells. An age-related increase in ERalpha number was also observed in human skin-derived fibroblasts and directly in dermal biopsies from women not on hormone replacement therapy. The results demonstrate ligand concentration-dependent ERalpha induction and indicate a loss of receptor regulation and diminution of ligand-receptor signal transduction with increasing donor age. PMID:9677341

  14. Influence of substrate curvature on osteoblast orientation and extracellular matrix deposition

    PubMed Central

    2013-01-01

    Background The effects of microchannel diameter in hydroxyapatite (HAp) substrates on osteoblast behavior were investigated in this study. Microchannels of 100, 250 and 500 μm diameter were created on hydroxyapatite disks. The changes in osteoblast precursor growth, differentiation, extra cellular matrix (ECM) secretion and cell attachment/orientation were investigated as a function of microchannel diameter. Results Curvature did not impact cellular differentiation, however organized cellular orientation was achieved within the 100 and 250 μm microchannels (mc) after 6 days compared to the 12 days it took for the 500mc group, while the flat substrate remained disorganized. Moreover, the 100, 250 and 500mc groups expressed a specific shift in orientation of 17.45°, 9.05°, and 22.86° respectively in 24 days. The secreted/mineralized ECM showed the 100 and 250mc groups to have higher modulus (E) and hardness (h) (E = 42.6GPa; h = 1.6GPa) than human bone (E = 13.4-25.7GPa; h = 0.47-0.74GPa), which was significantly greater than the 500mc and control groups (p < 0.05). It was determined that substrate curvature affects the cell orientation, the time required for initial response, and the shift in orientation with time. Conclusions These findings demonstrate the ability of osteoblasts to organize and mineralize differentially in microchannels similar to those found in the osteons of compact bone. These investigations could lead to the development of osteon-like scaffolds to support the regeneration of organized bone. PMID:24090183

  15. Comparison of Predictable Smooth Ocular and Combined Eye-Head Tracking Behaviour in Patients with Lesions Affecting the Brainstem and Cerebellum

    NASA Technical Reports Server (NTRS)

    Grant, Michael P.; Leigh, R. John; Seidman, Scott H.; Riley, David E.; Hanna, Joseph P.

    1992-01-01

    We compared the ability of eight normal subjects and 15 patients with brainstem or cerebellar disease to follow a moving visual stimulus smoothly with either the eyes alone or with combined eye-head tracking. The visual stimulus was either a laser spot (horizontal and vertical planes) or a large rotating disc (torsional plane), which moved at one sinusoidal frequency for each subject. The visually enhanced Vestibulo-Ocular Reflex (VOR) was also measured in each plane. In the horizontal and vertical planes, we found that if tracking gain (gaze velocity/target velocity) for smooth pursuit was close to 1, the gain of combined eye-hand tracking was similar. If the tracking gain during smooth pursuit was less than about 0.7, combined eye-head tracking was usually superior. Most patients, irrespective of diagnosis, showed combined eye-head tracking that was superior to smooth pursuit; only two patients showed the converse. In the torsional plane, in which optokinetic responses were weak, combined eye-head tracking was much superior, and this was the case in both subjects and patients. We found that a linear model, in which an internal ocular tracking signal cancelled the VOR, could account for our findings in most normal subjects in the horizontal and vertical planes, but not in the torsional plane. The model failed to account for tracking behaviour in most patients in any plane, and suggested that the brain may use additional mechanisms to reduce the internal gain of the VOR during combined eye-head tracking. Our results confirm that certain patients who show impairment of smooth-pursuit eye movements preserve their ability to smoothly track a moving target with combined eye-head tracking.

  16. Deletion of dopamine D1 and D3 receptors differentially affects spontaneous behaviour and cocaine-induced locomotor activity, reward and CREB phosphorylation.

    PubMed

    Karasinska, Joanna M; George, Susan R; Cheng, Regina; O'Dowd, Brian F

    2005-10-01

    Co-localization of dopamine D1 and D3 receptors in striatal neurons suggests that these two receptors interact at a cellular level in mediating dopaminergic function including psychostimulant-induced behaviour. To study D1 and D3 receptor interactions in cocaine-mediated effects, cocaine-induced locomotion and reward in mice lacking either D1, D3 or both receptors were analysed. Spontaneous locomotor activity was increased in D1-/- and D1-/-D3-/- mice and D1-/-D3-/- mice did not exhibit habituation of spontaneous rearing activity. Cocaine (20 mg/kg) increased locomotor activity in wild-type and D3-/- mice, failed to stimulate activity in D1-/- mice and reduced activity in D1-/-D3-/- mice. In the conditioned place preference, all groups exhibited reward at 5, 10 and 20 mg/kg of cocaine. D1-/-D3-/- mice did not demonstrate preference at 2.5 mg/kg of cocaine although preference was observed in wild-type, D1-/- and D3-/- mice. The transcription factor cAMP-responsive element binding protein (CREB) is activated by phosphorylation in striatal regions following dopamine receptor activation. Striatal pCREB levels following acute cocaine were increased in wild-type and D3-/- mice and decreased in D1-/- and D1-/-D3-/- mice. After repeated administration of 2.5 mg/kg of cocaine, D1-/- mice had lower pCREB levels in caudate-putamen and nucleus accumbens. Our findings suggest that, although spontaneous and cocaine-induced horizontal activity depended mainly on the presence of the D1 receptor, there may be crosstalk between D1 and D3 receptors in rearing habituation and the perception of cocaine reward at low doses of the drug. Furthermore, alterations in pCREB levels were associated with changes in cocaine-induced locomotor activity but not reward. PMID:16197514

  17. How does Australia's largest dolphin-watching industry affect the behaviour of a small and resident population of Indo-Pacific bottlenose dolphins?

    PubMed

    Steckenreuter, Andre; Möller, Luciana; Harcourt, Robert

    2012-04-30

    The small, genetically distinct population of Indo-Pacific bottlenose dolphins (Tursiops aduncus) in Port Stephens, New South Wales (NSW), is the target of the largest dolphin-watching industry in Australia and is located within the Port Stephens - Great Lakes Marine Park that was created in 2005. The effects of this industry have been identified as of significant management importance by the Marine Parks Authority NSW. Accordingly, the impact of commercial dolphin-watching boats was investigated from boat-based surveys from August 2008 to August 2009. Presence of dolphin-watching boats altered both the dolphins' behavioural states and activity budgets. Dolphins spent 66.5% less time feeding and 44.2% less time socialising, spent four times more milling, and were never observed to rest in the presence of dolphin-watching boats. Moreover, dolphin groups were more cohesive during dolphin-watching boat encounters and dolphins tended to avoid tour boats. These effects were exacerbated as the number of boats increased and the distance from boats decreased. The rate of approach was high with boats approaching each dolphin group three times per day in winter and six times in summer. Moreover, groups of dolphins with newborns were approached closer than state regulated minimum approach distances in nine out of ten encounters. Globally, dolphin-watching industries frequent small resident groups of coastal dolphins and effects are likely to be similar. We suggest that existing controls are inadequate and that these together with additional regulations be enforced by a regular presence of authorities. We suggest no more than one dolphin-watching boat within 50 m of a group of dolphins, or 100 m if calves are present. Operating times of dolphin-watching boats should be restricted in numbers after 1 pm, i.e., during preferred foraging times for dolphins. Additionally, exclusion zones should be considered to reduce pressure on dolphins undertaking critical activities such as

  18. AGEs Induce Apoptosis in Rat Osteoblast Cells by Activating the Caspase-3 Signaling Pathway Under a High-Glucose Environment In Vitro.

    PubMed

    Liu, Jiaqiang; Mao, Jing; Jiang, Yi; Xia, Lunguo; Mao, Lixia; Wu, Yong; Ma, Pan; Fang, Bing

    2016-03-01

    Advanced glycation end products (AGEs) accumulate under high-glucose conditions and affect the healing of bone damage through various pathways; however, the detail mechanisms underlying these changes are unknown. In this study, we investigated the effects of AGEs on the apoptosis of in vitro-cultured rat osteoblasts under high-glucose conditions and explored the underlying mechanisms of these effects. First, we cultured rat osteoblasts and determined the accumulation of AGEs in the culture medium under high-glucose conditions. Then, we cultured rat osteoblasts under a high glucose concentration (35 mM), a normal glucose concentration (5.5 mM), and a normal glucose concentration (5.5 mM) in the presence of AGEs. We examined the effects of high glucose and AGEs on the apoptosis of rat osteoblasts at different time points and further analyzed the activity and changes in the levels of procaspase-3, caspase-3, and the caspase-3 substrate poly ADP-ribose polymerase (PARP). Finally, we added sRAGE (soluble RAGE) (an AGE inhibitor) or DEVD (a caspase-3 inhibitor) to each culture group and examined apoptosis under each culture condition and the changes in the levels of procaspase-3, caspase-3, and its substrate PARP. The results showed that the high-glucose condition and the addition of AGEs increased the apoptosis of rat osteoblast cells and simultaneously increased the activity and quantity of caspase-3. These increases could be inhibited by the AGE inhibitor sRAGE or the caspase-3 inhibitor DEVD. The above results demonstrate that high-glucose conditions lead to the accumulation of AGEs and activation of the caspase-3 signaling pathway, resulting in the increased apoptosis of cultured rat osteoblast cells. PMID:26573666

  19. Apolipoprotein A-1 regulates osteoblast and lipoblast precursor cells in mice.

    PubMed

    Blair, Harry C; Kalyvioti, Elena; Papachristou, Nicholaos I; Tourkova, Irina L; Syggelos, Spryros A; Deligianni, Despina; Orkoula, Malvina G; Kontoyannis, Christos G; Karavia, Eleni A; Kypreos, Kyriakos E; Papachristou, Dionysios J

    2016-07-01

    Imbalances in lipid metabolism affect bone homeostasis, altering bone mass and quality. A link between bone mass and high-density lipoprotein (HDL) has been proposed. Indeed, it has been recently shown that absence of the HDL receptor scavenger receptor class B type I (SR-B1) causes dense bone mediated by increased adrenocorticotropic hormone (ACTH). In the present study we aimed at further expanding the current knowledge as regards the fascinating bone-HDL connection studying bone turnover in apoA-1-deficient mice. Interestingly, we found that bone mass was greatly reduced in the apoA-1-deficient mice compared with their wild-type counterparts. More specifically, static and dynamic histomorphometry showed that the reduced bone mass in apoA-1(-/-) mice reflect decreased bone formation. Biochemical composition and biomechanical properties of ApoA-1(-/-) femora were significantly impaired. Mesenchymal stem cell (MSC) differentiation from the apoA-1(-/-) mice showed reduced osteoblasts, and increased adipocytes, relative to wild type, in identical differentiation conditions. This suggests a shift in MSC subtypes toward adipocyte precursors, a result that is in line with our finding of increased bone marrow adiposity in apoA-1(-/-) mouse femora. Notably, osteoclast differentiation in vitro and osteoclast surface in vivo were unaffected in the knock-out mice. In whole bone marrow, PPARγ was greatly increased, consistent with increased adipocytes and committed precursors. Further, in the apoA-1(-/-) mice marrow, CXCL12 and ANXA2 levels were significantly decreased, whereas CXCR4 were increased, consistent with reduced signaling in a pathway that supports MSC homing and osteoblast generation. In keeping, in the apoA-1(-/-) animals the osteoblast-related factors Runx2, osterix, and Col1a1 were also decreased. The apoA-1(-/-) phenotype also included augmented CEPBa levels, suggesting complex changes in growth and differentiation that deserve further investigation. We

  20. Jagged1 is essential for osteoblast development during maxillary ossification.

    PubMed

    Hill, Cynthia R; Yuasa, Masato; Schoenecker, Jonathan; Goudy, Steven L

    2014-05-01

    Maxillary hypoplasia occurs due to insufficient maxillary intramembranous ossification, leading to poor dental occlusion, respiratory obstruction and cosmetic deformities. Conditional deletion of Jagged1 (Jag1) in cranial neural crest (CNC) cells using Wnt1-cre; Jagged1(f/f) (Jag1CKO) led to maxillary hypoplasia characterized by intrinsic differences in bone morphology and density using μCT evaluation. Jag1CKO maxillas revealed altered collagen deposition, delayed ossification, and reduced expression of early and late determinants of osteoblast development during maxillary ossification. In vitro bone cultures on Jag1CKO mouse embryonic maxillary mesenchymal (MEMM) cells demonstrated decreased mineralization that was also associated with diminished induction of osteoblast determinants. BMP receptor expression was dysregulated in the Jag1CKO MEMM cells suggesting that these cells were unable to respond to BMP-induced differentiation. JAG1-Fc rescued in vitro mineralization and osteoblast gene expression changes. These data suggest that JAG1 signaling in CNC-derived MEMM cells is required for osteoblast development and differentiation during maxillary ossification. PMID:24491691

  1. PRIMITIVE ADULT HEMATOPOIETIC STEM CELLS CAN FUNCTION AS OSTEOBLAST PRECURSORS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Osteoblasts are continually recruited from stem cell pools to maintain bone. Although their immediate precursor is a plastic-adherent mesenchymal stem cell able to generate tissues other than bone, increasing evidence suggests the existence of a more primitive cell that can differentiate to both hem...

  2. Genomic approaches to identifying transcriptional regulators of osteoblast differentiation

    NASA Technical Reports Server (NTRS)

    Stains, Joseph P.; Civitelli, Roberto

    2003-01-01

    Recent microarray studies of mouse and human osteoblast differentiation in vitro have identified novel transcription factors that may be important in the establishment and maintenance of differentiation. These findings help unravel the pattern of gene-expression changes that underly the complex process of bone formation.

  3. Galectin-3 Inhibits Osteoblast Differentiation through Notch Signaling12

    PubMed Central

    Nakajima, Kosei; Kho, Dhong Hyo; Yanagawa, Takashi; Harazono, Yosuke; Gao, Xiaoge; Hogan, Victor; Raz, Avraham

    2014-01-01

    Patients with bone cancer metastasis suffer from unbearable pain and bone fractures due to bone remodeling. This is caused by tumor cells that disturb the bone microenvironment. Here, we have investigated the role of tumor-secreted sugar-binding protein, i.e., galectin-3, on osteoblast differentiation and report that it downregulates the expression of osteoblast differentiation markers, e.g., RUNX2, SP7, ALPL, COL1A1, IBSP, and BGLAP, of treated human fetal osteoblast (hFOB) cells. Co-culturing of hFOB cells with human breast cancer BT-549 and prostate cancer LNCaP cells harboring galectin-3 has resulted in inhibition of osteoblast differentiation by the secreted galectin-3 into culture medium. The inhibitory effect of galectin-3 was found to be through its binding to Notch1 in a sugar-dependent manner that has led to accelerated Notch1 cleavage and activation of Notch signaling. Taken together, our findings show that soluble galectin-3 in the bone microenvironment niche regulates bone remodeling through Notch signaling, suggesting a novel bone metastasis therapeutic target. PMID:25425968

  4. Nanostructured magnesium has fewer detrimental effects on osteoblast function

    PubMed Central

    Weng, Lucy; Webster, Thomas J

    2013-01-01

    Efforts have been made recently to implement nanoscale surface features on magnesium, a biodegradable metal, to increase bone formation. Compared with normal magnesium, nanostructured magnesium has unique characteristics, including increased grain boundary properties, surface to volume ratio, surface roughness, and surface energy, which may influence the initial adsorption of proteins known to promote the function of osteoblasts (bone-forming cells). Previous studies have shown that one way to increase nanosurface roughness on magnesium is to soak the metal in NaOH. However, it has not been determined if degradation of magnesium is altered by creating nanoscale features on its surface to influence osteoblast density. The aim of the present in vitro study was to determine the influence of degradation of nanostructured magnesium, created by soaking in NaOH, on osteoblast density. Our results showed a less detrimental effect of magnesium degradation on osteoblast density when magnesium was treated with NaOH to create nanoscale surface features. The detrimental degradation products of magnesium are of significant concern when considering use of magnesium as an orthopedic implant material, and this study identified a surface treatment, ie, soaking in NaOH to create nanoscale features for magnesium that can improve its use in numerous orthopedic applications. PMID:23674891

  5. Osteoblast maturation occurs in overlapping proximal-distal compartments during fin regeneration in zebrafish.

    PubMed

    Brown, Andrew M; Fisher, Shannon; Iovine, M Kathryn

    2009-11-01

    During fin regeneration, osteoblasts must continually differentiate for outgrowth of the bony fin rays. Bone maturity increases in a distal-proximal manner, and osteoblast maturation can be detected similarly when following gene expression. We find that early markers for osteoblast differentiation are expressed in a discrete domain at the distal end of the fin, just proximal to the adjacent germinal compartment of dividing cells. Matrix genes, required at later stages developmentally, are expressed in a population of cells proximally to the early genes. A marker for mature osteoblasts is expressed in cells further proximal. These domains of gene expression are partially overlapping, perhaps revealing additional levels of osteoblast maturity. We suggest a model for growth where new cells are continually added to the distal-most osteoblast compartment, while osteoblasts in more proximal locations differentiate, thus translating developmental time to location on the proximal-distal axis. PMID:19842180

  6. Osteoblast maturation occurs in overlapping proximal-distal compartments during fin regeneration in zebrafish

    PubMed Central

    Brown, Andrew M.; Fisher, Shannon; Iovine, M. Kathryn

    2009-01-01

    During fin regeneration, osteoblasts must continually differentiate for outgrowth of the bony fin rays. Bone maturity increases in a distal-proximal manner, and osteoblast maturation can be detected similarly when following gene expression. We find that early markers for osteoblast differentiation are expressed in a discrete domain at the distal end of the fin, just proximal to the adjacent germinal compartment of dividing cells. Matrix genes, required at later stages developmentally, are expressed in a population of cells proximally to the early genes. A marker for mature osteoblasts is expressed in cells further proximal. These domains of gene expression are partially overlapping, perhaps revealing additional levels of osteoblast maturity. We suggest a model for growth where new cells are continually added to the distal-most osteoblast compartment, while osteoblasts in more proximal locations differentiate, thus translating developmental time to location on the proximal-distal axis. PMID:19842180

  7. Tenofovir treatment of primary osteoblasts alters gene expression profiles: implications for bone mineral density loss

    PubMed Central

    Grigsby, Iwen F.; Pham, Lan; Mansky, Louis M.; Gopalakrishnan, Raj; Carlson, Ann E.; Mansky, Kim C.

    2010-01-01

    There is strong clinical evidence that implicates tenofovir in the loss of bone mineral density during treatment of human immunodeficiency virus infection. In this study, we sought to test the hypothesis that tenofovir treatment of osteoblasts causes changes in the gene expression profile that would impact osteoblast function during bone formation. Primary osteoblasts were isolated and then treated with the tenofovir prodrug, tenofovir disoproxil fumarate (TDF). Total RNA from TDF-treated and untreated osteoblasts were extracted and used for microarray analysis to assess TDF-associated changes in the gene expression profile. Strikingly, the changes in gene expression profiles involved in cell signaling, cell cycle and amino acid metabolism, which would likely impact osteoblast function in bone formation. Our findings demonstrate for the first time that tenofovir treatment of primary osteoblasts results in gene expression changes that implicate loss of osteoblast function in tenofovir-associated bone mineral density loss. PMID:20171173

  8. Osteoblasts extracellular matrix induces vessel like structures through glycosylated collagen I

    SciTech Connect

    Palmieri, D.; Valli, M.; Viglio, S.; Ferrari, N.; Ledda, B.; Volta, C.; Manduca, P.

    2010-03-10

    Extracellular matrix (ECM) plays a fundamental role in angiogenesis affecting endothelial cells proliferation, migration and differentiation. Vessels-like network formation in vitro is a reliable test to study the inductive effects of ECM on angiogenesis. Here we utilized matrix deposed by osteoblasts as substrate where the molecular and structural complexity of the endogenous ECM is preserved, to test if it induces vessel-like network formation by endothelial cells in vitro. ECM is more similar to the physiological substrate in vivo than other substrates previously utilized for these studies in vitro. Osteogenic ECM, prepared in vitro from mature osteoblasts at the phase of maximal deposition and glycosylation of collagen I, induces EAhy926, HUVEC, and HDMEC endothelial cells to form vessels-like structures and promotes the activation of metalloproteinase-2 (MMP-2); the functionality of the p-38/MAPK signaling pathway is required. Osteogenic ECM also induces a transient increase of CXCL12 and a decrease of the receptor CXCR4. The induction of vessel-like networks is dependent from proper glycosylation of collagens and does not occur on osteogenic ECMs if deglycosylated by -galactosidase or on less glycosylated ECMs derived from preosteoblasts and normal fibroblasts, while is sustained on ECM from osteogenesis imperfecta fibroblasts only when their mutation is associated with over-glycosylation of collagen type I. These data support that post-translational glycosylation has a role in the induction in endothelial cells in vitro of molecules conductive to self-organization in vessels-like structures.

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

    NASA Technical Reports Server (NTRS)

    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.

  10. Rac1 GTPase silencing counteracts microgravity-induced effects on osteoblastic cells.

    PubMed

    Guignandon, Alain; Faure, Céline; Neutelings, Thibaut; Rattner, Aline; Mineur, Pierre; Linossier, Marie-Thérèse; Laroche, Norbert; Lambert, Charles; Deroanne, Christophe; Nusgens, Betty; Demets, René; Colige, Alain; Vico, Laurence

    2014-09-01

    Bone cells exposed to real microgravity display alterations of their cytoskeleton and focal adhesions, two major mechanosensitive structures. These structures are controlled by small GTPases of the Ras homology (Rho) family. We investigated the effects of RhoA, Rac1, and Cdc42 modulation of osteoblastic cells under microgravity conditions. Human MG-63 osteoblast-like cells silenced for RhoGTPases were cultured in the automated Biobox bioreactor (European Space Agency) aboard the Foton M3 satellite and compared to replicate ground-based controls. The cells were fixed after 69 h of microgravity exposure for postflight analysis of focal contacts, F-actin polymerization, vascular endothelial growth factor (VEGF) expression, and matrix targeting. We found that RhoA silencing did not affect sensitivity to microgravity but that Rac1 and, to a lesser extent, Cdc42 abrogation was particularly efficient in counteracting the spaceflight-related reduction of the number of focal contacts [-50% in silenced, scrambled (SiScr) controls vs. -15% for SiRac1], the number of F-actin fibers (-60% in SiScr controls vs. -10% for SiRac1), and the depletion of matrix-bound VEGF (-40% in SiScr controls vs. -8% for SiRac1). Collectively, these data point out the role of the VEGF/Rho GTPase axis in mechanosensing and validate Rac1-mediated signaling pathways as potential targets for counteracting microgravity effects. PMID:24903274

  11. Resveratrol augments the canonical Wnt signaling pathway in promoting osteoblastic differentiation of multipotent mesenchymal cells

    SciTech Connect

    Zhou, Haibin; Shang, Linshan; Li, Xi; Zhang, Xiyu; Gao, Guimin; Guo, Chenhong; Chen, Bingxi; Liu, Qiji; Gong, Yaoqin; Shao, Changshun

    2009-10-15

    Resveratrol has been shown to possess many health-benefiting effects, including the promotion of bone formation. In this report we investigated the mechanism by which resveratrol promotes osteoblastic differentiation from pluripotent mesenchymal cells. Since Wnt signaling is well documented to induce osteoblastogenesis and bone formation, we characterized the factors involved in Wnt signaling in response to resveratrol treatment. Resveratrol treatment of mesenchymal cells led to an increase in stabilization and nuclear accumulation of {beta}-catenin dose-dependently and time-dependently. As a consequence of the increased nuclear accumulation of {beta}-catenin, the ability to activate transcription of {beta}-catenin-TCF/LEF target genes that are required for osteoblastic differentiation was upregulated. However, resveratrol did not affect the initial step of the Wnt signaling pathway, as resveratrol was as effective in upregulating the activity of {beta}-catenin in cells in which Lrp5 was knocked down as in control cells. In addition, while conditioned medium enriched in Wnt signaling antagonist Dkk1 was able to inhibit Wnt3a-induced {beta}-catenin upregulation, this inhibitory effect can be abolished in resveratrol-treated cells. Furthermore, we showed that the level of glycogen synthase kinase 3{beta} (GSK-3{beta}), which phosphorylates and destabilizes {beta}-catenin, was reduced in response to resveratrol treatment. The phosphorylation of GSK-3{beta} requires extracellular signal-regulated kinase (ERK)1/2. Together, our data indicate that resveratrol promotes osteoblastogenesis and bone formation by augmenting Wnt signaling.

  12. Phenolic Compounds in Extra Virgin Olive Oil Stimulate Human Osteoblastic Cell Proliferation.

    PubMed

    García-Martínez, Olga; De Luna-Bertos, Elvira; Ramos-Torrecillas, Javier; Ruiz, Concepción; Milia, Egle; Lorenzo, María Luisa; Jimenez, Brigida; Sánchez-Ortiz, Araceli; Rivas, Ana

    2016-01-01

    In this study, we aimed to clarify the effects of phenolic compounds and extracts from different extra virgin olive oil (EVOO) varieties obtained from fruits of different ripening stages on osteoblast cells (MG-63) proliferation. Cell proliferation was increased by hydroxytyrosol, luteolin, apigenin, p-coumaric, caffeic, and ferulic acids by approximately 11-16%, as compared with controls that were treated with one vehicle alone, while (+)-pinoresinol, oleuropein, sinapic, vanillic acid and derivative (vanillin) did not affect cell proliferation. All phenolic extracts stimulated MG-63 cell growth, and they induced higher cell proliferation rates than individual compounds. The most effective EVOO phenolic extracts were those obtained from the Picual variety, as they significantly increased cell proliferation by 18-22%. Conversely, Arbequina phenolic extracts increased cell proliferation by 9-13%. A decline in osteoblast proliferation was observed in oils obtained from olive fruits collected at the end of the harvest period, as their total phenolic content decreases at this late stage. Further research on the signaling pathways of olive oil phenolic compounds involved in the processes and their metabolism should be carried out to develop new interventions and adjuvant therapies using EVOO for bone health (i.e.osteoporosis) in adulthood and the elderly. PMID:26930190

  13. Phenolic Compounds in Extra Virgin Olive Oil Stimulate Human Osteoblastic Cell Proliferation

    PubMed Central

    García-Martínez, Olga; De Luna-Bertos, Elvira; Ramos-Torrecillas, Javier; Ruiz, Concepción; Milia, Egle; Lorenzo, María Luisa; Jimenez, Brigida; Sánchez-Ortiz, Araceli; Rivas, Ana

    2016-01-01

    In this study, we aimed to clarify the effects of phenolic compounds and extracts from different extra virgin olive oil (EVOO) varieties obtained from fruits of different ripening stages on osteoblast cells (MG-63) proliferation. Cell proliferation was increased by hydroxytyrosol, luteolin, apigenin, p-coumaric, caffeic, and ferulic acids by approximately 11–16%, as compared with controls that were treated with one vehicle alone, while (+)-pinoresinol, oleuropein, sinapic, vanillic acid and derivative (vanillin) did not affect cell proliferation. All phenolic extracts stimulated MG-63 cell growth, and they induced higher cell proliferation rates than individual compounds. The most effective EVOO phenolic extracts were those obtained from the Picual variety, as they significantly increased cell proliferation by 18–22%. Conversely, Arbequina phenolic extracts increased cell proliferation by 9–13%. A decline in osteoblast proliferation was observed in oils obtained from olive fruits collected at the end of the harvest period, as their total phenolic content decreases at this late stage. Further research on the signaling pathways of olive oil phenolic compounds involved in the processes and their metabolism should be carried out to develop new interventions and adjuvant therapies using EVOO for bone health (i.e.osteoporosis) in adulthood and the elderly. PMID:26930190

  14. Calciotropic hormones raise the chemically detectable [Pi] in UMR 106-06 osteoblast-like cells.

    PubMed

    Ahmado, A; Khouja, H I; Kemp, G J; Guilland-Cumming, D F; Russell, R G; Bevington, A

    1993-03-01

    Uptake of orthophosphate (Pi) by osteoblast-like cells is known to be stimulated by parathyroid hormone (PTH), but effects on intracellular [Pi] have not been investigated. Here we show in rat osteoblast-like cells (UMR 106-06) that PTH (10(-11) to 10(-7) M) increases both 32Pi uptake and cellular [Pi] by up to 50 per cent. 1,25 Dihydroxyvitamin D3 (1,25D) (10(-12) to 10(-6) M) and salmon calcitonin (CT) (10(-12) to 10(-6) g ml-1) also increased cellular [Pi] (by up to 60 per cent), but the percentage increases in total cellular 32Pi uptake were smaller. The effects of 1,25D were transient (observable at 80 min and 6 h but not 24 h), and were also observed with 24,25 dihydroxy- and 25 hydroxyvitamin D3. Transient degradation of organic phosphorus pools to Pi might contribute to this increased [Pi]. These pools remain to be identified but were not shown to be phospholipids. Foetal bovine serum also affected cellular [Pi]. Care is therefore needed in distinguishing direct hormonal effects on cellular [Pi] from indirect effects arising from changes in the rate of cell growth. PMID:8453734

  15. Neural mechanisms underlying the evolvability of behaviour

    PubMed Central

    Katz, Paul S.

    2011-01-01

    The complexity of nervous systems alters the evolvability of behaviour. Complex nervous systems are phylogenetically constrained; nevertheless particular species-specific behaviours have repeatedly evolved, suggesting a predisposition towards those behaviours. Independently evolved behaviours in animals that share a common neural architecture are generally produced by homologous neural structures, homologous neural pathways and even in the case of some invertebrates, homologous identified neurons. Such parallel evolution has been documented in the chromatic sensitivity of visual systems, motor behaviours and complex social behaviours such as pair-bonding. The appearance of homoplasious behaviours produced by homologous neural substrates suggests that there might be features of these nervous systems that favoured the repeated evolution of particular behaviours. Neuromodulation may be one such feature because it allows anatomically defined neural circuitry to be re-purposed. The developmental, genetic and physiological mechanisms that contribute to nervous system complexity may also bias the evolution of behaviour, thereby affecting the evolvability of species-specific behaviour. PMID:21690127

  16. Fabrication of poly(ethylene glycol) hydrogel micropatterns with osteoinductive growth factors and evaluation of the effects on osteoblast activity and function

    NASA Astrophysics Data System (ADS)

    Subramani, K.; Birch, M. A.

    2006-09-01

    The aims of this study were to fabricate poly(ethylene glycol) (PEG) hydrogel micropatterns on a biomaterial surface to guide osteoblast behaviour and to study how incorporating vascular endothelial growth factor (VEGF) within the adhered hydrogel influenced cell morphology. Standard photolithographic procedures or photopolymerization through a poly(dimethyl siloxane) (PDMS) mould were used to fabricate patterned PEG hydrogels on the surface of silanized silicon wafers. Hydrogel patterns were evaluated by light microscopy and surface profilometry. Rat osteoblasts were cultured on these surfaces and cell morphology investigated by fluorescence microscopy, scanning electron microscopy (SEM) and atomic force microscopy (AFM). Release of protein trapped in the polymerized PEG was evaluated and VEGF-PEG surfaces were characterized for their ability to support cell growth. These studies show that photopolymerized PEG can be used to create anti-adhesive structures on the surface of silicon that completely control where cell interaction with the substrate takes place. Using conventional lithography, structures down to 50 µm were routinely fabricated with the boundaries exhibiting sloping sides. Using the PDMS mould approach, structures were fabricated as small as 10 µm and boundaries were very sharp and vertical. Osteoblasts exhibiting typical morphology only grew on the silicon wafer surface that was not coated with PEG. Adding BSA to the monomer solution showed that protein could be released from the hydrogel for up to 7 days in vitro. Incorporating VEGF in the hydrogel produced micropatterns that dramatically altered osteoblast behaviour. At boundaries with the VEGF-PEG hydrogel, there was striking formation of cellular processes and membrane ruffling indicative of a change in cell morphology. This study has explored the morphogenetic properties of VEGF and the applications of nano/microfabrication techniques for guided tissue (bone) regeneration in dental and

  17. Megakaryocytes are mechanically responsive and influence osteoblast proliferation and differentiation

    PubMed Central

    Soves, Constance P.; Miller, Joshua D.; Begun, Dana L.; Taichman, Russell S.; Hankenson, Kurt D.; Goldstein, Steven A.

    2014-01-01

    Maintenance of bone mass and geometry is influenced by mechanical stimuli. Paradigms suggest that osteocytes embedded within the mineralized matrix and osteoblasts on the bone surfaces are the primary responders to physical forces. However, other cells within the bone marrow cavity, such as megakaryocytes (MKs), are also subject to mechanical forces. Recent studies have highlighted the potent effects of MKs on osteoblast proliferation as well as bone formation in vivo. We hypothesize that MKs are capable of responding to physical forces and that the interactions between these cells and osteoblasts can be influenced by mechanical stimulation. In this study, we demonstrate that two MK cell lines respond to fluid shear stress in culture. Furthermore, using laser capture microdissection, we isolated MKs from histologic sections of murine tibiae that were exposed to compressive loads in vivo. C-fos, a transcription factor shown to be upregulated in response to load in various tissue types, was increased in MKs from loaded relative to non-loaded limbs at a level comparable to that of osteocytes from the same limbs. We also developed a co-culture system to address whether mechanical stimulation of MKs in culture would impact osteoblast proliferation and differentiation. The presence of MKs in co-culture, but not conditioned media, had dramatic effects on proliferation of preosteoblast MC3T3-E1 cells in culture. Our data suggests a minimal decrease in proliferation as well as an increase in mineralization capacity of osteoblasts co-cultured with MKs exposed to shear compared to co-cultures with unstimulated MKs. PMID:24882736

  18. Downregulation of taurine transport by calcium blockers in osteoblast cells.

    PubMed

    Kang, Young-Sook

    2009-01-01

    Taurine is found in a high concentration in bone cells and is thought to help enhance bone tissue formation and inhibit bone loss. It is mainly transported by a sodium and chloride ion dependent taurine transporter (TauT), which is expressed in a variety of tissues, such as brain, retina, and placenta, but in bone the transporter has not yet been identified. The purpose of this study is to clarify the uptake mechanism of taurine in osteoblasts using mouse osteoblast cell lines. Mouse stromal ST2 cells and mouse osteoblast-like MC3T3-E1 cells were used as osteoblast cell lines. Detection of TauT mRNA expression in these cells was performed by RT-PCR. The activity of the taurine transporter was assessed by measuring the uptake of [3H]taurine in cell lines in the presence and absence of inhibitors. TauT mRNA was detected in ST2 and MC3T3-E1 cells. [3H]Taurine uptake by these cells exhibited a time dependent increase that was linear for at least 10 min. [3H]Taurine uptake was dependent on the presence of extracellular sodium and chloride ions, and was inhibited by unlabeled taurine, beta-alanine and gamma-amino-n-butyric acid. Moreover, uptake of [3H]taurine by these cells was dependent on the presence of extracellular calcium. The uptake of [3H]taurine in ST2 cells treated with 4 mM calcium was increased 1.7-fold. The initial rate of [3H]taurine uptake was significantly inhibited by 100 microM nifedipine and 100 microM verapamil. These results suggest that in mouse osteoblast cell lines taurine transport is controlled by extracellular calcium. PMID:19239183

  19. Megakaryocytes are mechanically responsive and influence osteoblast proliferation and differentiation.

    PubMed

    Soves, Constance P; Miller, Joshua D; Begun, Dana L; Taichman, Russell S; Hankenson, Kurt D; Goldstein, Steven A

    2014-09-01

    Maintenance of bone mass and geometry is influenced by mechanical stimuli. Paradigms suggest that osteocytes embedded within the mineralized matrix and osteoblasts on the bone surfaces are the primary responders to physical forces. However, other cells within the bone marrow cavity, such as megakaryocytes (MKs), are also subject to mechanical forces. Recent studies have highlighted the potent effects of MKs on osteoblast proliferation as well as bone formation in vivo. We hypothesize that MKs are capable of responding to physical forces and that the interactions between these cells and osteoblasts can be influenced by mechanical stimulation. In this study, we demonstrate that two MK cell lines respond to fluid shear stress in culture. Furthermore, using laser capture microdissection, we isolated MKs from histologic sections of murine tibiae that were exposed to compressive loads in vivo. C-fos, a transcription factor shown to be upregulated in response to load in various tissue types, was increased in MKs from loaded relative to non-loaded limbs at a level comparable to that of osteocytes from the same limbs. We also developed a co-culture system to address whether mechanical stimulation of MKs in culture would impact osteoblast proliferation and differentiation. The presence of MKs in co-culture, but not conditioned media, had dramatic effects on proliferation of preosteoblast MC3T3-E1 cells in culture. Our data suggests a minimal decrease in proliferation as well as an increase in mineralization capacity of osteoblasts co-cultured with MKs exposed to shear compared to co-cultures with unstimulated MKs. PMID:24882736

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

    PubMed

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

    2014-01-01

    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. PMID:25277146

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

    NASA Astrophysics Data System (ADS)

    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.

  2. The basic helix loop helix transcription factor Twist1 is a novel regulator of ATF4 in osteoblasts.

    PubMed

    Danciu, Theodora E; Li, Yan; Koh, Amy; Xiao, Guozhi; McCauley, Laurie K; Franceschi, Renny T

    2012-01-01

    Parathyroid hormone (PTH) is an essential regulator of endochondral bone formation and an important anabolic agent for the reversal of bone loss. PTH mediates its functions in part by regulating binding of the bone-related activating transcription factor 4 (ATF4) to the osteoblast-specific gene, osteocalcin. The basic helix-loop-helix (bHLH) factors Twist1 and Twist2 also regulate osteocalcin transcription in part through the interaction of the C-terminal "box" domain in these factors and Runx2. In this study, we discovered a novel function of PTH: its ability to dramatically decrease Twist1 transcription. Since ATF4 is a major regulator of the PTH response in osteoblasts, we assessed the mutual regulation between these factors and determined that Twist proteins and ATF4 physically interact in a manner that affects ATF4 DNA binding function. We mapped the interaction domain of Twist proteins to the C-terminal "box" domain and of ATF4, to the N-terminus. Furthermore, we demonstrate that Twist1 overexpression in osteoblasts attenuates ATF4 binding to the osteocalcin promoter in response to PTH. This study thus identifies Twist proteins as novel inhibitory binding partners of ATF4 and explores the functional significance of this interaction. PMID:21866569

  3. The basic helix loop helix transcription factor Twist1 is a novel regulator of ATF4 in osteoblasts

    PubMed Central

    Danciu, Theodora E.; Li, Yan; Koh, Amy; Xiao, Guozhi; McCauley, Laurie K.; Franceschi, Renny T.

    2011-01-01

    Parathyroid hormone (PTH) is an essential regulator of endochondral bone formation and an important anabolic agent for the reversal of bone loss. PTH mediates its functions in part by regulating binding of the bone-related activating transcription factor 4 (ATF4) to the osteoblast-specific gene, osteocalcin. The basic helix-loop-helix (bHLH) factors Twist1 and Twist2 also regulate osteocalcin transcription in part through the interaction of the C-terminal “box” domain in these factors and Runx2. In this study, we discovered a novel function of PTH: its ability to dramatically decrease Twist1 transcription. Since ATF4 is a major regulator of the PTH response in osteoblasts, we assessed the mutual regulation between these factors and determined that Twist proteins and ATF4 physically interact in a manner that affects ATF4 DNA binding function. We mapped the interaction domain of Twist proteins to the C-terminal “box” domain and of ATF4, to the N-terminus. Furthermore, we demonstrate that Twist1 overexpression in osteoblasts attenuates ATF4 binding to the osteocalcin promoter in response to PTH. This study thus identifies Twist proteins as novel inhibitory binding partners of ATF4 and explores the functional significance of this interaction. PMID:21866569

  4. Forskolin Regulates L-Type Calcium Channel through Interaction between Actinin 4 and β3 Subunit in Osteoblasts

    PubMed Central

    Guo, Lin; Hei, Hongya; Tian, Lulu; Peng, Wen; Cai, Hui

    2015-01-01

    Voltage-dependent L-type calcium channels that permit cellular calcium influx are essential in calcium-mediated modulation of cellular signaling. Although the regulation of voltage-dependent L-type calcium channels is linked to many factors including cAMP-dependent protein kinase A (PKA) activity and actin cytoskeleton, little is known about the detailed mechanisms underlying the regulation in osteoblasts. Our present study investigated the modulation of L-type calcium channel activities through the effects of forskolin on actin reorganization and on its functional interaction with actin binding protein actinin 4. The results showed that forskolin did not significantly affect the trafficking of pore forming α1c subunit and its interaction with actin binding protein actinin 4, whereas it significantly increased the expression of β3 subunit and its interaction with actinin 4 in osteoblast cells as assessed by co-immunoprecipitation, pull-down assay, and immunostaining. Further mapping showed that the ABD and EF domains of actinin 4 were interaction sites. This interaction is independent of PKA phosphorylation. Knockdown of actinin 4 significantly decreased the activities of L-type calcium channels. Our study revealed a new aspect of the mechanisms by which the forskolin activation of adenylyl cyclase - cAMP cascade regulates the L-type calcium channel in osteoblast cells, besides the PKA mediated phosphorylation of the channel subunits. These data provide insight into the important role of interconnection among adenylyl cyclase, cAMP, PKA, the actin cytoskeleton, and the channel proteins in the regulation of voltage-dependent L-type calcium channels in osteoblast cells. PMID:25902045

  5. Effects of transforming growth factor type beta on expression of cytoskeletal proteins in endosteal mouse osteoblastic cells

    SciTech Connect

    Lomri, A.; Marie, P.J. )

    1990-01-01

    Transforming growth factor beta (TGF beta) has been shown to influence the growth and differentiation of many cell types in vitro. We have examined the effects of TGF beta on cell morphology and cytoskeletal organization in relation to parameters of cell proliferation and differentiation in endosteal osteoblastic cells isolated from mouse caudal vertebrae. Treatment of mouse osteoblastic cells cultured in serum free medium for 24 hours with TGF beta (1.5-30 ng/mL) slightly (-23%) inhibited alkaline phosphatase activity. In parallel, TGF beta (0.5-30 ng/mL, 24 hours) greatly increased cell replication as evaluated by (3H)-thymidine incorporation into DNA (157% to 325% of controls). At a median dose (1.5 ng/mL) that affected both alkaline phosphatase and DNA synthesis (235% of controls) TGF beta induced rapid (six hours) cell respreading of quiescent mouse osteoblastic cells. This effect was associated with increased polymerization of actin, alpha actinin, and tubulins, as evaluated by both biochemical and immunofluorescence methods. In addition, TGF beta (1.5 ng/mL) increased the de novo biosynthesis of actin, alpha actinin, vimentin, and tubulins, as determined by {sup 35}S methionine labeling and fractionation of cytoskeletal proteins using two-dimensional gel electrophoresis. These effects were rapid and transient, as they occurred at six hours and were reversed after 24 hours of TGF beta exposure. The results indicate that the stimulatory effect of TGF beta on DNA synthesis in endosteal mouse osteoblastic cells is associated with a transient increase in cell spreading associated with enhanced polymerization and synthesis of cytoskeletal proteins.

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

    PubMed

    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

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

    PubMed Central

    Wan, Qiaoqiao; Cho, Eunhye; Yokota, Hiroki; Na, Sungsoo

    2013-01-01

    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 both Rac1 and Cdc42 GTPases are critical regulators in shear stress-driven β-catenin signaling in osteoblasts. PMID:23524265

  8. EB1 Levels Are Elevated in Ascorbic Acid (AA)-stimulated Osteoblasts and Mediate Cell-Cell Adhesion-induced Osteoblast Differentiation*

    PubMed Central

    Pustylnik, Sofia; Fiorino, Cara; Nabavi, Noushin; Zappitelli, Tanya; da Silva, Rosa; Aubin, Jane E.; Harrison, Rene E.

    2013-01-01

    Osteoblasts are differentiated mesenchymal cells that function as the major bone-producing cells of the body. Differentiation cues including ascorbic acid (AA) stimulation provoke intracellular changes in osteoblasts leading to the synthesis of the organic portion of the bone, which includes collagen type I α1, proteoglycans, and matrix proteins, such as osteocalcin. During our microarray analysis of AA-stimulated osteoblasts, we observed a significant up-regulation of the microtubule (MT) plus-end binding protein, EB1, compared with undifferentiated osteoblasts. EB1 knockdown significantly impaired AA-induced osteoblast differentiation, as detected by reduced expression of osteoblast differentiation marker genes. Intracellular examination of AA-stimulated osteoblasts treated with EB1 siRNA revealed a reduction in MT stability with a concomitant loss of β-catenin distribution at the cell cortex and within the nucleus. Diminished β-catenin levels in EB1 siRNA-treated osteoblasts paralleled an increase in phospho-β-catenin and active glycogen synthase kinase 3β, a kinase known to target β-catenin to the proteasome. EB1 siRNA treatment also reduced the expression of the β-catenin gene targets, cyclin D1 and Runx2. Live immunofluorescent imaging of differentiated osteoblasts revealed a cortical association of EB1-mcherry with β-catenin-GFP. Immunoprecipitation analysis confirmed an interaction between EB1 and β-catenin. We also determined that cell-cell contacts and cortically associated EB1/β-catenin interactions are necessary for osteoblast differentiation. Finally, using functional blocking antibodies, we identified E-cadherin as a major contributor to the cell-cell contact-induced osteoblast differentiation. PMID:23740245

  9. The effect of variation in physical properties of porous bioactive glass on the expression and maintenance of the osteoblastic phenotype

    NASA Astrophysics Data System (ADS)

    Effah Kaufmann, Elsie Akosua Biraa

    pore sizes and porosity and determined the effect of substrate properties on the expression and maintenance of the osteoblastic phenotype, using an in vitro culture of osteoblast-like cells. Our data showed that porous bioactive glass substrates support the proliferation and maturation of osteoblast-like cells. Within the conditions of the experiment, we also found that at a given porosity of 44% the pore size of bioactive glass neither directs nor modulates the in vitro expression of the osteoblastic phenotype. On the other hand, at an average pore size of 92 mum, when cultures are maintained for 14 days, cell activity is greatly affected by the substrate porosity. As the porosity increases from 35% to 59%, osteoblast activity is adversely affected. (Abstract shortened by UMI.)

  10. The Retinoblastoma Tumor Suppressor Transcriptionally Represses Pak1 in Osteoblasts

    PubMed Central

    Sosa-García, Bernadette; Vázquez-Rivera, Viviana; González-Flores, Jonathan N.; Engel, Brienne E.; Cress, W. Douglas; Santiago-Cardona, Pedro G.

    2015-01-01

    We previously characterized the retinoblastoma tumor suppressor protein (Rb) as a regulator of adherens junction assembly and cell-to-cell adhesion in osteoblasts. This is a novel function since Rb is predominantly known as a cell cycle repressor. Herein, we characterized the molecular mechanisms by which Rb performs this function, hypothesizing that Rb controls the activity of known regulators of adherens junction assembly. We found that Rb represses the expression of the p21-activated protein kinase (Pak1), an effector of the small Rho GTPase Rac1. Rac1 is a well-known regulator of adherens junction assembly whose increased activity in cancer is linked to perturbations of intercellular adhesion. Using nuclear run-on and luciferase reporter transcription assays, we found that Pak1 repression by Rb is transcriptional, without affecting Pak1 mRNA and protein stability. Pak1 promoter bioinformatics showed multiple E2F1 binding sites within 155 base pairs of the transcriptional start site, and a Pak1-promoter region containing these E2F sites is susceptible to transcriptional inhibition by Rb. Chromatin immunoprecipitations showed that an Rb-E2F complex binds to the region of the Pak1 promoter containing the E2F1 binding sites, suggesting that Pak1 is an E2F target and that the repressive effect of Rb on Pak1 involves blocking the trans-activating capacity of E2F. A bioinformatics analysis showed elevated Pak1 expression in several solid tumors relative to adjacent normal tissue, with both Pak1 and E2F increased relative to normal tissue in breast cancer, supporting a cancer etiology for Pak1 up-regulation. Therefore, we propose that by repressing Pak1 expression, Rb prevents Rac1 hyperactivity usually associated with cancer and related to cytoskeletal derangements that disrupt cell adhesion, consequently enhancing cancer cell migratory capacity. This de-regulation of cell adhesion due to Rb loss could be part of the molecular events associated with cancer progression

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

    SciTech Connect

    Kawano, Michinao; Ariyoshi, Wataru; Iwanaga, Kenjiro; Okinaga, Toshinori; Habu, Manabu; Yoshioka, Izumi; Tominaga, Kazuhiro; Nishihara, Tatsuji

    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

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

    NASA Astrophysics Data System (ADS)

    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

  13. Edaravone protects osteoblastic cells from dexamethasone through inhibiting oxidative stress and mPTP opening.

    PubMed

    Sun, Wen-xiao; Zheng, Hai-ya; Lan, Jun

    2015-11-01

    Existing evidences have emphasized an important role of oxidative stress in dexamethasone (Dex)-induced osteoblastic cell damages. Here, we investigated the possible anti-Dex activity of edaravone in osteoblastic cells, and studied the underlying mechanisms. We showed that edaravone dose-dependently attenuated Dex-induced death and apoptosis of established human or murine osteoblastic cells. Further, Dex-mediated damages to primary murine osteoblasts were also alleviated by edaravone. In osteoblastic cells/osteoblasts, Dex induced significant oxidative stresses, tested by increased levels of reactive oxygen species and lipid peroxidation, which were remarkably inhibited by edaravone. Meanwhile, edaravone repressed Dex-induced mitochondrial permeability transition pore (mPTP) opening, or mitochondrial membrane potential reduction, in osteoblastic cells/osteoblasts. Significantly, edaravone-induced osteoblast-protective activity against Dex was alleviated with mPTP inhibition through cyclosporin A or cyclophilin-D siRNA. Together, we demonstrate that edaravone protects osteoblasts from Dex-induced damages probably through inhibiting oxidative stresses and following mPTP opening. PMID:26179849

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

    SciTech Connect

    Yang, Lei; Wu, Zhong; Yin, Gang; Liu, Haifeng; Guan, Xiaojun; Zhao, Xiaoqiang; Wang, Jianguang; Zhu, Jianguo

    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.

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

    PubMed

    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

  16. Establishment of Immortalized BMP2/4 Double Knock-Out Osteoblastic Cells Is Essential for Study of Osteoblast Growth, Differentiation, and Osteogenesis.

    PubMed

    Wu, Li-An; Wang, Feng; Donly, Kevin J; Baker, Andrew; Wan, Chunyan; Luo, Daoshu; MacDougall, Mary; Chen, Shuo

    2016-06-01

    Bone morphogenetic proteins 2 and 4 (BMP2/4) are essential for osteoblast differentiation and osteogenesis. Generation of a BMP2/4 dual knock-out ((ko/ko)) osteoblastic cell line is a valuable asset for studying effects of BMP2/4 on skeletal development. In this study, our goal was to create immortalized mouse deleted BMP2/4 osteoblasts by infecting adenoviruses with Cre recombinase and green fluorescent protein genes into immortalized murine floxed BMP2/4 osteoblasts. Transduced BMP2/4(ko/ko) cells were verified by green immunofluorescence and PCR. BMP2/4(ko/ko) osteoblasts exhibited small size, slow cell proliferation rate and cell growth was arrested in G1 and G2 phases. Expression of bone-relate genes was reduced in the BMP2/4(ko/ko) cells, resulting in delay of cell differentiation and mineralization. Importantly, extracellular matrix remodeling was impaired in the BMP2/4(ko/ko) osteoblasts as reflected by decreased Mmp-2 and Mmp-9 expressions. Cell differentiation and mineralization were rescued by exogenous BMP2 and/or BMP4. Therefore, we for the first time described establishment of an immortalized deleted BMP2/4 osteoblast line useful for study of mechanisms in regulating osteoblast lineages. PMID:26595646

  17. Cancer–Osteoblast Interaction Reduces Sost Expression in Osteoblasts and Up-Regulates lncRNA MALAT1 in Prostate Cancer

    PubMed Central

    Sebastian, Aimy; Hum, Nicholas R.; Hudson, Bryan D.; Loots, Gabriela G.

    2015-01-01

    Dynamic interaction between prostate cancer and the bone microenvironment is a major contributor to metastasis of prostate cancer to bone. In this study, we utilized an in vitro co-culture model of PC3 prostate cancer cells and osteoblasts followed by microarray based gene expression profiling to identify previously unrecognized prostate cancer–bone microenvironment interactions. Factors secreted by PC3 cells resulted in the up-regulation of many genes in osteoblasts associated with bone metabolism and cancer metastasis, including Mmp13, Il-6 and Tgfb2, and down-regulation of Wnt inhibitor Sost. To determine whether altered Sost expression in the bone microenvironment has an effect on prostate cancer metastasis, we co-cultured PC3 cells with Sost knockout (SostKO) osteoblasts and wildtype (WT) osteoblasts and identified several genes differentially regulated between PC3-SostKO osteoblast co-cultures and PC3-WT osteoblast co-cultures. Co-culturing PC3 cells with WT osteoblasts up-regulated cancer-associated long noncoding RNA (lncRNA) MALAT1 in PC3 cells. MALAT1 expression was further enhanced when PC3 cells were co-cultured with SostKO osteoblasts and treatment with recombinant Sost down-regulated MALAT1 expression in these cells. Our results suggest that reduced Sost expression in the tumor microenvironment may promote bone metastasis by up-regulating MALAT1 in prostate cancer.

  18. Establishment of Immortalized BMP2/4 Double Knock‐Out Osteoblastic Cells Is Essential for Study of Osteoblast Growth, Differentiation, and Osteogenesis

    PubMed Central

    Wu, Li‐An; Wang, Feng; Donly, Kevin J.; Baker, Andrew; Wan, Chunyan; Luo, Daoshu; MacDougall, Mary

    2015-01-01

    Bone morphogenetic proteins 2 and 4 (BMP2/4) are essential for osteoblast differentiation and osteogenesis. Generation of a BMP2/4 dual knock‐out (ko/ko) osteoblastic cell line is a valuable asset for studying effects of BMP2/4 on skeletal development. In this study, our goal was to create immortalized mouse deleted BMP2/4 osteoblasts by infecting adenoviruses with Cre recombinase and green fluorescent protein genes into immortalized murine floxed BMP2/4 osteoblasts. Transduced BMP2/4ko/ko cells were verified by green immunofluorescence and PCR. BMP2/4ko/ko osteoblasts exhibited small size, slow cell proliferation rate and cell growth was arrested in G1 and G2 phases. Expression of bone‐relate genes was reduced in the BMP2/4ko/ko cells, resulting in delay of cell differentiation and mineralization. Importantly, extracellular matrix remodeling was impaired in the BMP2/4ko/ko osteoblasts as reflected by decreased Mmp‐2 and Mmp‐9 expressions. Cell differentiation and mineralization were rescued by exogenous BMP2 and/or BMP4. Therefore, we for the first time described establishment of an immortalized deleted BMP2/4 osteoblast line useful for study of mechanisms in regulating osteoblast lineages. J. Cell. Physiol. 231: 1189–1198, 2016. © 2015 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc. PMID:26595646

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

    PubMed

    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

  20. The potential role of the osteoblast in the development of periprosthetic osteolysis: review of in vitro osteoblast responses to wear debris, corrosion products, and cytokines and growth factors.

    PubMed

    Vermes, C; Glant, T T; Hallab, N J; Fritz, E A; Roebuck, K A; Jacobs, J J

    2001-12-01

    Limited information is available on the responses of osteoblasts to wear debris, corrosion products, and cytokines and on the roles of altered osteoblast functions in the development of periprosthetic bone loss. Wear debris-challenged osteoblasts exhibit altered functions resulting in the loss of their capacity to produce bone matrix and to replace the resorbed bone. Also, osteoblasts may secrete cytokines, which act in a paracrine fashion to recruit inflammatory cells into the periprosthetic space and to stimulate osteoclastic bone resorption. These effects may be mediated in part by ionic metal dissolution products. We review the mechanisms by which altered osteoblast functions, in response to particulate wear debris, corrosion products, and cytokines and growth factors, may contribute to the development and the progression of periprosthetic osteolysis. PMID:11742458

  1. Cytocompatibility of Ti-6Al-4V and Ti-5Al-2.5Fe alloys according to three surface treatments, using human fibroblasts and osteoblasts.

    PubMed

    Bordji, K; Jouzeau, J Y; Mainard, D; Payan, E; Netter, P; Rie, K T; Stucky, T; Hage-Ali, M

    1996-05-01

    Titanium alloys are well known for their superior mechanical properties as well as for their good biocompatibility, making them desirable as surgical implant materials. However, these alloys have been proven to behave poorly in friction since wear particles were often detected in tissues and organs associated with titanium implants. In this paper, three surface treatments were investigated in order to improve the wear resistance and the hardness of Ti-6Al-4V and Ti-5Al-2.5Fe: (a) glow discharge nitrogen implantation (10(17) atoms cm-2), (b) plasma nitriding by plasma diffusion treatment (PDT) and (c) deposition of TiN layer by plasma-assisted chemical vapour deposition (PACVD) additionally to PDT. Surface characterization after the different treatments showed considerable improvement in surface hardness, especially after the two nitriding processes. Moreover, the good corrosion resistance of untreated alloys was maintained. A cell culture model using human cells was chosen to study the effect of such treatments on the cytocompatibility of these materials. The results showed that Ti-5Al-2.5Fe alloy was as cytocompatible as the Ti-6Al-4V alloy and the same surface treatment led to identical biological consequences on both alloys. Nitrogen implantation did not modify at all the cellular behaviour observed on untreated samples. After the two nitriding treatments, cell proliferation and viability appeared to be significantly reduced and the scanning electron microscopy study revealed somewhat irregular surface states. However, osteoblast phenotype expression and protein synthesis capacity were not affected. PDT and PACVD may be interesting alternatives to the physical vapour deposition technique. PMID:8718939

  2. Effects of low level laser therapy (LLLT) on pressured human osteoblasts: A histomorphologic and quantitative study

    NASA Astrophysics Data System (ADS)

    Pyo, S. J.; Song, W. W.; Kim, I. R.; Park, B. S.; Kim, C. H.; Kim, S. S.; Chung, I. K.; Kim, Y. D.

    2012-03-01

    Previous research has investigated the effects of LLLT during titanium implantation, tooth movement and bone graft using deproteinized bovine bone and recognized that these circumstances were nothing more than intentional controlled overpressure against static cells since this controlled trauma could affect cell function/malfunction, or cell recovery/apoptosis. The present preliminary study was conducted to prove if LLL would influence cell viability and cell function after excessive damage, which is enough to diminish cell numbers and distort the features of cells. Our aim is to evaluate whether low level laser irradiation (LLLi) could be helpful in the recovery of traumatized osteoblasts (pressure damaged cells) by observing the morphology and the survival rate of those cells. This model used bone cell cultures which were traumatized by a pressure with 250 G of centripetal force and observed their response to such trauma and low level laser irradiation. In this experiment, a Ga-Al-As diode LLL (IMPRA-ORT, NDLux, Seoul, KOREA) was used with a wavelength of 808 nm, a focus of 14 × 24 mm, which was wide enough to cover the whole dish surface or well within at least 2 times radiation, and an output of 100 mW. Statistical analysis showed a higher recovery rate of damaged osteoblasts in the radiation group than the non-radiation group ( p < 0.05). The nonradiation group had a very poor proliferation rate in comparison to the control group ( p < 0.05) in every time period. In the control group, actin filaments showed a random orientation and cell process branched variously around each cell. In contrast, compressed cells, these patterns were turned into thicker and shorter cytoskeletons. As time progressed, every living cell recovered from the severe stress and recovered both form and function. In summary, the present study showed the capacity of LLLT to aid the recovery of the cell skeleton and affect cell viability on overpressured osteoblasts. These results may

  3. Mechanical regulation of osteoclastic genes in human osteoblasts

    SciTech Connect

    Kreja, Ludwika Liedert, Astrid; Hasni, Sofia; Claes, Lutz; Ignatius, Anita

    2008-04-11

    Bone adaptation to mechanical load is accompanied by changes in gene expression of bone-forming cells. Less is known about mechanical effects on factors controlling bone resorption by osteoclasts. Therefore, we studied the influence of mechanical loading on several key genes modulating osteoclastogenesis. Human osteoblasts were subjected to various cell stretching protocols. Quantitative RT-PCR was used to evaluate gene expression. Cell stretching resulted in a significant up-regulation of receptor activator of nuclear factor-{kappa}B ligand (RANKL) immediate after intermittent loading (3 x 3 h, 3 x 6 h, magnitude 1%). Continuous loading, however, had no effect on RANKL expression. The expression of osteoprotegerin (OPG), macrophage-colony stimulating factor (M-CSF), and osteoclast inhibitory lectin (OCIL) was not significantly altered. The data suggested that mechanical loading could influence osteoclasts recruitment by modulating RANKL expression in human osteoblasts and that the effects might be strictly dependent on the quality of loading.

  4. Arachidonic acid and prostaglandin E2 influence human osteoblast (MG63) response to titanium surface roughness.

    PubMed

    Dean, David D; Campbell, Casey M; Gruwell, Scott F; Tindall, John W M; Chuang, Hui-Hsiu; Zhong, Weinan; Schmitz, John P; Sylvia, Victor L

    2008-01-01

    Prior studies have shown that implant surface roughness affects osteoblast proliferation, differentiation, matrix synthesis, and local factor production. Further, cell response is modulated by systemic factors, such as 1,25(OH)2D3 and estrogen as well as mechanical forces. Based on the fact that peri-implant bone healing occurs in a site containing elevated amounts of prostaglandin E2 (PGE2), the hypothesis of the current study is that PGE2 and arachidonic acid (AA), the substrate used by cyclooxygenase to form PGE2, influence osteoblast response to implant surface roughness. To test this hypothesis, 4 different types of commercially pure titanium (cpTi) disks with surfaces of varying roughness (smooth Ti, R(a) 0.30 microm; smooth and acid etched Ti [SAE Ti], R(a) 0.40 microm; rough Ti, R(a) 4.3 microm; rough and acid etched Ti [RAE Ti], R(a) 4.15 (microm) were prepared. MG63 osteoblasts were seeded onto the surfaces, cultured to confluence, and then treated for the last 24 hours of culture with AA (0, 0.1, 1, and 10 nM), PGE2 (0, 1, 10, 25, and 100 nM), or the general cyclooxygenase inhibitor indomethacin (0 or 100 nM). At harvest, the effect of treatment on cell proliferation was assessed by measuring cell number and [3H]-thymidine incorporation, and the effect on cell differentiation was determined by measuring alkaline phosphatase (ALP) specific activity. The effect of AA and PGE2 on cell number was somewhat variable but showed a general decrease on plastic and smooth surfaces and an increase on rough surfaces. In contrast, [3H]-thymidine incorporation was uniformly decreased with treatment on all surfaces. ALP demonstrated the most prominent effect of treatment. On smooth surfaces, AA and PGE2 dose-dependently increased ALP, while on rough surfaces, treatment dose-dependently decreased enzyme specific activity. Indomethacin treatment had either no effect or a slightly inhibitory effect on [3H]-thymidine incorporation on all surfaces. In contrast, indomethacin

  5. Porous ongrowth surfaces alter osteoblast maturation and mineralization.

    PubMed

    Ninomiya, James T; Struve, Janine A; Krolikowski, John; Hawkins, Michael; Weihrauch, Dorothee

    2015-01-01

    Implant fixation through osseointegration is essential for the success of uncemented total joint arthroplasty, and nature and composition of implant surface play a critical role in this process. Despite widespread use of uncemented implants, the extent of bone ingrowth into implants is generally only a small percentage of the total implant surface. An understanding of the processes whereby bone cells grow into and multiply on porous surfaces is critical for the design and manufacture of implants that maximize ingrowth and implant fixation. A wide variety of implant materials are currently utilized for uncemented total joint arthroplasty, including titanium mesh, cobalt chromium beads, and tantalum deposited on a carbon network. Despite differences in physical and chemical properties of these materials, all have functioned well clinically. Therefore, the goals of this study were to compare and contrast the effects of these materials on the proliferation, phenotypic maturation, and mineralization of osteoblasts. Disks of porous tantalum, titanium mesh, and cobalt chromium beaded surfaces were fabricated and processed employing the same methods used to produce implants, including packaging and sterilization. Preosteoblasts were plated on disks, cellular morphology was evaluated by scanning electron microscopy. Osteoblast proliferation was significantly higher on the porous tantalum compared to other implant surfaces. Alkaline phosphatase activity, osteocalcin secretion, and upregulation of RUNX2 were inversely proportional to the rate of proliferation. Mineralization of osteoblasts paralleled the rate of proliferation. These findings suggest that proliferation of osteoblasts into the interstices of implant materials along with delayed maturation were favorable for increased bone ongrowth and ultimately implant stabilization. PMID:24677492

  6. Development of osteoblast colonies on new bioactive coatings

    NASA Astrophysics Data System (ADS)

    Legoux, J. G.; Chellat, F.; Lima, R. S.; Marple, B. R.; Bureau, M. N.; Shen, H.; Candeliere, G. A.

    2006-12-01

    The aging baby boomer population coupled with an increase in life expectancy is leading to a rising number of active elderly persons in occidental countries. As a result, the orthopedic implant industry is facing numerous challenges such as the need to extend implant life, reduce the incidence of revision surgery, and improve implant performance. This paper reports results of an investigation on the bioperformance of newly developed coating-substrate systems. Hydroxyapatite (HA) and nano-titania (nano-TiO2) coatings were produced on Ti-6Al-4V and fiber reinforced polymer composite substrates. In vitro studies were conducted to determine the capacity of bioactive coatings developed to sustain osteoblast cells (fetal rat calvaria) adherence, growth, and differentiation. As revealed by scanning electron microscopy (SEM) observations and alkaline phosphatase activity, cell adhesion and proliferation demonstrated that HA coatings over a polymer composite are at least as good as HA coatings made over Ti-6Al-4V substrate in terms of osteoblast cell activity. Nano-TiO2 coatings produced by high-velocity oxyfuel (HVOF) spraying led to different results. For short-term cell culture (4.5 and 24 h), the osteoblasts appeared more flattened when grown on nano-TiO2 than on HA. The surface cell coverage after seven days of incubation was also more complete on nano-TiO2 than HA. Preliminary results indicate that osteoblast activity after 15 days of incubation on nano-TiO2 is equivalent to or greater than that observed on HA.

  7. Are cementoblasts a subpopulation of osteoblasts or a unique phenotype?

    PubMed

    Bosshardt, D D

    2005-05-01

    Experimental studies have shown a great potential for periodontal regeneration. The limitations of periodontal regeneration largely depend on the regenerative potential at the root surface. Cellular intrinsic fiber cementum (CIFC), so-called bone-like tissue, may form instead of the desired acellular extrinsic fiber cementum (AEFC), and the interfacial tissue bonding may be weak. The periodontal ligament harbors progenitor cells that can differentiate into periodontal ligament fibroblasts, osteoblasts, and cementoblasts, but their precise location is unknown. It is also not known whether osteoblasts and cementoblasts arise from a common precursor cell line, or whether distinct precursor cell lines exist. Thus, there is limited knowledge about how cell diversity evolves in the space between the developing root and the alveolar bone. This review supports the hypothesis that AEFC is a unique tissue, while CIFC and bone share some similarities. Morphologically, functionally, and biochemically, however, CIFC is distinctly different from any bone type. There are several lines of evidence to propose that cementoblasts that produce both AEFC and CIFC are unique phenotypes that are unrelated to osteoblasts. Cementum attachment protein appears to be cementum-specific, and the expression of two proteoglycans, fibromodulin and lumican, appears to be stronger in CIFC than in bone. A theory is presented that may help explain how cell diversity evolves in the periodontal ligament. It proposes that Hertwig's epithelial root sheath and cells derived from it play an essential role in the development and maintenance of the periodontium. The role of enamel matrix proteins in cementoblast and osteoblast differentiation and their potential use for tissue engineering are discussed. PMID:15840773

  8. Platelet-rich plasma stimulates osteoblastic differentiation in the presence of BMPs

    SciTech Connect

    Tomoyasu, Akihiro; Higashio, Kanji; Kanomata, Kazuhiro; Goto, Masaaki; Kodaira, Kunihiko; Serizawa, Hiroko; Suda, Tatsuo; Nakamura, Atsushi; Nojima, Junya; Fukuda, Toru; Katagiri, Takenobu . E-mail: katagiri@saitama-med.ac.jp

    2007-09-14

    Platelet-rich plasma (PRP) is clinically used as an autologous blood product to stimulate bone formation in vivo. In the present study, we examined the effects of PRP on proliferation and osteoblast differentiation in vitro in the presence of bone morphogenetic proteins (BMPs). PRP and its soluble fraction stimulated osteoblastic differentiation of myoblasts and osteoblastic cells in the presence of BMP-2, BMP-4, BMP-6 or BMP-7. The soluble PRP fraction stimulated osteoblastic differentiation in 3D cultures using scaffolds made of collagen or hydroxyapatite. Moreover, heparin-binding fractions obtained from serum also stimulated osteoblastic differentiation in the presence of BMP-4. These results suggested that platelets contain not only growth factors for proliferation but also novel potentiator(s) for BMP-dependent osteoblastic differentiation.

  9. Mechanoresponses of human primary osteoblasts grown on carbon nanotubes.

    PubMed

    Kroustalli, A; Kotsikoris, V; Karamitri, A; Topouzis, S; Deligianni, D

    2015-03-01

    Bone mechanotransduction is strongly influenced by the biomaterial properties. A good understanding of these mechanosensory mechanisms in bone has the potential to provide new strategies in the highly evolving field of bone tissue engineering. The aim of the present investigation was to study the interactive effects of local mechanical stimuli on multiwalled carbon nanotubes (MWCNTs)/osteoblast interface, using an in vitro model that allows the study of cell growth, attachment and differentiation. Strain was applied at physiological levels [strain magnitudes 500 microstrain (μɛ), at frequency of load application 0.5 Hz]. The effect of mechanical strain and substrate was thus studied by measuring the messenger RNA expression of alkaline phosphatase, vinculin, collagen 1A, and integrins β1, β3, α4, and αv, using real-time quantitative polymerase chain reaction. The osteoblasts grown on MWCNTs displayed quick adaptation to the new environment by modulating the expression of key adhesion integrins. Furthermore, the addition of mechanical strain interplayed with the extracellular matrix and was efficiently transduced by cells grown on MWCNTs, providing stronger adhesion and survival. MWCNTs are therefore a material perfectly compatible with osteoblast differentiation, adhesion, and growth, and should be further evaluated, to derive new-generation biomaterial scaffolds for the treatment of skeletal defects which require bone reconstruction. PMID:24910375

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

    PubMed

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

    2016-08-14

    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. PMID:27412794

  11. Microcracks induce osteoblast alignment and maturation on hydroxyapatite scaffolds

    NASA Astrophysics Data System (ADS)

    Shu, Yutian

    Physiological bone tissue is a mineral/collagen composite with a hierarchical structure. The features in bone, such as mineral crystals, fibers, and pores can range from the nanometer to the centimeter in size. Currently available bone tissue scaffolds primarily address the chemical composition, pore size, and pore size distribution. While these design parameters are extensively investigated for mimicking bone function and inducing bone regeneration, little is known about microcracks, which is a prevalent feature found in fractured bone in vivo and associated with fracture healing and repair. Since the purpose of bone tissue engineering scaffold is to enhance bone regeneration, the coincidence of microcracks and bone densification should not be neglected but rather be considered as a potential parameter in bone tissue engineering scaffold design. The purpose of this study is to test the hypothesis that microcracks enhance bone healing. In vitro studies were designed to investigate the osteoblast (bone forming cells) response to microcracks in dense (94%) hydroxyapatite substrates. Microcracks were introduced using a well-established Vickers indentation technique. The results of our study showed that microcracks induced osteoblast alignment, enhanced osteoblast attachment and more rapid maturation. These findings may provide insight into fracture healing mechanism(s) as well as improve the design of bone tissue engineering orthopedic scaffolds for more rapid bone regeneration.

  12. Osteoblast growth behavior on porous-structure titanium surface

    NASA Astrophysics Data System (ADS)

    Tian, Yuan; Ding, Siyang; Peng, Hui; Lu, Shanming; Wang, Guoping; Xia, Lu; Wang, Peizhi

    2012-11-01

    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.

  13. Biodistribution of locally or systemically transplanted osteoblast-like cells

    PubMed Central

    Okabe, Y. T.; Kondo, T.; Mishima, K.; Hayase, Y.; Kato, K.; Mizuno, M.; Ishiguro, N.; Kitoh, H.

    2014-01-01

    Objectives In order to ensure safety of the cell-based therapy for bone regeneration, we examined in vivo biodistribution of locally or systemically transplanted osteoblast-like cells generated from bone marrow (BM) derived mononuclear cells. Methods BM cells obtained from a total of 13 Sprague-Dawley (SD) green fluorescent protein transgenic (GFP-Tg) rats were culture-expanded in an osteogenic differentiation medium for three weeks. Osteoblast-like cells were then locally transplanted with collagen scaffolds to the rat model of segmental bone defect. Donor cells were also intravenously infused to the normal Sprague-Dawley (SD) rats for systemic biodistribution. The flow cytometric and histological analyses were performed for cellular tracking after transplantation. Results Locally transplanted donor cells remained within the vicinity of the transplantation site without migrating to other organs. Systemically administered large amounts of osteoblast-like cells were cleared from various organ tissues within three days of transplantation and did not show any adverse effects in the transplanted rats. Conclusions We demonstrated a precise assessment of donor cell biodistribution that further augments prospective utility of regenerative cell therapy. PMID:24652780

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

    PubMed

    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

  15. Defective osteoblast function in ICAP-1-deficient mice

    PubMed Central

    Bouvard, Daniel; Aszodi, Attila; Kostka, Günter; Block, Marc R.; Albigès-Rizo, Corinne; Fässler, Reinhard

    2007-01-01

    SUMMARY The integrin receptor family plays important roles in cell-to-cell and cell-to-extracellular matrix (ECM) interactions through the recruitment of accessory molecules. One of them is the integrin cytoplasmic domain-associated protein-1 (ICAP-1), which specifically interacts with the cytoplasmic domain of β1 integrin subunit and negatively regulates its function in vitro. To address the role of ICAP-1 in vivo, we ablated the Icap-1 gene in mice. Here we report an unexpected role of ICAP-1 for osteoblast function during bone development. Icap-1-deficient mice suffer from a reduced osteoblast proliferation and delayed bone mineralization, giving rise to a retarded formation of bone sutures. In vitro studies revealed that primary and immortalized Icap-1-null osteoblasts display enhanced adhesion and spreading on extracellular matrix substrates likely due to an increase in β1 integrin activation. Finally, we provide evidence that ICAP-1 promotes differentiation of osteoprogenitors by supporting their condensation through modulating the integrin high affinity state. PMID:17567669

  16. Estrogen Receptor α Regulates Dlx3-Mediated Osteoblast Differentiation

    PubMed Central

    Lee, Sung Ho; Oh, Kyo-Nyeo; Han, Younho; Choi, You Hee; Lee, Kwang-Youl

    2016-01-01

    Estrogen receptor α (ER-α), which is involved in bone metabolism and breast cancer, has been shown to have transcriptional targets. Dlx3 is essential for the skeletal development and plays an important role in osteoblast differentiation. Various osteogenic stimulators and transcription factors can induce the protein expression of Dlx3. However, the regulatory function of ER-α in the Dlx3 mediated osteogenic process remains unknown. Therefore, we investigated the regulation of Dlx3 and found that ER-α is a positive regulator of Dlx3 transcription in BMP2-induced osteoblast differentiation. We also found that ER-α interacts with Dlx3 and increases its transcriptional activity and DNA binding affinity. Furthermore, we demonstrated that the regulation of Dlx3 activity by ER-α is independent of the ligand (estradiol) binding domain. These results indicate that Dlx3 is a novel target of ER-α, and that ER-α regulates the osteoblast differentiation through modulation of Dlx3 expression and/or interaction with Dlx3. PMID:26674964

  17. Tailoring nanocrystalline diamond coated on titanium for osteoblast adhesion.

    PubMed

    Pareta, Rajesh; Yang, Lei; Kothari, Abhishek; Sirinrath, Sirivisoot; Xiao, Xingcheng; Sheldon, Brian W; Webster, Thomas J

    2010-10-01

    Diamond coatings with superior chemical stability, antiwear, and cytocompatibility properties have been considered for lengthening the lifetime of metallic orthopedic implants for over a decade. In this study, an attempt to tailor the surface properties of diamond films on titanium to promote osteoblast (bone forming cell) adhesion was reported. The surface properties investigated here included the size of diamond surface features, topography, wettability, and surface chemistry, all of which were controlled during microwave plasma enhanced chemical-vapor-deposition (MPCVD) processes using CH4-Ar-H2 gas mixtures. The hardness and elastic modulus of the diamond films were also determined. H2 concentration in the plasma was altered to control the crystallinity, grain size, and topography of the diamond coatings, and specific plasma gases (O2 and NH3) were introduced to change the surface chemistry of the diamond coatings. To understand the impact of the altered surface properties on osteoblast responses, cell adhesion tests were performed on the various diamond-coated titanium. The results revealed that nanocrystalline diamond (grain sizes <100 nm) coated titanium dramatically increased surface hardness, and the introduction of O2 and NH3 during the MPCVD process promoted osteoblast adhesion on diamond and, thus, should be further studied for improving orthopedic applications. PMID:20540097

  18. Circadian rhythm of mechanically mediated differentiation of osteoblasts.

    PubMed

    Roberts, W E; Klingler, E; Mozsary, P G

    1984-01-01

    Rats entrained to alternating 12 h light/dark periods were sacrificed at hourly intervals over one complete circadian cycle. Each animal was injected with 3H-Thymidine 1 h before death. Autoradiographs of serial sections of maxillary first molar periodontal ligament (PDL) were prepared. Nuclear volume was determined for labeled fibroblastlike PDL cells along a physiological bone forming surface. Preosteoblasts (large nuclei), the immediate proliferating precursors of osteoblasts, were found to synthesize DNA primarily during the environmental light period and divide during the subsequent dark cycle. Less differentiated precursor cells (small nuclei), the proliferating predecessors of preosteoblasts, were in S phase primarily during the dark period and divided in the following light cycle. Since previous studies have indicated, the stress/strain-mediated increase in nuclear size to form preosteoblasts also requires about 8-12 h, the least complex osteoblast differentiation model, which is consistent with the present data, is a 60 h sequence involving at least four cell types and five alternating dark/light cycles. The principal rate-limiting step in osteoblast differentiation is the mechanically related shift in nuclear size (change in genomic expression) associated with formation of preosteoblasts. PMID:6430524

  19. Inhibition of fatty acid biosynthesis prevents adipocyte lipotoxicity on human osteoblasts in vitro.

    PubMed

    Elbaz, Alexandre; Wu, Xiying; Rivas, Daniel; Gimble, Jeffrey M; Duque, Gustavo

    2010-04-01

    Although increased bone marrow fat in age-related bone loss has been associated with lower trabecular mass, the underlying mechanism responsible remains unknown. We hypothesized that marrow adipocytes exert a lipotoxic effect on osteoblast function and survival through the reversible biosynthesis of fatty acids (FA) into the bone marrow microenvironment. We have used a two-chamber system to co-culture normal human osteoblasts (NHOst) with differentiating pre-adipocytes in the absence or presence of an inhibitor of FA synthase (cerulenin) and separated by an insert that allowed unidirectional trafficking of soluble factors only and prevented direct cell-cell contact. Supernatants were assayed for the presence of FA using mass spectophotometry. After 3 weeks in co-culture, NHOst showed significantly lower levels of differentiation and function based on lower mineralization and expression of alkaline phosphatase, osterix, osteocalcin and Runx2. In addition, NHOst survival was affected by the presence of adipocytes as determined by MTS-formazan and TUNEL assays as well as higher activation of caspases 3/7. These toxic effects were inhibited by addition of cerulenin. Furthermore, culture of NHOst with either adipocyte-conditioned media alone in the absence of adipocytes themselves or with the addition of the most predominant FA (stearate or palmitate) produced similar toxic results. Finally, Runx2 nuclear binding was affected by addition of either adipocyte conditioned media or FA into the osteogenic media. We conclude that the presence of FA within the marrow milieu can contribute to the age-related changes in bone mass and can be prevented by the inhibition of FA synthase. PMID:19382912

  20. Inhibition of fatty acid biosynthesis prevents adipocyte lipotoxicity on human osteoblasts in vitro

    PubMed Central

    Elbaz, Alexandre; Wu, Xiying; Rivas, Daniel; Gimble, Jeffrey M; Duque, Gustavo

    2010-01-01

    Abstract Although increased bone marrow fat in age-related bone loss has been associated with lower trabecular mass, the underlying mechanism responsible remains unknown. We hypothesized that marrow adipocytes exert a lipotoxic effect on osteoblast function and survival through the reversible biosynthesis of fatty acids (FA) into the bone marrow microenvironment. We have used a two-chamber system to co-culture normal human osteoblasts (NHOst) with differentiating pre-adipocytes in the absence or presence of an inhibitor of FA synthase (cerulenin) and separated by an insert that allowed unidirectional trafficking of soluble factors only and prevented direct cell–cell contact. Supernatants were assayed for the presence of FA using mass spectophotometry. After 3 weeks in co-culture, NHOst showed significantly lower levels of differentiation and function based on lower mineralization and expression of alkaline phosphatase, osterix, osteocalcin and Runx2. In addition, NHOst survival was affected by the presence of adipocytes as determined by MTS-formazan and TUNEL assays as well as higher activation of caspases 3/7. These toxic effects were inhibited by addition of cerulenin. Furthermore, culture of NHOst with either adipocyte-conditioned media alone in the absence of adipocytes themselves or with the addition of the most predominant FA (stearate or palmitate) produced similar toxic results. Finally, Runx2 nuclear binding was affected by addition of either adipocyte conditioned media or FA into the osteogenic media. We conclude that the presence of FA within the marrow milieu can contribute to the age-related changes in bone mass and can be prevented by the inhibition of FA synthase. PMID:19382912

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

    NASA Technical Reports Server (NTRS)

    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

  2. Individual differences in behavioural plasticities.

    PubMed

    Stamps, Judy A

    2016-05-01

    Interest in individual differences in animal behavioural plasticities has surged in recent years, but research in this area has been hampered by semantic confusion as different investigators use the same terms (e.g. plasticity, flexibility, responsiveness) to refer to different phenomena. The first goal of this review is to suggest a framework for categorizing the many different types of behavioural plasticities, describe examples of each, and indicate why using reversibility as a criterion for categorizing behavioural plasticities is problematic. This framework is then used to address a number of timely questions about individual differences in behavioural plasticities. One set of questions concerns the experimental designs that can be used to study individual differences in various types of behavioural plasticities. Although within-individual designs are the default option for empirical studies of many types of behavioural plasticities, in some situations (e.g. when experience at an early age affects the behaviour expressed at subsequent ages), 'replicate individual' designs can provide useful insights into individual differences in behavioural plasticities. To date, researchers using within-individual and replicate individual designs have documented individual differences in all of the major categories of behavioural plasticities described herein. Another important question is whether and how different types of behavioural plasticities are related to one another. Currently there is empirical evidence that many behavioural plasticities [e.g. contextual plasticity, learning rates, IIV (intra-individual variability), endogenous plasticities, ontogenetic plasticities) can themselves vary as a function of experiences earlier in life, that is, many types of behavioural plasticity are themselves developmentally plastic. These findings support the assumption that differences among individuals in prior experiences may contribute to individual differences in behavioural

  3. The Transcription Factor EB (TFEB) Regulates Osteoblast Differentiation Through ATF4/CHOP-Dependent Pathway.

    PubMed

    Yoneshima, Erika; Okamoto, Kuniaki; Sakai, Eiko; Nishishita, Kazuhisa; Yoshida, Noriaki; Tsukuba, Takayuki

    2016-06-01

    Osteoblasts are bone-forming cells that produce large amounts of collagen type I and various bone matrix proteins. Although osteoblast differentiation is highly regulated by various factors, it remains unknown whether lysosomes are directly involved in osteoblast differentiation. Here, we demonstrate the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, modulates osteoblast differentiation. The expression levels of TFEB as well as those of endosomal/lysosomal proteins were up-regulated during osteoblast differentiation using mouse osteoblastic MC3T3-E1 cells. By gene knockdown (KD) experiments with small interfering RNA (siRNA), TFEB depletion caused markedly reduced osteoblast differentiation as compared with the control cells. Conversely, overexpression (OE) of TFEB resulted in strikingly enhanced osteoblastogenesis compared to the control cells. By analysis of down-stream effector molecules, TFEB KD was found to cause marked up-regulation of activating transcription factor 4 (ATF4) and CCAAT/enhancer-binding protein homologous protein (CHOP), both of which are essential factors for osteoblastogenesis. In contrast, TFEB OE promoted osteoblast differentiation through reduced expression of ATF4 and CHOP without differentiation agents. Given the importance of ATF4 and CHOP in osteoblastogenesis, it is clear that the TFEB-regulated signaling pathway for osteoblast differentiation is involved in ATF4/CHOP-dependent signaling pathway. PMID:26519689

  4. 17-β-estradiol up-regulates apolipoprotein genes expression during osteoblast differentiation in vitro.

    PubMed

    Gui, Yuyan; Chu, Nan; Qiu, Xuemin; Tang, Wei; Gober, Hans-Jürgen; Li, Dajin; Wang, Ling

    2016-05-23

    Apolipoproteins are of great physiological importance and are associated with different diseases. Many independent studies of patterns of gene expression during osteoblast differentiation have been described, and some apolipoproteins have been induced during this process. 17-β-estradiol (E2) may enhance osteoblast physiological function. However, no studies have indicated whether E2 can modulate the expression of apolipoproteins during osteoblast differentiation in vitro. The aim of the current study was to observe the regulation of apolipoprotein mRNA expression by E2 during this process. Primary osteoblasts were collected from the calvaria of newborn mice and were subjected to osteoblast differentiation in vitro with serial concentrations of E2. RNA was isolated on days 0, 5, and 25 of differentiation. Real-time PCR was performed to analyze the levels of apolipoprotein mRNA. Results showed that during osteoblast differentiation all of the apolipoprotein genes were up-regulated by E2 in a dose-dependent manner. Moreover, only ApoE was strongly induced during the mineralization of cultured osteoblasts. This result suggests that ApoE might be involved in osteoblast differentiation. The hypothesis is that E2 promotes osteoblast differentiation by up-regulating ApoE gene expression, though further study is needed to confirm this hypothesis. PMID:27074899

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

    PubMed

    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

  6. Soy Protein Isolate Inhibits High-Fat Diet-Induced Senescence Pathways in Osteoblasts to Maintain Bone Acquisition in Male Rats

    PubMed Central

    Lazarenko, Oxana P.; Blackburn, Michael L.; Badger, Thomas M.; Ronis, Martin J. J.

    2015-01-01

    Chronic consumption by experimental animals of a typical Western diet high in saturated fats and cholesterol during postnatal life has been demonstrated to impair skeletal development. However, the underlying mechanism by which high-fat, energy-dense diets affect bone-forming cell phenotypes is poorly understood. Here, we show that male weanling rats fed a diet containing 45% fat and 0.5% cholesterol made with casein (HF-Cas) for 6 weeks displayed lower bone mineral density and strength compared with those of AIN-93G–fed dietary controls. Substitution of casein with soy protein isolate (SPI) in the high-fat diet (HF-SPI) prevented these effects. The bone-sparing effects of SPI were associated with prevention of HF-Cas–induced osteoblast senescence pathways through suppression of the p53/p21 signaling pathways. HF-Cas–fed rats had increased caveolin-1 and down-regulated Sirt1, leading to activations of peroxisome proliferator–activated receptor γ (PPARγ) and p53/p21, whereas rats fed HF-SPI suppressed caveolin-1 and activated Sirt1 to deacetylate PPARγ and p53 in bone. Treatment of osteoblastic cells with nonesterified free fatty acid (NEFA) increased cell senescence signaling pathways. Isoflavones significantly blocked activations of senescence-associated β-galactosidase and PPARγ/p53/p21 by NEFA. Finally, replicative senescent osteoblastic cells and bone marrow mesenchymal ST2 cells exhibited behavior similar to that of cells treated with NEFA and in vivo bone cells in rats fed the HF-Cas diet. These results suggest that (1) high concentrations of NEFA occurring with HF intake are mediators of osteoblast cell senescence leading to impairment of bone development and acquisition and (2) the molecular mechanisms underlying the SPI-protective effects involve isoflavone-induced inhibition of osteoblastic cell senescence to prevent HF-induced bone impairments. PMID:25490147

  7. Contribution of human osteoblasts and macrophages to bone matrix degradation and proinflammatory cytokine release after exposure to abrasive endoprosthetic wear particles

    PubMed Central

    Jonitz-Heincke, Anika; Lochner, Katrin; Schulze, Christoph; Pohle, Diana; Pustlauk, Wera; Hansmann, Doris; Bader, Rainer

    2016-01-01

    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 concomitant

  8. Expression of precerebellins in cultured rat calvaria osteoblast-like cells.

    PubMed

    Rucinski, Marcin; Zok, Agnieszka; Guidolin, Diego; De Caro, Raffaele; Malendowicz, Ludwik K

    2008-10-01

    Cerebellin (CER), originally isolated from rat cerebellum, is a hexadecapeptide derived from the larger precursor called precerebellin 1 (Cbln1). At present 4 propeptides designated as Cbln1, Cbln2, Cbln3 and Cbln4 are recognized. They belong to precerebellin subfamily of the C1q family proteins. Precerebellins act as transneuronal regulators of synapse development and synaptic plasticity in various brain regions. Initially CER was thought to be a cerebellum specific peptide, however subsequent studies revealed its presence in other brain regions as well as in extraneuronal tissues. We investigated whether precerebellins are expressed and involved in regulation of cultured rat calvarial osteoblast-like (ROB) cells. Classic RT-PCR revealed the presence of Cbln1 and Cbln3 mRNA in fragments of rat calvaria, in freshly isolated ROB cells and in ROB cells cultured for 7, 14 and 21 days. Cbln2 and Cbln4 mRNA, on the other hand, could not be demonstrated in ROB cells but was found to be present in the brain. In freshly isolated ROB cells expression of Cbln1 gene was very low and gradually increased in relation to the duration of culture. Expression of Cbln3, on the other hand, was very low in fragments of rat calvaria, and increased notably after digestion with collagenase-I. The highest expression of this precerebellin was observed at day 14 of culture while at days 7 and 21 levels of expressions were notably lower. Neither Cbln2 nor Cbln4 was found to be expressed in the ROB cells. Neither CER nor des-Ser1-CER (10(-10)-10(-6)M) affect osteocalcin production and proliferation rate of studied cells. The above findings suggest that CER, which theoretically would be derived from Cbln1, modulate neither differentiated (osteocalcin secretion) nor basic (proliferation) functions of cultured rat osteoblast-like cells. The obtained data raise an intriguing hypothesis that precerebellins may be involved in regulating of spatial organization of osteoblastic niches in the bone

  9. Developmental constraints on behavioural flexibility

    PubMed Central

    Holekamp, Kay E.; Swanson, Eli M.; Van Meter, Page E.

    2013-01-01

    We suggest that variation in mammalian behavioural flexibility not accounted for by current socioecological models may be explained in part by developmental constraints. From our own work, we provide examples of constraints affecting variation in behavioural flexibility, not only among individuals, but also among species and higher taxonomic units. We first implicate organizational maternal effects of androgens in shaping individual differences in aggressive behaviour emitted by female spotted hyaenas throughout the lifespan. We then compare carnivores and primates with respect to their locomotor and craniofacial adaptations. We inquire whether antagonistic selection pressures on the skull might impose differential functional constraints on evolvability of skulls and brains in these two orders, thus ultimately affecting behavioural flexibility in each group. We suggest that, even when carnivores and primates would theoretically benefit from the same adaptations with respect to behavioural flexibility, carnivores may nevertheless exhibit less behavioural flexibility than primates because of constraints imposed by past adaptations in the morphology of the limbs and skull. Phylogenetic analysis consistent with this idea suggests greater evolutionary lability in relative brain size within families of primates than carnivores. Thus, consideration of developmental constraints may help elucidate variation in mammalian behavioural flexibility. PMID:23569298

  10. Developmental constraints on behavioural flexibility.

    PubMed

    Holekamp, Kay E; Swanson, Eli M; Van Meter, Page E

    2013-05-19

    We suggest that variation in mammalian behavioural flexibility not accounted for by current socioecological models may be explained in part by developmental constraints. From our own work, we provide examples of constraints affecting variation in behavioural flexibility, not only among individuals, but also among species and higher taxonomic units. We first implicate organizational maternal effects of androgens in shaping individual differences in aggressive behaviour emitted by female spotted hyaenas throughout the lifespan. We then compare carnivores and primates with respect to their locomotor and craniofacial adaptations. We inquire whether antagonistic selection pressures on the skull might impose differential functional constraints on evolvability of skulls and brains in these two orders, thus ultimately affecting behavioural flexibility in each group. We suggest that, even when carnivores and primates would theoretically benefit from the same adaptations with respect to behavioural flexibility, carnivores may nevertheless exhibit less behavioural flexibility than primates because of constraints imposed by past adaptations in the morphology of the limbs and skull. Phylogenetic analysis consistent with this idea suggests greater evolutionary lability in relative brain size within families of primates than carnivores. Thus, consideration of developmental constraints may help elucidate variation in mammalian behavioural flexibility. PMID:23569298

  11. [Affective dependency].

    PubMed

    Scantamburlo, G; Pitchot, W; Ansseau, M

    2013-01-01

    Affective dependency is characterized by emotional distress (insecure attachment) and dependency to another person with a low self-esteem and reassurance need. The paper proposes a reflection on the definition of emotional dependency and the confusion caused by various denominations. Overprotective and authoritarian parenting, cultural and socio-environmental factors may contribute to the development of dependent personality. Psychological epigenetic factors, such as early socio-emotional trauma could on neuronal circuits in prefronto-limbic regions that are essential for emotional behaviour.We also focus on the interrelations between dependent personality, domestic violence and addictions. The objective for the clinician is to propose a restoration of self-esteem and therapeutic strategies focused on autonomy. PMID:23888587

  12. Chondrocytes transdifferentiate into osteoblasts in endochondral bone during development, postnatal growth and fracture healing in mice.

    PubMed

    Zhou, Xin; von der Mark, Klaus; Henry, Stephen; Norton, William; Adams, Henry; de Crombrugghe, Benoit

    2014-12-01

    One of the crucial steps in endochondral bone formation is the replacement of a cartilage matrix produced by chondrocytes with bone trabeculae made by osteoblasts. However, the precise sources of osteoblasts responsible for trabecular bone formation have not been fully defined. To investigate whether cells derived from hypertrophic chondrocytes contribute to the osteoblast pool in trabecular bones, we genetically labeled either hypertrophic chondrocytes by Col10a1-Cre or chondrocytes by tamoxifen-induced Agc1-CreERT2 using EGFP, LacZ or Tomato expression. Both Cre drivers were specifically active in chondrocytic cells and not in perichondrium, in periosteum or in any of the osteoblast lineage cells. These in vivo experiments allowed us to follow the fate of cells labeled in Col10a1-Cre or Agc1-CreERT2 -expressing chondrocytes. After the labeling of chondrocytes, both during prenatal development and after birth, abundant labeled non-chondrocytic cells were present in the primary spongiosa. These cells were distributed throughout trabeculae surfaces and later were present in the endosteum, and embedded within the bone matrix. Co-expression studies using osteoblast markers indicated that a proportion of the non-chondrocytic cells derived from chondrocytes labeled by Col10a1-Cre or by Agc1-CreERT2 were functional osteoblasts. Hence, our results show that both chondrocytes prior to initial ossification and growth plate chondrocytes before or after birth have the capacity to undergo transdifferentiation to become osteoblasts. The osteoblasts derived from Col10a1-expressing hypertrophic chondrocytes represent about sixty percent of all mature osteoblasts in endochondral bones of one month old mice. A similar process of chondrocyte to osteoblast transdifferentiation was involved during bone fracture healing in adult mice. Thus, in addition to cells in the periosteum chondrocytes represent a major source of osteoblasts contributing to endochondral bone formation in vivo

  13. Nano rough micron patterned titanium for directing osteoblast morphology and adhesion

    PubMed Central

    Puckett, Sabrina; Pareta, Rajesh; Webster, Thomas J

    2008-01-01

    Previous studies have demonstrated greater functions of osteoblasts (bone-forming cells) on nanophase compared with conventional metals. Nanophase metals possess a biologically inspired nanostructured surface that mimics the dimensions of constituent components in bone, including collagen and hydroxyapatite. Not only do these components possess dimensions on the nanoscale, they are aligned in a parallel manner creating a defined orientation in bone. To date, research has yet to evaluate the effect that organized nanosurface features can have on the interaction of osteoblasts with material surfaces. Therefore, to determine if surface orientation of features can mediate osteoblast adhesion and morphology, this study investigated osteoblast function on patterned titanium substrates containing alternating regions of micron rough and nano rough surfaces prepared by novel electron beam evaporation techniques. This study was also interested in determining whether or not the size of the patterned regions had an effect on osteoblast behavior and alignment. Results indicated early controlled osteoblast alignment on these patterned materials as well as greater osteoblast adhesion on the nano rough regions of these patterned substrates. Interestingly, decreasing the width of the nano rough regions (from 80 μm to 22 μm) on these patterned substrates resulted in a decreased number of osteoblasts adhering to these areas. Changes in the width of the nano rough regions also resulted in changes in osteoblast morphology, thus, suggesting there is an optimal pattern dimension that osteoblasts prefer. In summary, results of this study provided evidence that aligned nanophase metal features on the surface of titanium improved early osteoblast functions (morphology and adhesion) promising for their long term functions, criteria necessary to improve orthopedic implant efficacy. PMID:18686782

  14. CGRP may regulate bone metabolism through stimulating osteoblast differentiation and inhibiting osteoclast formation.

    PubMed

    He, Haitao; Chai, Jianshen; Zhang, Shengfu; Ding, Linlin; Yan, Peng; Du, Wenjun; Yang, Zhenzhou

    2016-05-01

    Calcitonin-gene-related peptide (CGRP) is a neuropeptide, which is widely distributed throughout the central and peripheral nervous systems. Numerous mechanisms underlying the action of CGRP in osteoblast-associated cells have been suggested for bone growth and metabolism. The present study was designed to closely investigate the osteoblast‑ and osteoclast-associated mechanisms of the effect of CGRP administration on bone metabolism in primary osteoblasts. Primary osteoblasts were obtained from newborn rabbit calvaria and incubated with different concentrations of human CGRP (hCGRP), hCGRP and hCGRP (8‑37), or without treatment as a control. Intracellular calcium (Ca2+) and cyclic adenosine monophosphate (cAMP) were detected following treatment, as well as the expression levels of osteoblast differentiation markers, including activating transcription factor‑4 (ATF4) and osteocalcin (OC), and receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG). The isolated primary osteoblasts were found to stain positively for ALP. hCGRP treatment had no significant effect on transient intracellular Ca2+ in the osteoblasts. Treatment of the osteoblasts with hCGRP led to elevations in the expression levels of cAMP, ATF4 and OPG, and downregulation in the expression of RANKL, in a dose‑dependent manner. These effects were markedly reversed by the addition of hCGRP (8‑37). The results of the present study demonstrated that CGRP administration not only stimulated osteoblast differentiation, as demonstrated by upregulated expression levels of ATF4 and OC in the hCGRP‑treated osteoblasts, but also inhibited OPG/RANKL‑regulated osteoclastogenesis. CGRP may act as a modulator of bone metabolism through osteoblast and osteoclast-associated mechanisms, which result in osteoblast formation with subsequent activation of bone formation. PMID:27035229

  15. Chondrocytes Transdifferentiate into Osteoblasts in Endochondral Bone during Development, Postnatal Growth and Fracture Healing in Mice

    PubMed Central

    Zhou, Xin; von der Mark, Klaus; Henry, Stephen; Norton, William; Adams, Henry; de Crombrugghe, Benoit

    2014-01-01

    One of the crucial steps in endochondral bone formation is the replacement of a cartilage matrix produced by chondrocytes with bone trabeculae made by osteoblasts. However, the precise sources of osteoblasts responsible for trabecular bone formation have not been fully defined. To investigate whether cells derived from hypertrophic chondrocytes contribute to the osteoblast pool in trabecular bones, we genetically labeled either hypertrophic chondrocytes by Col10a1-Cre or chondrocytes by tamoxifen-induced Agc1-CreERT2 using EGFP, LacZ or Tomato expression. Both Cre drivers were specifically active in chondrocytic cells and not in perichondrium, in periosteum or in any of the osteoblast lineage cells. These in vivo experiments allowed us to follow the fate of cells labeled in Col10a1-Cre or Agc1-CreERT2 -expressing chondrocytes. After the labeling of chondrocytes, both during prenatal development and after birth, abundant labeled non-chondrocytic cells were present in the primary spongiosa. These cells were distributed throughout trabeculae surfaces and later were present in the endosteum, and embedded within the bone matrix. Co-expression studies using osteoblast markers indicated that a proportion of the non-chondrocytic cells derived from chondrocytes labeled by Col10a1-Cre or by Agc1-CreERT2 were functional osteoblasts. Hence, our results show that both chondrocytes prior to initial ossification and growth plate chondrocytes before or after birth have the capacity to undergo transdifferentiation to become osteoblasts. The osteoblasts derived from Col10a1-expressing hypertrophic chondrocytes represent about sixty percent of all mature osteoblasts in endochondral bones of one month old mice. A similar process of chondrocyte to osteoblast transdifferentiation was involved during bone fracture healing in adult mice. Thus, in addition to cells in the periosteum chondrocytes represent a major source of osteoblasts contributing to endochondral bone formation in vivo

  16. Disruption of Kif3a in osteoblasts results in defective bone formation and osteopenia

    PubMed Central

    Qiu, Ni; Xiao, Zhousheng; Cao, Li; Buechel, Meagan M.; David, Valentin; Roan, Esra; Quarles, L. Darryl

    2012-01-01

    We investigated whether Kif3a in osteoblasts has a direct role in regulating postnatal bone formation. We conditionally deleted Kif3a in osteoblasts by crossing osteocalcin (Oc; also known as Bglap)–Cre with Kif3aflox/null mice. Conditional Kif3a-null mice (Kif3aOc-cKO) had a 75% reduction in Kif3a transcripts in bone and osteoblasts. Conditional deletion of Kif3a resulted in the reduction of primary cilia number by 51% and length by 27% in osteoblasts. Kif3aOc-cKO mice developed osteopenia by 6 weeks of age unlike Kif3aflox/+ control mice, as evidenced by reductions in femoral bone mineral density (22%), trabecular bone volume (42%) and cortical thickness (17%). By contrast, Oc-Cre;Kif3aflox/+ and Kif3aflox/null heterozygous mice exhibited no skeletal abnormalities. Loss of bone mass in Kif3aOc-cKO mice was associated with impaired osteoblast function in vivo, as reflected by a 54% reduction in mineral apposition rate and decreased expression of Runx2, osterix (also known as Sp7 transcription factor 7; Sp7), osteocalcin and Dmp1 compared with controls. Immortalized osteoblasts from Kif3aOc-cKO mice exhibited increased cell proliferation, impaired osteoblastic differentiation, and enhanced adipogenesis in vitro. Osteoblasts derived from Kif3aOc-cKO mice also had lower basal cytosolic calcium levels and impaired intracellular calcium responses to fluid flow shear stress. Sonic hedgehog-mediated Gli2 expression and Wnt3a-mediated β-catenin and Axin2 expression were also attenuated in Kif3aOc-cKO bone and osteoblast cultures. These data indicate that selective deletion of Kif3a in osteoblasts disrupts primary cilia formation and/or function and impairs osteoblast-mediated bone formation through multiple pathways including intracellular calcium, hedgehog and Wnt signaling. PMID:22357948

  17. Soy protein isolate down-regulates caveolin-1 expression to suppress osteoblastic cell senescence pathways.

    PubMed

    Zhang, Jian; Lazarenko, Oxana P; Blackburn, Michael L; Badger, Thomas M; Ronis, Martin J J; Chen, Jin-Ran

    2014-07-01

    It has been suggested that the beneficial effects of soy protein isolate (SPI) on bone quality are due to either stimulation of estrogenic signaling via isoflavones or through a novel and as yet uncharacterized nonestrogenic pathway. In our study, SPI-fed rat serum inhibited the osteoblastic cell senescence pathway. This effect was accompanied by stimulation of cell differentiation, proliferation, and significant restoration of replicative senescent bone marrow mesenchymal ST2 cells (passaged 30 times). These effects were reproduced in bone from 5-wk-old intact and 10-wk-old ovariectomized female rats fed SPI diets. Caveolin-1 and p53 expression was decreased in bone in SPI-fed, but not in 17β-estradiol (E2)-treated rats. In cell culture studies, membranous caveolin-1 and nuclear p53 expression was greater in replicative senescent ST2 cell cultures than in earlier passaged cells. SPI-fed rat serum significantly down-regulated both caveolin-1 and p53 in senescent and nonsenescent cells. Replicative senescent ST2 cells exhibited a strong association among caveolin-1, p53, and mouse double minute 2 homologue (mdm2), which was inhibited by SPI-fed rat serum. Overexpression of caveolin-1 in ST2 cells resulted in increased expression of p53 and p21, whereas, knockdown of caveolin-1 using shRNA led to increases in mdm2 and eliminated SPI-fed rat serum's effects on p53 and p21 expression. In contrast, manipulation of caveolin-1 expression did not affect the actions of E2 or isoflavones on p53 expression in either ST2 or OB6 cells. These results suggest that caveolin-1 is a mediator of nonestrogenic SPI effects on bone cells.-Zhang, J., Lazarenko, O. P., Blackburn, M. L., Badger, T. M., Ronis, M. J. J., Chen, J.-R. Soy protein isolate down-regulates caveolin-1 expression to suppress osteoblastic cell senescence pathways. PMID:24719353

  18. Evaluation of epigallocatechin-3-gallate (EGCG) cross-linked collagen membranes and concerns on osteoblasts.

    PubMed

    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

  19. Fibrillar assembly and stability of collagen coating on titanium for improved osteoblast responses.

    PubMed

    Kim, Hae-Won; Li, Long-Hao; Lee, Eun-Jung; Lee, Su-Hee; Kim, Hyoun-Ee

    2005-12-01

    Collagen, as a major constituent of human connective tissues, has been regarded as one of the most important biomaterials. As a coating moiety on Ti hard-tissue implants, the collagen has recently attracted a great deal of attention. This article reports the effects of fibrillar assembly and crosslinking of collagen on its chemical stability and the subsequent osteoblastic responses. The fibrillar self-assembly of collagen was carried out by incubating acid-dissolved collagen in an ionic-buffered medium at 37 degrees C. The degree of assembly was varied with the incubation time and monitored by the turbidity change. The differently assembled collagen was coated on the Ti and crosslinked with a carbodiimide derivative. The partially assembled collagen contained fibrils with varying diameters as well as nonfibrillar aggregates. On the other hand, the fully assembled collagen showed the complete formation of fibrils with uniform diameters of approximately 100-200 nm with periodic stain patterns within the fibrils, which are typical of native collagen fibers. Through this fibrillar assembly, the collagen coating had significantly improved chemical stability in both the saline and collagenase media. The subsequent crosslinking step also improved the stability of the collagen coating, particularly in the unassembled collagen. The fibrillar assembly and the crosslinking of collagen significantly influenced the osteoblastic cell responses. Without the assembly, the collagen layer on Ti adversely affected the cell attachment and proliferation. However, those cellular responses were improved significantly when the collagen was assembled to fibrils and the assembly degree was increased. After crosslinking the collagen coating, these cellular responses were significantly enhanced in the case of the unassembled collagen but were not altered much in the assembled collagen. Based on these observations, it is suggested that the fibrillar assembly and the crosslinking of collagen

  20. Cloning and regulation of rat tissue inhibitor of metalloproteinases-2 in osteoblastic cells

    NASA Technical Reports Server (NTRS)

    Cook, T. F.; Burke, J. S.; Bergman, K. D.; Quinn, C. O.; Jeffrey, J. J.; Partridge, N. C.

    1994-01-01

    Rat tissue inhibitor of metalloproteinases-2 (TIMP-2) was cloned from a UMR 106-01 rat osteoblastic osteosarcoma cDNA library. The 969-bp full-length clone demonstrates 98 and 86% sequence identity to human TIMP-2 at the amino acid and nucleic acid levels, respectively. Parathyroid hormone (PTH), at 10(-8) M, stimulates an approximately twofold increase in both the 4.2- and 1.0-kb transcripts over basal levels in UMR cells after 24 h of exposure. The PTH stimulation of TIMP-2 transcripts was not affected by the inhibitor of protein synthesis, cycloheximide (10(-5) M), suggesting a primary effect of the hormone. This is in contradistinction to regulation of interstitial collagenase (matrix metalloproteinase-1) by PTH in these same cells. Nuclear run-on assays demonstrate that PTH causes an increase in TIMP-2 transcription that parallels the increase in message levels. Parathyroid hormone, in its stimulation of TIMP-2 mRNA, appears to act through a signal transduction pathway involving protein kinase A (PKA) since the increase in TIMP-2 mRNA is reproduced by treatment with the cAMP analogue, 8-bromo-cAMP (5 x 10(-3) M). The protein kinase C and calcium pathways do not appear to be involved due to the lack of effect of phorbol 12-myristate 13-acetate (2.6 x 10(-6) M) and the calcium ionophore, ionomycin (10(-7) M), on TIMP-2 transcript abundance. In this respect, regulation of TIMP-2 and collagenase in osteoblastic cells by PTH are similar. However, we conclude that since stimulation of TIMP-2 transcription is a primary event, the PKA pathway must be responsible for a direct increase in transcription of this gene.

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

    PubMed

    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

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

    PubMed Central

    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

  3. Deletion of connexin43 in osteoblasts/osteocytes leads to impaired muscle formation in mice

    PubMed Central

    Shen, Hua; Grimston, Susan; Civitelli, Roberto; Thomopoulos, Stavros

    2015-01-01

    It is well-established that muscle forces are necessary for bone development as well as proper bone modeling and remodeling. Recent work has also suggested that bone acts as an endocrine organ that can influence the development of other organs. Connexin43 (Cx43), a gap junction protein that transduces mechanical signals, is an important determinant of cortical bone modeling. Using an osteoblast/osteocyte-specific ablation of the Cx43 gene (Gja1) driven by the 2.3 kb Col1α1 promoter (cKO) in the mouse, this study confirmed reduced cortical bone thickness and density with expanded bone marrow cavity in the cKO humerus. Surprisingly, Gja1 deletion in bone cells also affected skeletal muscle development, resulting in lower fast muscle weight, grip strength, and maximum absolute and specific tetanic forces (60–80%, 85%, and 50%, respectively, of WT mice). The normally fast twitch extensor digitorium longus (EDL) muscle exhibited increased slow twitch fibers in cKO mice. These muscle defects were accompanied by a 40–60% reduction in mRNA abundance for genes encoding osteocalcin in the humerus, relative to WT mice. Accordingly, both carboxylated and undercarboxylated isoforms of osteocalcin were reduced by over 30% in the circulation of cKO mice. Moreover, the active, undercarboxylated isoform of osteocalcin (glu-OC) promoted myotube formation in C2C12 myoblast cultures, and glu-OC injections to cKO mice rescued EDL muscle cross section area and grip strength in vivo. These findings demonstrate that Cx43 in osteoblasts/osteocytes indirectly modulates skeletal muscle growth and function, potentially via an endocrine effect of glu-OC. PMID:25348938

  4. Hypergravity Stimulates Osteoblast Proliferation Via Matrix-Integrin-Signaling Pathways

    NASA Technical Reports Server (NTRS)

    Vercoutere, W.; Parra, M.; Roden, C.; DaCosta, M.; Wing, A.; Damsky, C.; Holton, E.; Searby, N.; Globus, R.; Almeida, E.

    2003-01-01

    Extensive characterizations of the physiologic consequences of microgravity and gravity indicate that lack of weight-bearing may cause tissue atrophy through cellular and subcellular level mechanisms. We hypothesize that gravity is needed for the efficient transduction of cell growth and survival signals from the extra-cellular matrix (ECM) in mechanosensitive tissues. Recent work from our laboratory and from others shows that an increase of gravity increases bone cell growth and survival. We found that 50-g hypergravity stimulation increased osteoblast proliferation for cells grown on Collagen Type I and Fibronectin, but not on Laminin or uncoated plastic. This may be a tissue-specific response, because 50-g hypergravity stimulation caused no increase in proliferation for primary rat fibroblasts. These results combined with RT-PCR for all possible integrins indicate that beta1 integrin subunit may be involved. The osteoblast proliferation response on Collagen Type I was greater at 25-g than at 10-g or 50-g; 24-h duration of hypergravity was necessary to see an increase in proliferation. Survival was enhanced during hypergravity stimulation by the presence of matrix. Flow cytometry analysis indicated that cell cycle may be altered; BrdU incorporation in proliferating cells showed an increase in the number of actively dividing cells from about 60% at 1-g to over 90% at 25-g. To further investigate the molecular components involved, we applied fluorescence labeling of cytoskeletal and signaling molecules to cells after 2 to 30 minutes of hypergravity stimulation. While structural components did not appear to be altered, phosphorylation increased, indicating that signaling pathways may be activated. These data indicate that gravity mechanostimulation of osteoblast proliferation involves specific matrix-integrin signaling pathways which are sensitive to duration and g-level.

  5. Vibrational force alters mRNA expression in osteoblasts

    NASA Technical Reports Server (NTRS)

    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.

  6. Palmitate attenuates osteoblast differentiation of fetal rat calvarial cells

    SciTech Connect

    Yeh, Lee-Chuan C.; Ford, Jeffery J.; Lee, John C.; Adamo, Martin L.

    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.

  7. Compressive forces induce osteogenic gene expression in calvarial osteoblasts.

    PubMed

    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

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

    SciTech Connect

    Wan, Qiaoqiao; Cho, Eunhye; Yokota, Hiroki; Na, Sungsoo

    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

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

    PubMed Central

    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

  10. Protein Phosphatase 2A Mediates Oxidative Stress Induced Apoptosis in Osteoblasts.

    PubMed

    Huang, Chong-xin; Lv, Bo; Wang, Yue

    2015-01-01

    Osteoporosis is one of the most common bone diseases, which is characterized by a systemic impairment of bone mass and fragility fractures. Age-related oxidative stress is highly associated with impaired osteoblastic dysfunctions and subsequent osteoporosis. In osteoblasts (bone formation cells), reactive oxygen species (ROS) are continuously generated and further cause lipid peroxidation, protein damage, and DNA lesions, leading to osteoblastic dysfunctions, dysdifferentiations, and apoptosis. Although much progress has been made, the mechanism responsible for oxidative stress induced cellular alternations and osteoblastic toxicity is still not fully elucidated. Here, we demonstrate that protein phosphatase 2A (PP2A), a major protein phosphatase in mammalian cells, mediates oxidative stress induced apoptosis in osteoblasts. Our results showed that lipid peroxidation products (4-HNE) may induce dramatic oxidative stress, inflammatory reactions, and apoptosis in osteoblasts. These oxidative stress responses may ectopically activate PP2A phosphatase activity, which may be mediated by inactivation of AKT/mTOR pathway. Moreover, inhibition of PP2A activity by okadaic acid might partly prevent osteoblastic apoptosis under oxidative conditions. These findings may reveal a novel mechanism to clarify the role of oxidative stress for osteoblastic apoptosis and provide new possibilities for the treatment of related bone diseases, such as osteoporosis. PMID:26538836

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

    PubMed

    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

  12. Osteoblast-Targeting-Peptide Modified Nanoparticle for siRNA/microRNA Delivery.

    PubMed

    Sun, Yao; Ye, Xiongzhen; Cai, Mingxiang; Liu, Xiangning; Xiao, Jia; Zhang, Chenyang; Wang, Yayu; Yang, Li; Liu, Jiafan; Li, Shannai; Kang, Chen; Zhang, Bin; Zhang, Qi; Wang, Zuolin; Hong, An; Wang, Xiaogang

    2016-06-28

    Antiosteoporosis gene-based drug development strategies are presently focused on targeting osteoblasts to either suppress bone loss or increase bone mass. Although siRNA/microRNA-based gene therapy has enormous potential, it is severely limited by the lack of specific cell-targeting delivery systems. We report an osteoblast-targeting peptide (SDSSD) that selectively binds to osteoblasts via periostin. We developed SDSSD-modified polyurethane (PU) nanomicelles encapsulating siRNA/microRNA that delivers drugs to osteoblasts; the data showed that SDSSD-PU could selectively target not only bone-formation surfaces but also osteoblasts without overt toxicity or eliciting an immune response in vivo. We used the SDSSD-PU delivery system to deliver anti-miR-214 to osteoblasts and our results showed increased bone formation, improved bone microarchitecture, and increased bone mass in an ovariectomized osteoporosis mouse model. SDSSD-PU may be a useful osteoblast-targeting small nucleic acid delivery system that could be used as an anabolic strategy to treat osteoblast-induced bone diseases. PMID:27176123

  13. Autophagy plays a protective role in cell death of osteoblasts exposure to lead chloride.

    PubMed

    Lv, Xiao-hua; Zhao, Da-hang; Cai, Shi-zhong; Luo, Shi-ying; You, Tingting; Xu, Bi-lian; Chen, Ke

    2015-12-01

    Lead (Pb) is a toxic heavy metal widespreadly used in industrial field. Prior studies showed that Pb exposure had detrimental effects on osteoblasts. The mechanisms underlying Pb-induced damage are complex. Autophagy can protect cells from various cytotoxic stimuli. In the present study, the aim of our research was to investigate whether Pb could activate autophagy to play a protective role against osteoblasts apoptosis. Our results indicated that PbCl2 induced autophagy and autophagic flux in MC3T3-E1 murine osteoblastic cell by RT-PCR, western blot, as well as fluorescence microscopy analysis of GFP-LC3, AO and MDC staining. Pb increased the apoptosis of osteoblasts, evidenced by western blot and Hoechst 33258 staining assessment. In addition, inhibiting autophagy by 3-MA further increased the osteoblasts apoptosis after Pb exposure, showed by flow cytometry and Hoechst 33258 staining. Furthermore, phosphorylation of mTOR and p70S6K was inhibited by Pb exposure, indicating that Pb might induce autophagy in osteoblasts via inhibiting mTOR pathway. Altogether, these evidence suggested that Pb exporsure promoted autophagy flux in osteoblasts. The activation of autophagy by Pb played a protective role in osteoblasts apoptosis, which might be mediated through the mTOR pathway. PMID:26383630

  14. Gliotoxin potentiates osteoblast differentiation by inhibiting nuclear factor-κB signaling

    PubMed Central

    WANG, GUANGYE; ZHANG, XIAOHAI; YU, BAOQING; REN, KE

    2015-01-01

    The differentiation of pluripotent mesenchymal stem cells to mature osteoblasts is crucial for the maintenance of the adult skeleton. In rheumatic arthritis, osteoblast differentiation is impaired by the overproduction of cytokine tumor necrosis factor (TNF)-α. It has been demonstrated that TNF-α is able to inhibit osteoblast differentiation through the activation of nuclear factor (NF)-κB signaling. As a result of the critical role of TNF-α and NF-κB in the pathogenesis of bone-loss associated diseases, these factors are regarded as key targets for the development of therapeutic agents. In the current study, the role of the NF-κB inhibitor gliotoxin (GTX) in the regulation of osteoblast differentiation was evaluated. The non-toxic GTX doses were determined to be ≤3 μg/ml. It was revealed that GTX was able to block TNF-α-induced inhibition of osteoblast differentiation, as indicated by alkaline phosphatase (ALP) activity and ALP staining assays, as well as the expression levels of osteoblast-associated genes Col I, Ocn, Bsp, Runx2, Osx and ATF4. Additionally, it was identified that gliotoxin directly promoted bone morphoge-netic protein-2-induced osteoblast differentiation. GTX was found to inhibit the accumulation of NF-κB protein p65 in the nucleus and reduce NF-κB transcriptional activity, suggesting that GTX potentiated osteoblast differentiation via the suppression of NF-κB signaling. PMID:25816130

  15. Focal Adhesion of Osteoblastic Cells on Titanium Surface with Amine Functionalities Formed by Plasma Polymerization

    NASA Astrophysics Data System (ADS)

    Song, Heesang; Jung, Sang Chul; Kim, Byung Hoon

    2012-08-01

    To enhance the focal adhesion of osteoblastic cells on a titanium surface, plasma polymerized allyl amine (AAm) thin films were deposited by plasma polymerization. This plasma polymer functionalization of titanium is advantageous for osteoblastic focal adhesion formation. Such Ti surfaces are useful for the fabrication of titanium-based dental implants for enhancement of osseointegration.

  16. Protein Phosphatase 2A Mediates Oxidative Stress Induced Apoptosis in Osteoblasts

    PubMed Central

    Huang, Chong-xin; Lv, Bo; Wang, Yue

    2015-01-01

    Osteoporosis is one of the most common bone diseases, which is characterized by a systemic impairment of bone mass and fragility fractures. Age-related oxidative stress is highly associated with impaired osteoblastic dysfunctions and subsequent osteoporosis. In osteoblasts (bone formation cells), reactive oxygen species (ROS) are continuously generated and further cause lipid peroxidation, protein damage, and DNA lesions, leading to osteoblastic dysfunctions, dysdifferentiations, and apoptosis. Although much progress has been made, the mechanism responsible for oxidative stress induced cellular alternations and osteoblastic toxicity is still not fully elucidated. Here, we demonstrate that protein phosphatase 2A (PP2A), a major protein phosphatase in mammalian cells, mediates oxidative stress induced apoptosis in osteoblasts. Our results showed that lipid peroxidation products (4-HNE) may induce dramatic oxidative stress, inflammatory reactions, and apoptosis in osteoblasts. These oxidative stress responses may ectopically activate PP2A phosphatase activity, which may be mediated by inactivation of AKT/mTOR pathway. Moreover, inhibition of PP2A activity by okadaic acid might partly prevent osteoblastic apoptosis under oxidative conditions. These findings may reveal a novel mechanism to clarify the role of oxidative stress for osteoblastic apoptosis and provide new possibilities for the treatment of related bone diseases, such as osteoporosis. PMID:26538836

  17. Identification of the molecular mechanisms contributing to polarized trafficking in osteoblasts.

    PubMed

    Prêle, Cecilia M; Horton, Michael A; Caterina, Paul; Stenbeck, Gudrun

    2003-01-01

    The directionality of matrix deposition in vivo is governed by the ability of a cell to direct vesicularflow to a specific target site. Osteoblastic cells direct newly synthesized bone matrix proteins toward the bone surface. In this study, we dissect the molecular mechanisms underlying the polarized trafficking of matrix protein in osteoblasts. We demonstrate using TEM, immunocytochemistry, and cDNA analysis, the ability of osteoblastic cells in culture to form tight junction-like structures and report the expression of the tight junction associated proteins occludin and claudins 1-3 in these cells. We identify intercellular contact sites and the leading edge of migratory osteoblasts as major target sites of vesicular trafficking in osteoblasts. Proteins required for this process, rsec6, NSF, VAMP1, and syntaxin 4, as well as the bone matrix protein, osteopontin, localize to these sites. We demonstrate that osteoblasts in vivo possess VAMP1 and, furthermore, report the expression of two VAMP1 splice variants in these cells. In addition, osteoblasts express the NSF attachment protein alpha-SNAP and the t-SNARE SNAP23. Thus, cell-to-cell contact sites and the leading edge of migratory osteoblasts contain a unique complement of proteins required for SNARE mediated membrane fusion. PMID:12490191

  18. Osteoblastic cells trigger gate currents on nanocrystalline diamond transistor.

    PubMed

    Izak, Tibor; Krátká, Marie; Kromka, Alexander; Rezek, Bohuslav

    2015-05-01

    We show the influence of osteoblastic SAOS-2 cells on the transfer characteristics of nanocrystalline diamond solution-gated field-effect transistors (SGFET) prepared on glass substrates. Channels of these fully transparent SGFETs are realized by hydrogen termination of undoped diamond film. After cell cultivation, the transistors exhibit about 100× increased leakage currents (up to 10nA). During and after the cell delamination, the transistors return to original gate currents. We propose a mechanism where this triggering effect is attributed to ions released from adhered cells, which depends on the cell adhesion morphology, and could be used for cell culture monitoring. PMID:25835144

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

    SciTech Connect

    Muthukumaran, Padmalosini; Lim, Chwee Teck; Lee, Taeyong

    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

  20. Hypergravity-induced enrichment of β1 integrin on the cell membranes of osteoblast-like cells via caveolae-dependent endocytosis.

    PubMed

    Zhou, Shuai; Zu, Yan; Zhuang, Fengyuan; Yang, Chun

    2015-08-01

    In bone cells, integrins on the cellular surface are the primary sensors of their mechanical environment. Although gravitational changes are known to affect the adhesion and functions of bone cells, whether integrins respond to hypergravity in osteoblasts remains unclear. In this work, we demonstrate that exposure to a hypergravitational environment (20 × g via centrifugation) resulted in the concentration of β1, but not β3, integrin on the cell membrane of osteoblast-like (MC3T3-E1) cells. Notably, the total expression of both integrins was unaffected by the hypergravitational environment. In addition, caveolin-dependent endocytosis was discovered to be involved in the regulation of the enrichment of β1 integrin on the cell surface after stimulation by hypergravity. These findings could aid in the improvement of our understanding of the mechanisms underlying the effects of different gravitational forces on the human body. PMID:26071356

  1. Assessing the osteoblast transcriptome in a model of enhanced bone formation due to constitutive G{sub s}–G protein signaling in osteoblasts

    SciTech Connect

    Wattanachanya, Lalita; Wang, Liping; Millard, Susan M.; Lu, Wei-Dar; O’Carroll, Dylan; Hsiao, Edward C.; Conklin, Bruce R.; Nissenson, Robert A.

    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

  2. Anticipated affective reactions and prevention of AIDS.

    PubMed

    Richard, R; van der Pligt, J; de Vries, N

    1995-03-01

    Controlling the AIDs epidemic may depend largely upon health education aimed at adolescents. A number of approaches have been applied to human immunodeficiency virus (HIV) preventive behaviour in adolescents, including the health belief model (Becker, 1974), protection motivation theory (Rogers, 1983), and the theory of planned behaviour (Ajzen, 1985, 1991). Since sexual behaviour is heavily influenced by emotions, a possible shortcoming of these models is that little attention is given to affective processes. In this study we investigated the role of anticipated, post-behavioural, affective reactions to (un)safe sexual behaviours in the context of the theory of planned behaviour (TPB). The results showed that anticipated affective reactions such as worry and regret predicted behavioural expectations over and above the components of the TPB. The implications for our understanding of adolescent sexual behaviour and for campaigns aimed at the reduction of risky sexual practices will be discussed. PMID:7735735

  3. Immortalization and characterization of mouse floxed Bmp2/4 osteoblasts

    SciTech Connect

    Wu, Li-An; Yuan, Guohua; Yang, Guobin; Ortiz-Gonzalez, Iris; Yang, Wuchen; Cui, Yong; MacDougall, Mary; Harris, Stephen; Chen, Shuo

    2009-08-14

    Generation of a floxed Bmp2/4 osteoblast cell line is a valuable tool for studying the modulatory effects of Bmp2 and Bmp4 on osteoblast differentiation as well as relevant molecular events. In this study, primary floxed Bmp2/4 mouse osteoblasts were cultured and transfected with simian virus 40 large T-antigen. Transfection was verified by polymerase chain reaction (PCR) and immunohistochemistry. To examine the characteristics of the transfected cells, morphology, proliferation and mineralization were analyzed, expression of cell-specific genes including Runx2, ATF4, Dlx3, Osx, dentin matrix protein 1, bone sialoprotein, osteopontin, osteocalcin, osteonectin and collagen type I was detected. These results show that transfected floxed Bmp2/4 osteoblasts bypassed senescence with a higher proliferation rate, but retain the genotypic and phenotypic characteristics similar to the primary cells. Thus, we for the first time demonstrate the establishment of an immortalized mouse floxed Bmp2/4 osteoblast cell line.

  4. Nuclear size as a cell-kinetic marker for osteoblast differentiation

    NASA Technical Reports Server (NTRS)

    Roberts, W. E.; Mozsary, P. G.; Klingler, E.

    1982-01-01

    A nuclear morphometric assay for preosteoblasts is introduced as a cell-kinetic technique, applicable to routine histological preparations of mineralized tissue. Because this method is a morphological marker for osteoblast precursor cell differentiation, it provides a new dimension for determining the mechanism of osteoblast histogenesis. Osteoblast precursors of the periodontal ligament are a mixed population of progenitors, kinetically separable into two distinct groups according to nuclear size. Preosteoblasts, the immediate proliferating precursors of osteoblasts, have large nuclei (greater than 170 micrometers3) and are derived from relatively undifferentiated fibroblastlike cells, which have smaller nuclei (less than 80 micrometers3). Increase in nuclear volume, during G1 phase of the cell cycle, is apparently a morphological manifestation of change in genomic expression. This key event in preosteoblast differentiation is related to mechanical stress/strain and may be an important rate-limiting step in osteoblast histogenesis.

  5. How Osteoblasts Sense their E