Sample records for factors affecting osteoblastic

  1. 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.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ehnert, Sabrina, E-mail: sabrina.ehnert@gmail.com; Freude, Thomas, E-mail: tfreude@bgu-tuebingen.de; Ihle, Christoph, E-mail: cihle@bgu-tuebingen.de

    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.more » 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.« less

  3. Plasma Membrane Factor XIIIA Transglutaminase Activity Regulates Osteoblast Matrix Secretion and Deposition by Affecting Microtubule Dynamics

    PubMed Central

    Al-Jallad, Hadil F.; Myneni, Vamsee D.; Piercy-Kotb, Sarah A.; Chabot, Nicolas; Mulani, Amina; Keillor, Jeffrey W.; Kaartinen, Mari T.

    2011-01-01

    Transglutaminase activity, arising potentially from transglutaminase 2 (TG2) and Factor XIIIA (FXIIIA), has been linked to osteoblast differentiation where it is required for type I collagen and fibronectin matrix deposition. In this study we have used an irreversible TG-inhibitor to ‘block –and-track’ enzyme(s) targeted during osteoblast differentiation. We show that the irreversible TG-inhibitor is highly potent in inhibiting osteoblast differentiation and mineralization and reduces secretion of both fibronectin and type I collagen and their release from the cell surface. Tracking of the dansyl probe by Western blotting and immunofluorescence microscopy demonstrated that the inhibitor targets plasma membrane-associated FXIIIA. TG2 appears not to contribute to crosslinking activity on the osteoblast surface. Inhibition of FXIIIA with NC9 resulted in defective secretory vesicle delivery to the plasma membrane which was attributable to a disorganized microtubule network and decreased microtubule association with the plasma membrane. NC9 inhibition of FXIIIA resulted in destabilization of microtubules as assessed by cellular Glu-tubulin levels. Furthermore, NC9 blocked modification of Glu-tubulin into 150 kDa high-molecular weight Glu-tubulin form which was specifically localized to the plasma membrane. FXIIIA enzyme and its crosslinking activity were colocalized with plasma membrane-associated tubulin, and thus, it appears that FXIIIA crosslinking activity is directed towards stabilizing the interaction of microtubules with the plasma membrane. Our work provides the first mechanistic cues as to how transglutaminase activity could affect protein secretion and matrix deposition in osteoblasts and suggests a novel function for plasma membrane FXIIIA in microtubule dynamics. PMID:21283799

  4. Gallium modulates osteoclastic bone resorption in vitro without affecting osteoblasts

    PubMed Central

    Verron, Elise; Masson, Martial; Khoshniat, Solmaz; Duplomb, Laurence; Wittrant, Yohann; Baud'huin, Marc; Badran, Zahi; Bujoli, Bruno; Janvier, Pascal; Scimeca, Jean-Claude; Bouler, Jean-Michel; Guicheux, Jérôme

    2010-01-01

    Background and purpose: Gallium (Ga) has been shown to be effective in the treatment of disorders associated with accelerated bone loss, including cancer-related hypercalcemia and Paget's disease. These clinical applications suggest that Ga could reduce bone resorption. However, few studies have studied the effects of Ga on osteoclastic resorption. Here, we have explored the effects of Ga on bone cells in vitro. Experimental approach: In different osteoclastic models [osteoclasts isolated from long bones of neonatal rabbits (RBC), murine RAW 264.7 cells and human CD14-positive cells], we have performed resorption activity tests, staining for tartrate resistant acid phosphatase (TRAP), real-time polymerase chain reaction analysis, viability and apoptotic assays. We also evaluated the effect of Ga on osteoblasts in terms of proliferation, viability and activity by using an osteoblastic cell line (MC3T3-E1) and primary mouse osteoblasts. Key results: Gallium dose-dependently (0–100 µM) inhibited the in vitro resorption activity of RBC and induced a significant decrease in the expression level of transcripts coding for osteoclastic markers in RAW 264.7 cells. Ga also dramatically reduced the formation of TRAP-positive multinucleated cells. Ga down-regulated in a dose-dependant manner the expression of the transcription factor NFATc1. However, Ga did not affect the viability or activity of primary and MC3T3-E1 osteoblasts. Conclusions and implications: Gallium exhibits a dose-dependent anti-osteoclastic effect by reducing in vitro osteoclastic resorption, differentiation and formation without negatively affecting osteoblasts. We provide evidence that this inhibitory mechanism involves down-regulation of NFATc1 expression, a master regulator of RANK-induced osteoclastic differentiation. PMID:20397300

  5. Wnt3a induces the expression of acetylcholinesterase during osteoblast differentiation via the Runx2 transcription factor.

    PubMed

    Xu, Miranda L; Bi, Cathy W C; Liu, Etta Y L; Dong, Tina T X; Tsim, Karl W K

    2017-07-28

    Acetylcholinesterase (AChE) hydrolyzes acetylcholine to terminate cholinergic transmission in neurons. Apart from this AChE activity, emerging evidence suggests that AChE could also function in other, non-neuronal cells. For instance, in bone, AChE exists as a proline-rich membrane anchor (PRiMA)-linked globular form in osteoblasts, in which it is proposed to play a noncholinergic role in differentiation. However, this hypothesis is untested. Here, we found that in cultured rat osteoblasts, AChE expression was increased in parallel with osteoblastic differentiation. Because several lines of evidence indicate that AChE activity in osteoblast could be triggered by Wnt/β-catenin signaling, we added recombinant human Wnt3a to cultured osteoblasts and found that this addition induced expression of the ACHE gene and protein product. This Wnt3a-induced AChE expression was blocked by the Wnt-signaling inhibitor Dickkopf protein-1 (DKK-1). We hypothesized that the Runt-related transcription factor 2 (Runx2), a downstream transcription factor in Wnt/β-catenin signaling, is involved in AChE regulation in osteoblasts, confirmed by the identification of a Runx2-binding site in the ACHE gene promoter, further corroborated by ChIP. Of note, Runx2 overexpression in osteoblasts induced AChE expression and activity of the ACHE promoter tagged with the luciferase gene. Moreover, deletion of the Runx2-binding site in the ACHE promoter reduced its activity during osteoblastic differentiation, and addition of 5-azacytidine and trichostatin A to differentiating osteoblasts affected AChE expression, suggesting epigenetic regulation of the ACHE gene. We conclude that AChE plays a role in osteoblastic differentiation and is regulated by both Wnt3a and Runx2. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Rough titanium alloys regulate osteoblast production of angiogenic factors.

    PubMed

    Olivares-Navarrete, Rene; Hyzy, Sharon L; Gittens, Rolando A; Schneider, Jennifer M; Haithcock, David A; Ullrich, Peter F; Slosar, Paul J; Schwartz, Zvi; Boyan, Barbara D

    2013-11-01

    Polyether-ether-ketone (PEEK) and titanium-aluminum-vanadium (titanium alloy) are used frequently in lumbar spine interbody fusion. Osteoblasts cultured on microstructured titanium generate an environment characterized by increased angiogenic factors and factors that inhibit osteoclast activity mediated by integrin α2β1 signaling. It is not known if this is also true of osteoblasts on titanium alloy or PEEK. The purpose of this study was to determine if osteoblasts generate an environment that supports angiogenesis and reduces osteoclastic activity when grown on smooth titanium alloy, rough titanium alloy, or PEEK. This in vitro study compared angiogenic factor production and integrin gene expression of human osteoblast-like MG63 cells cultured on PEEK or titanium-aluminum-vanadium (titanium alloy). MG63 cells were grown on PEEK, smooth titanium alloy, or rough titanium alloy. Osteogenic microenvironment was characterized by secretion of osteoprotegerin and transforming growth factor beta-1 (TGF-β1), which inhibit osteoclast activity and angiogenic factors including vascular endothelial growth factor A (VEGF-A), fibroblast growth factor 2 (FGF-2), and angiopoietin-1 (ANG-1). Expression of integrins, transmembrane extracellular matrix recognition proteins, was measured by real-time polymerase chain reaction. Culture on titanium alloy stimulated osteoprotegerin, TGF-β1, VEGF-A, FGF-2, and angiopoietin-1 production, and levels were greater on rough titanium alloy than on smooth titanium alloy. All factors measured were significantly lower on PEEK than on smooth or rough titanium alloy. Culture on titanium alloy stimulated expression of messenger RNA for integrins that recognize Type I collagen in comparison with PEEK. Rough titanium alloy stimulated cells to create an osteogenic-angiogenic microenvironment. The osteogenic-angiogenic responses to titanium alloy were greater than PEEK and greater on rough titanium alloy than on smooth titanium alloy. Surface

  7. 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.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Schindeler, Aaron; Little, David G.; Discipline of Paediatrics and Child Health, Faculty of Medicine, University of Sydney, Sydney

    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 themore » 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.« less

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tang Lin; Lin Zhu; Li Yongming

    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 andmore » 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.« less

  10. Signaling by Fibroblast Growth Factors (Fgf) and Fibroblast Growth Factor Receptor 2 (Fgfr2)–Activating Mutations Blocks Mineralization and Induces Apoptosis in Osteoblasts

    PubMed Central

    Mansukhani, Alka; Bellosta, Paola; Sahni, Malika; Basilico, Claudio

    2000-01-01

    Fibroblast growth factors (FGF) play a critical role in bone growth and development affecting both chondrogenesis and osteogenesis. During the process of intramembranous ossification, which leads to the formation of the flat bones of the skull, unregulated FGF signaling can produce premature suture closure or craniosynostosis and other craniofacial deformities. Indeed, many human craniosynostosis disorders have been linked to activating mutations in FGF receptors (FGFR) 1 and 2, but the precise effects of FGF on the proliferation, maturation and differentiation of the target osteoblastic cells are still unclear. In this report, we studied the effects of FGF treatment on primary murine calvarial osteoblast, and on OB1, a newly established osteoblastic cell line. We show that FGF signaling has a dual effect on osteoblast proliferation and differentiation. FGFs activate the endogenous FGFRs leading to the formation of a Grb2/FRS2/Shp2 complex and activation of MAP kinase. However, immature osteoblasts respond to FGF treatment with increased proliferation, whereas in differentiating cells FGF does not induce DNA synthesis but causes apoptosis. When either primary or OB1 osteoblasts are induced to differentiate, FGF signaling inhibits expression of alkaline phosphatase, and blocks mineralization. To study the effect of craniosynostosis-linked mutations in osteoblasts, we introduced FGFR2 carrying either the C342Y (Crouzon syndrome) or the S252W (Apert syndrome) mutation in OB1 cells. Both mutations inhibited differentiation, while dramatically inducing apoptosis. Furthermore, we could also show that overexpression of FGF2 in transgenic mice leads to increased apoptosis in their calvaria. These data provide the first biochemical analysis of FGF signaling in osteoblasts, and show that FGF can act as a cell death inducer with distinct effects in proliferating and differentiating osteoblasts. PMID:10851026

  11. Commensal Microbiota Enhance Both Osteoclast and Osteoblast Activities.

    PubMed

    Uchida, Yoko; Irie, Koichiro; Fukuhara, Daiki; Kataoka, Kota; Hattori, Takako; Ono, Mitsuaki; Ekuni, Daisuke; Kubota, Satoshi; Morita, Manabu

    2018-06-23

    Recent studies suggest that the commensal microbiota affects not only host energy metabolism and development of immunity but also bone remodeling by positive regulation of osteoclast activity. However, the mechanism of regulation of bone cells by the commensal microbiota has not been elucidated. In this study, 8-week-old specific pathogen-free (SPF) and germ-free (GF) mice were compared in terms of alveolar bones and primary osteoblasts isolated from calvarias. Micro-CT analysis showed that SPF mice had larger body size associated with lower bone mineral density and bone volume fraction in alveolar bones compared with GF mice. Greater numbers of osteoclasts in alveolar bone and higher serum levels of tartrate-resistant acid phosphatase 5b were observed in SPF mice. Tissue extracts from SPF alveolar bone showed higher levels of cathepsin K, indicating higher osteoclast activity. SPF alveolar extracts also showed elevated levels of γ-carboxylated glutamic acid⁻osteocalcin as a marker of mature osteoblasts compared with GF mice. Polymerase chain reaction (PCR) array analysis of RNA directly isolated from alveolar bone showed that in SPF mice, expression of mRNA of osteocalcin , which also acts as an inhibitor of bone mineralization, was strongly enhanced compared with GF mice. Cultured calvarial osteoblasts from SPF mice showed reduced mineralization but significantly enhanced expression of mRNAs of osteocalcin, alkaline phosphatase, insulin-like growth factor-I/II , and decreased ratio of osteoprotegerin/receptor activator of nuclear factor-kappa B ligand compared with GF mice. Furthermore, PCR array analyses of transcription factors in cultured calvarial osteoblasts showed strongly upregulated expression of Forkhead box g1 . In contrast, Gata-binding protein 3 was strongly downregulated in SPF osteoblasts. These results suggest that the commensal microbiota prevents excessive mineralization possibly by stimulating osteocalcin expression in osteoblasts, and

  12. Acetyl-11-Keto-β-Boswellic Acid Promotes Osteoblast Differentiation by Inhibiting Tumor Necrosis Factor-α and Nuclear Factor-κB Activity.

    PubMed

    Bai, Fan; Chen, Xuewu; Yang, Hui; Xu, Hong-Guang

    2018-06-20

    Tumor necrosis factor (TNF) -α plays a crucial role in rheumatoid arthritis (RA)-related bone loss disease. The main mechanism of action of RA induced bone loss is the significant inhibitory effect of TNF-α on osteoblast differentiation. TNF-α inhibits osteoblast differentiation mainly by activating nuclear factor (NF) -κB signaling pathway. Owing to the crucial role of TNF-α and NF-κB in the inhibition of osteoblast differentiation, they are considered as targets for the development of therapeutic drugs. In the present study, we evaluated the NF-κB inhibitor Boswellic acid (BA) and its derivatives in the regulation of osteoblast differentiation and the molecular mechanism. Based on the cell model of TNF-α induced inhibition of osteoblast differentiation of MC3T3-E1, the regulatory role of BAs was studied. The result of MTT assay indicated that bone morphogenetic protein (BMP) -2, TNF-α, or acetyl-11-keto-β-BA (AKBA) impact no significant effect for cell viability of MC3T3-E1. The results of alkaline phosphatase (ALP activity assay and real-time polymerase chain reaction indicated that AKBA blocked TNF-α-induced inhibition of the expression of osteoblast markers, suggesting that AKBA rescued osteoblast differentiation from TNF-α-induced inhibition. Additionally, AKBA stimulated the BMP-2-induced expression of osteoblast markers, suggesting that AKBA promotes osteoblast differentiation directly. The results of western blotting and luciferase assay indicated that N-κB signaling was activated by TNF-α. The overexpression of NF-κB component p65 in MC3T3-E1 was found to attenuate the positive effect of AKBA in osteoblast differentiation, suggesting that AKBA potentiates osteoblast differentiation by inhibiting NF-κB signaling. Collectively, AKBA promotes osteoblast differentiation by inhibiting TNF-α and NF-κB. Our study revealed a new discovery of AKBA in regulating osteoblast differentiation, and demonstrated that AKBA may be a potential anabolic

  13. Osteoblast role in osteoarthritis pathogenesis.

    PubMed

    Maruotti, Nicola; Corrado, Addolorata; Cantatore, Francesco P

    2017-11-01

    Even if osteoarthritis pathogenesis is still poorly understood, numerous evidences suggest that osteoblasts dysregulation plays a key role in osteoarthritis pathogenesis. An abnormal expression of OPG and RANKL has been described in osteoarthritis osteoblasts, which is responsible for abnormal bone remodeling and decreased mineralization. Alterations in genes expression are involved in dysregulation of osteoblast function, bone remodeling, and mineralization, leading to osteoarthritis development. Moreover, osteoblasts produce numerous transcription factors, growth factors, and other proteic molecules which are involved in osteoarthritis pathogenesis. © 2017 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.

  14. 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. © 2014 New York Academy of Sciences.

  15. Microtubule actin crosslinking factor 1 promotes osteoblast differentiation by promoting β-catenin/TCF1/Runx2 signaling axis.

    PubMed

    Hu, Lifang; Su, Peihong; Yin, Chong; Zhang, Yan; Li, Runzhi; Yan, Kun; Chen, Zhihao; Li, Dijie; Zhang, Ge; Wang, Liping; Miao, Zhiping; Qian, Airong; Xian, Cory J

    2018-02-01

    Osteoblast differentiation is a multistep process delicately regulated by many factors, including cytoskeletal dynamics and signaling pathways. Microtubule actin crosslinking factor 1 (MACF1), a key cytoskeletal linker, has been shown to play key roles in signal transduction and in diverse cellular processes; however, its role in regulating osteoblast differentiation is still needed to be elucidated. To further uncover the functions and mechanisms of action of MACF1 in osteoblast differentiation, we examined effects of MACF1 knockdown (MACF1-KD) in MC3T3-E1 osteoblastic cells on their osteoblast differentiation and associated molecular mechanisms. The results showed that knockdown of MACF1 significantly suppressed mineralization of MC3T3-E1 cells, down-regulated the expression of key osteogenic genes alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2) and type I collagen α1 (Col Iα1). Knockdown of MACF1 dramatically reduced the nuclear translocation of β-catenin, decreased the transcriptional activation of T cell factor 1 (TCF1), and down-regulated the expression of TCF1, lymphoid enhancer-binding factor 1 (LEF1), and Runx2, a target gene of β-catenin/TCF1. In addition, MACF1-KD increased the active level of glycogen synthase kinase-3β (GSK-3β), which is a key regulator for β-catenin signal transduction. Moreover, the reduction of nuclear β-catenin amount and decreased expression of TCF1 and Runx2 were significantly reversed in MACF1-KD cells when treated with lithium chloride, an agonist for β-catenin by inhibiting GSK-3β activity. Taken together, these findings suggest that knockdown of MACF1 in osteoblastic cells inhibits osteoblast differentiation through suppressing the β-catenin/TCF1-Runx2 axis. Thus, a novel role of MACF1 in and a new mechanistic insight of osteoblast differentiation are uncovered. © 2017 Wiley Periodicals, Inc.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yang, Lei; Wu, Zhong; Yin, Gang

    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 littlemore » 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.« less

  17. 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.

  18. Nano hydroxyapatite-blasted titanium surface affects pre-osteoblast morphology by modulating critical intracellular pathways.

    PubMed

    Bezerra, Fábio; Ferreira, Marcel R; Fontes, Giselle N; da Costa Fernandes, Célio Jr; Andia, Denise C; Cruz, Nilson C; da Silva, Rodrigo A; Zambuzzi, Willian F

    2017-08-01

    Although, intracellular signaling pathways are proposed to predict the quality of cell-surface relationship, this study addressed pre-osteoblast behavior in response to nano hydroxyapatite (HA)-blasted titanium (Ti) surface by exploring critical intracellular pathways and pre-osteoblast morphological change. Physicochemical properties were evaluated by atomic force microscopy (AFM) and wettability considering water contact angle of three differently texturized Ti surfaces: Machined (Mac), Dual acid-etching (DAE), and nano hydroxyapatite-blasted (nHA). The results revealed critical differences in surface topography, impacting the water contact angle and later the osteoblast performance. In order to evaluate the effect of those topographical characteristics on biological responses, we have seeded pre-osteoblast cells on the Ti discs for up to 4 h and subjected the cultures to biological analysis. First, we have observed pre-osteoblasts morphological changes resulting from the interaction with the Ti texturized surfaces whereas the cells cultured on nHA presented a more advanced spreading process when compared with the cells cultured on the other surfaces. These results argued us for analyzing the molecular machinery and thus, we have shown that nHA promoted a lower Bax/Bcl2 ratio, suggesting an interesting anti-apoptotic effect, maybe explained by the fact that HA is a natural element present in bone composition. Thereafter, we investigated the potential effect of those surfaces on promoting pre-osteoblast adhesion and survival signaling by performing crystal violet and immunoblotting approaches, respectively. Our results showed that nHA promoted a higher pre-osteoblast adhesion supported by up-modulating FAK and Src activations, both signaling transducers involved during eukaryotic cell adhesion. Also, we have shown Ras-Erk stimulation by the all evaluated surfaces. Finally, we showed that all Ti-texturing surfaces were able to promote osteoblast differentiation

  19. Pulsed electromagnetic fields promote the proliferation and differentiation of osteoblasts by reinforcing intracellular calcium transients.

    PubMed

    Tong, Jie; Sun, Lijun; Zhu, Bin; Fan, Yun; Ma, Xingfeng; Yu, Liyin; Zhang, Jianbao

    2017-10-01

    Pulsed electromagnetic fields (PEMF) can be used to treat bone-related diseases, but the underlying mechanism remains unclear, especially the process by which PEMFs initiate biological effects. In this study, we demonstrated the effects of PEMF on proliferation and differentiation of osteoblasts using the model of calcium transients induced by high extracellular calcium. Our results showed that PEMF can increase both the percentage of responding cells and amplitude of intracellular calcium transients induced by high extracellular calcium stimulation. Compared with corresponding extracellular calcium levels, PEMF stimulation increased proliferation and differentiation of osteoblasts and related gene expressions, such as insulin-like growth factor 1 (IGF-1), alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), and osteocalcin (OCN), which can be completely abolished by BAPTA-AM. Moreover, PEMF did not affect proliferation and differentiation of osteoblasts if no intracellular calcium transient was present in osteoblasts during PEMF exposure. Our results revealed that PEMF affects osteoblast proliferation and differentiation through enhanced intracellular calcium transients, which provided a cue to treat bone-related diseases with PEMF. Bioelectromagnetics. 38:541-549, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  20. Differential responses of osteoblast lineage cells to nanotopographically-modified, microroughened titanium-aluminum-vanadium alloy surfaces.

    PubMed

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

    2012-12-01

    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. Copyright © 2012 Elsevier Ltd. All rights reserved.

  1. 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

  2. Appearance of cell-adhesion factor in osteoblast proliferation and differentiation of apatite coating titanium by blast coating method.

    PubMed

    Umeda, Hirotsugu; Mano, Takamitsu; Harada, Koji; Tarannum, Ferdous; Ueyama, Yoshiya

    2017-08-01

    We have already reported that the apatite coating of titanium by the blast coating (BC) method could show a higher rate of bone contact from the early stages in vivo, when compared to the pure titanium (Ti) and the apatite coating of titanium by the flame spraying (FS) method. However, the detailed mechanism by which BC resulted in satisfactory bone contact is still unknown. In the present study, we investigated the importance of various factors including cell adhesion factor in osteoblast proliferation and differentiation that could affect the osteoconductivity of the BC disks. Cell proliferation assay revealed that Saos-2 could grow fastest on BC disks, and that a spectrophotometric method using a LabAssay TM ALP kit showed that ALP activity was increased in cells on BC disks compared to Ti disks and FS disks. In addition, higher expression of E-cadherin and Fibronectin was observed in cells on BC disks than Ti disks and FS disks by relative qPCR as well as Western blotting. These results suggested that the expression of cell-adhesion factors, proliferation and differentiation of osteoblast might be enhanced on BC disks, which might result higher osteoconductivity.

  3. Mechanical unloading reduces microtubule actin crosslinking factor 1 expression to inhibit β-catenin signaling and osteoblast proliferation.

    PubMed

    Yin, Chong; Zhang, Yan; Hu, Lifang; Tian, Ye; Chen, Zhihao; Li, Dijie; Zhao, Fan; Su, Peihong; Ma, Xiaoli; Zhang, Ge; Miao, Zhiping; Wang, Liping; Qian, Airong; Xian, Cory J

    2018-07-01

    Mechanical unloading was considered a major threat to bone homeostasis, and has been shown to decrease osteoblast proliferation although the underlying mechanism is unclear. Microtubule actin crosslinking factor 1 (MACF1) is a cytoskeletal protein that regulates cellular processes and Wnt/β-catenin pathway, an essential signaling pathway for osteoblasts. However, the relationship between MACF1 expression and mechanical unloading, and the function and the associated mechanisms of MACF1 in regulating osteoblast proliferation are unclear. This study investigated effects of mechanical unloading on MACF1 expression levels in cultured MC3T3-E1 osteoblastic cells and in femurs of mice with hind limb unloading; and it also examined the role and potential action mechanisms of MACF1 in osteoblast proliferation in MACF1-knockdown, overexpressed or control MC3T3-E1 cells treated with or without the mechanical unloading condition. Results showed that the mechanical unloading condition inhibited osteoblast proliferation and MACF1 expression in MC3T3-E1 osteoblastic cells and mouse femurs. MACF1 knockdown decreased osteoblast proliferation, while MACF1 overexpression increased it. The inhibitory effect of mechanical unloading on osteoblast proliferation also changed with MACF1 expression levels. Furthermore, MACF1 was found to enhance β-catenin expression and activity, and mechanical unloading decreased β-catenin expression through MACF1. Moreover, β-catenin was found an important regulator of osteoblast proliferation, as its preservation by treatment with its agonist lithium attenuated the inhibitory effects of MACF1-knockdown or mechanical unloading on osteoblast proliferation. Taken together, mechanical unloading decreases MACF1 expression, and MACF1 up-regulates osteoblast proliferation through enhancing β-catenin signaling. This study has thus provided a mechanism for mechanical unloading-induced inhibited osteoblast proliferation. © 2017 Wiley Periodicals, Inc.

  4. 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.

  5. Bisphosphonate stimulation of osteoblasts and osteoblastic metastasis as a mechanism of hypocalcaemia.

    PubMed

    Ho, Joon Wee

    2012-03-01

    Bisphosphonates are used in the oncological setting to treat and prevent skeletal-related events and preserve bone mineral density. Bisphosphonates also possess a hypocalcaemic effect. When undesired, hypocalcaemia can result in increased morbidity and complications. The currently understood mechanism of bisphosphonate-induced hypocalcaemia is by osteoclast inhibition. The effect of bisphosphonates on osteoblasts is less well understood. Laboratory studies demonstrate that bisphosphonates increase osteoblast and osteoblastic metastases maturation, activity and bone mineralization. We hypothesize that where large populations of osteoblasts exist increased mineralization may result in hypocalcaemia. Consequently patients with bone-metastatic prostate cancer may be more susceptible to symptomatic hypocalcaemia following bisphosphonate therapy. We are currently designing a study to investigate our hypothesis and to identify the risk factors of hypocalcaemia. Copyright © 2011 Elsevier Ltd. All rights reserved.

  6. Physical activity and hypocaloric diet recovers osteoblasts homeostasis in women affected by abdominal obesity.

    PubMed

    Bimonte, Viviana M; Fittipaldi, Simona; Marocco, Chiara; Emerenziani, Gian Pietro; Fornari, Rachele; Guidetti, Laura; Poggiogalle, Eleonora; Nicolai, Emanuele; Di Luigi, Luigi; Donini, Lorenzo M; Baldari, Carlo; Lenzi, Andrea; Greco, Emanuela A; Migliaccio, Silvia

    2017-11-01

    Obesity is a multifactorial disease linked to metabolic chronic disorders such as diabetes, and hypertension. Also, it has recently been associated with skeletal alterations and low bone mineral density. We previously demonstrated that exposure of osteoblasts to sera of sedentary subjects affected by obesity alters cell homeostasis in vitro, leading to disruption of intracellular differentiation pathways and cellular activity. Thus, the purpose of the present study has been to evaluate whether sera of sedentary obese women, subjected to physical activity and hypocaloric diet, could recover osteoblast homeostasis in vitro as compared to the sera of same patients before intervention protocol. To this aim, obese women were evaluated at time 0 and after 4, 6, and 12 months of individualized prescribed physical activity and hypocaloric diet. Dual-energy-X-ray absorptiometry measurements were performed at each time point, as well as blood was collected at the same points. Cells were incubated with sera of subjects before and after physical activity as described: obese at baseline and after for 4, 6, and 12 months of physical activity and nutritional protocol intervention. Osteoblasts exposed to sera of patients, who displayed increased lean and decreased fat mass (from 55.5 ± 6.5 to 57.1 ± 5.6% p ≤ 0.05; from 44.5 ± 1.1 to 40.9 ± 2.6% p ≤ 0.01 respectively), showed a time-dependent increase of Wnt/β-catenin signaling, versus cells exposed to sera of obese patients before intervention protocol, suggesting recovery of osteoblast homeostasis upon improvement of body composition. An increase in β-catenin nuclear accumulation and nuclear translocation was also observed, accompanied by an increase in Adiponectin receptor 1 protein expression, suggesting positive effect on cell differentiation program. Furthermore, a decrease in sclerostin amount and an increase of type 1 procollagen amino-terminal-propeptide were depicted as compared to

  7. Osteoblast Role in Rheumatic Diseases.

    PubMed

    Corrado, Addolorata; Maruotti, Nicola; Cantatore, Francesco Paolo

    2017-06-15

    Alterations in osteoblast growth, differentiation and activity play a role in the pathogenesis of several rheumatic diseases, such as rheumatoid arthritis, spondyloarthritides, osteoarthritis, and osteoporosis. In fact, in these rheumatic diseases, abnormal activity of Wnt signaling, receptor activator of nuclear factor-κB (RANK)-RANK ligand (RANKL)-osteoprotegerin (OPG) signaling, bone morphogenetic proteins (BMPs) pathway and other mechanisms have been described in osteoblasts. This review article is focused on current knowledge on the role of osteoblast dysregulation occurring in rheumatic diseases.

  8. Osteoblast-specific transcription factor Osterix (Osx) is an upstream regulator of Satb2 during bone formation.

    PubMed

    Tang, Wanjin; Li, Yang; Osimiri, Lindsey; Zhang, Chi

    2011-09-23

    Osterix (Osx) is an osteoblast-specific transcription factor essential for osteoblast differentiation and bone formation. Osx knock-out mice lack bone completely. Satb2 is critical for osteoblast differentiation as a special AT-rich binding transcription factor. It is not known how Satb2 is transcriptionally regulated during bone formation. In this study, quantitative real-time RT-PCR results demonstrated that Satb2 was down-regulated in Osx-null calvaria. In stable C2C12 mesenchymal cells using the tetracycline (Tet)-Off system, overexpression of Osx stimulated Satb2 expression. Moreover, inhibition of Osx by siRNA led to repression of Satb2 expression in osteoblasts. These results suggest that Osx controls Satb2 expression. Transient transfection assay showed that Osx activated 1kb Satb2 promoter reporter activity in a dose-dependent manner. To define the region of Satb2 promoter responsive to Osx activation, a series of deletion mutants of Satb2 constructs were made, and the minimal region was narrowed down to the proximal 130 bp of the Satb2 promoter. Further point mutation studies found that two GC-rich region mutations disrupted the Satb2 130bp promoter activation by Osx, suggesting that these GC-rich binding sites were responsible for Satb2 activation by Osx. Gel shift assay showed that Osx bound to the Satb2 promoter sequence directly. ChIP assays indicated that endogenous Osx associated with the native Satb2 promoter in osteoblasts. Importantly, Satb2 siRNA significantly inhibited Osx-induced osteoblast marker gene expressions. Taken together, our findings indicate that Osx is an upstream regulator of Satb2 during bone formation. This reveals a new additional link of the transcriptional regulation mechanism that Osx controls bone formation.

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

    PubMed Central

    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

  10. 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.

  11. Mangiferin positively regulates osteoblast differentiation and suppresses osteoclast differentiation

    PubMed Central

    Sekiguchi, Yuusuke; Mano, Hiroshi; Nakatani, Sachie; Shimizu, Jun; Kataoka, Aya; Ogura, Kana; Kimira, Yoshifumi; Ebata, Midori; Wada, Masahiro

    2017-01-01

    Mangiferin is a polyphenolic compound present in Salacia reticulata. It has been reported to reduce bone destruction and inhibit osteoclastic differentiation. This study aimed to determine whether mangiferin directly affects osteoblast and osteoclast proliferation and differentiation, and gene expression in MC3T3-E1 osteoblastic cells and osteoclast-like cells derived from primary mouse bone marrow macrophage cells. Mangiferin induced significantly greater WST-1 activity, indicating increased cell proliferation. Mangiferin induced significantly increased alkaline phosphatase staining, indicating greater cell differentiation. Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated that mangiferin significantly increased the mRNA level of runt-related transcription factor 2 (RunX2), but did not affect RunX1 mRNA expression. Mangiferin significantly reduced the formation of tartrate-resistant acid phosphatase-positive multinuclear cells. RT-PCR demonstrated that mangiferin significantly increased the mRNA level of estrogen receptor β (ERβ), but did not affect the expression of other osteoclast-associated genes. Mangiferin may inhibit osteoclastic bone resorption by suppressing differentiation of osteoclasts and promoting expression of ERβ mRNA in mouse bone marrow macrophage cells. It also has potential to promote osteoblastic bone formation by promoting cell proliferation and inducing cell differentiation in preosteoblast MC3T3-E1 cells via RunX2. Mangiferin may therefore be useful in improving bone disease outcomes. PMID:28627701

  12. Mangiferin positively regulates osteoblast differentiation and suppresses osteoclast differentiation.

    PubMed

    Sekiguchi, Yuusuke; Mano, Hiroshi; Nakatani, Sachie; Shimizu, Jun; Kataoka, Aya; Ogura, Kana; Kimira, Yoshifumi; Ebata, Midori; Wada, Masahiro

    2017-08-01

    Mangiferin is a polyphenolic compound present in Salacia reticulata. It has been reported to reduce bone destruction and inhibit osteoclastic differentiation. This study aimed to determine whether mangiferin directly affects osteoblast and osteoclast proliferation and differentiation, and gene expression in MC3T3‑E1 osteoblastic cells and osteoclast‑like cells derived from primary mouse bone marrow macrophage cells. Mangiferin induced significantly greater WST‑1 activity, indicating increased cell proliferation. Mangiferin induced significantly increased alkaline phosphatase staining, indicating greater cell differentiation. Reverse transcription‑polymerase chain reaction (RT‑PCR) demonstrated that mangiferin significantly increased the mRNA level of runt‑related transcription factor 2 (RunX2), but did not affect RunX1 mRNA expression. Mangiferin significantly reduced the formation of tartrate‑resistant acid phosphatase‑positive multinuclear cells. RT‑PCR demonstrated that mangiferin significantly increased the mRNA level of estrogen receptor β (ERβ), but did not affect the expression of other osteoclast‑associated genes. Mangiferin may inhibit osteoclastic bone resorption by suppressing differentiation of osteoclasts and promoting expression of ERβ mRNA in mouse bone marrow macrophage cells. It also has potential to promote osteoblastic bone formation by promoting cell proliferation and inducing cell differentiation in preosteoblast MC3T3‑E1 cells via RunX2. Mangiferin may therefore be useful in improving bone disease outcomes.

  13. CONNECTIVE TISSUE GROWTH FACTOR IS A TARGET OF NOTCH SIGNALING IN CELLS OF THE OSTEOBLASTIC LINEAGE

    PubMed Central

    Canalis, Ernesto; Zanotti, Stefano; Smerdel-Ramoya, Anna

    2014-01-01

    Connective tissue growth factor (Ctgf) or CCN2 is a protein synthesized by osteoblasts necessary for skeletal homeostasis, although its overexpression inhibits osteogenic signals and bone formation. Ctgf is induced by bone morphogenetic proteins, transforming growth factor β and Wnt; and in the present studies, we explored whether Notch regulated Ctgf expression in osteoblasts. We employed RosaNotch mice, where the Notch intracellular domain (NICD) is expressed following the excision of a STOP cassette, placed between the Rosa26 promoter and NICD. Notch was activated by transduction of adenoviral vectors expressing Cre recombinase (Ad-CMV-Cre). Notch induced Ctgf mRNA levels in a time dependent manner and increased Ctgf heterogeneous nuclear RNA. Notch also destabilized Ctgf mRNA shortening its half-life from 13 h to 3 h. The effect of Notch on Ctgf expression was lost following Rbpjκ downregulation, demonstrating that it was mediated by Notch canonical signaling. However, downregulation of the classic Notch target genes Hes1, Hey1 and Hey2 did not modify the effect of Notch on Ctgf expression. Wild type osteoblasts exposed to immobilized Delta-like 1 displayed enhanced Notch signaling and increased Ctgf expression. In addition to the effects of Notch in vitro, Notch induced Ctgf in vivo, and calvariae and femurs from RosaNotch mice mated with transgenics expressing the Cre recombinase in cells of the osteoblastic lineage exhibited increased expression of Ctgf. In conclusion, Ctgf is a target of Notch canonical signaling in osteoblasts, and may act in concert with Notch to regulate skeletal homeostasis. PMID:24792956

  14. Effect of Granulocyte-Colony Stimulating Factor on Endothelial Cells and Osteoblasts

    PubMed Central

    Liu, Xi Ling; Hu, Xiang; Cai, Wei Xin; Lu, Weijia William; Zheng, Li Wu

    2016-01-01

    Objectives. Some animal studies showed that granulocyte-colony stimulating factor (G-CSF) provides beneficial environment for bone healing. It has been well documented that endothelial cells and osteoblasts play critical roles in multiple phases of bone healing. However, the biological effects of G-CSF on these cells remain controversial. This study aimed to investigate the influence of G-CSF at various concentrations on endothelial cells and osteoblasts. Materials and Methods. Human umbilical vein endothelial cells (HUVECs) and human osteoblasts (hOBs) were treated with G-CSF at 1000, 100, 10, and 0 ng/mL, respectively. The capacity of cell proliferation, migration, and tube formation of HUVECs was evaluated at 72, 8, and 6 hours after treatment, respectively. The capacity of proliferation, differentiation, and mineralization of hOBs was evaluated at 24 hours, 72 hours, and 21 days after treatment, respectively. Results. HUVECs treated with 100 and 1000 ng/mL G-CSF showed a significantly higher value comparing with controls in migration assay (p < 0.001, p < 0.01, resp.); the group treated with 1000 ng/mL G-CSF showed a significantly lower value on tube formation. No significant difference was detected in groups of hOBs. Conclusions. G-CSF showed favorable effects only on the migration of HUVECs, and no direct influence was found on hOBs. PMID:27006951

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

    PubMed

    Fan, J Z; Yang, X; Bi, Z G

    2015-07-01

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

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

    PubMed Central

    Fan, J.Z.; Yang, X.; Bi, Z.G.

    2015-01-01

    We investigated whether 6-gingerol affects the maturation and proliferation of osteoblast-like MG63 cells in vitro. Osteoblast-like MG63 cells were treated with 6-gingerol under control conditions, and experimental inflammation was induced by tumor necrosis factor-α (TNF-α). Expression of different osteogenic markers and cytokines was analyzed by real-time PCR, Western blotting, and enzyme-linked immunosorbent assay. In addition, alkaline phosphatase (ALP) enzyme activity and biomineralization as markers for differentiation were measured. Treatment with 6-gingerol resulted in insignificant effects on the proliferation rate. 6-Gingerol induced the differentiation of osteoblast-like cells with increased transcription levels of osteogenic markers, upregulated ALP enzyme activity, and enhanced mineralized nodule formation. Stimulation with TNF-α led to enhanced interleukin-6 and nuclear factor-κB expression and downregulated markers of osteoblastic differentiation. 6-Gingerol reduced the degree of inflammation in TNF-α-treated MG-63 cells. In conclusion, 6-gingerol stimulated osteoblast differentiation in normal physiological and inflammatory settings, and therefore, 6-gingerol represents a promising agent for treating osteoporosis or bone inflammation. PMID:25923459

  17. Lactoferrin promote primary rat osteoblast proliferation and differentiation via up-regulation of insulin-like growth factor-1 expression.

    PubMed

    Hou, Jian-ming; Wu, Man; Lin, Qing-ming; Lin, Fan; Xue, Ying; Lan, Xu-hua; Chen, En-yu; Wang, Mei-li; Yang, Hai-yan; Wang, Feng-xiong

    2014-08-01

    The aim of this study was to explore the effect of lactoferrin (LF) in primary fetal rat osteoblasts proliferation and differentiation and investigate the underlying molecular mechanisms. Primary rat osteoblasts were obtained from the calvarias of neonatal rats. Osteoblasts were treated with LF (0.1-1000 μg/mL), or OSI-906 [a selective inhibitor of insulin-like growth factor 1 (IGF-1) receptor and insulin receptor]. The IGF-1 was then knocked down by small hairpin RNA (shRNA) technology and then was treated with recombinant human IGF-1 or LF. Cell proliferation and differentiation were measured by MTT assay and alkaline phosphatase (ALP) assay, respectively. The expression of IGF-1 and IGF binding protein 2 (IGFBP2) mRNA were analyzed using real-time PCR. LF promotes the proliferation and differentiation of osteoblasts in a certain range (1-100 μg/mL) in time- and dose-dependent manner. The mRNA level of IGF-1 was significantly increased, while the expression of IGFBP2 was suppressed by LF treatment. Knockdown of IGF-1 by shRNA in primary rat osteoblast dramatically decreased the abilities of proliferation and differentiation of osteoblasts and blocked the proliferation and differentiation effect of LF in osteoblasts. OSI906 (5 μM) blocked the mitogenic and differentiation of LF in osteoblasts. Proliferation and differentiation of primary rat osteoblasts in response to LF are mediated in part by stimulating of IGF-1 gene expression and alterations in the gene expression of IGFBP2.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Nifuji, Akira, E-mail: nifuji-a@tsurumi-u.ac.jp; Department of Pharmacology, Tsurumi University School of Dental Medicine, Yokohama; Ideno, Hisashi

    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 NLKmore » 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.« less

  19. miR-764-5p promotes osteoblast differentiation through inhibition of CHIP/STUB1 expression.

    PubMed

    Guo, Junwei; Ren, Fangli; Wang, Yinyin; Li, Shan; Gao, Zhengrong; Wang, Xiaoyan; Ning, Hongxiu; Wu, Jianguo; Li, Yi; Wang, Zhao; Chim, Shek Man; Xu, Jiake; Chang, Zhijie

    2012-07-01

    Differentiation of committed precursor cells into the osteoblast lineage is tightly regulated by several factors, including Runx2 and BMP2. We previously reported that C terminus of Hsc70-interacting protein/STIP1 homology and U-Box containing protein 1 (CHIP/STUB1) negatively regulated osteoblast differentiation through promoting Runx2 protein degradation. However, how CHIP is regulated during osteoblast differentiation remains unknown. In this study, we found that miR-764-5p is up-expressed during the osteoblast differentiation in calvarial and osteoblast progenitor cells, coupled with down-expression of CHIP protein. We observed that forced expression or inhibition of miR-764-5p decreased or increased the CHIP protein level through affecting its translation by targeting the 3'-UTR region. Perturbation of miR-764-5p resulted in altered differentiation fate of osteoblast progenitor cells and the role of miR-764-5p was reversed by overexpression of CHIP, whereas depletion of CHIP impaired the effect of miR-764-5p. Our data showed that miR-764-5p positively regulates osteoblast differentiation from osteoblast progenitor cells by repressing the translation of CHIP protein. Copyright © 2012 American Society for Bone and Mineral Research.

  20. Static magnetic fields promote osteoblastic/cementoblastic differentiation in osteoblasts, cementoblasts, and periodontal ligament cells

    PubMed Central

    2017-01-01

    Purpose Although static magnetic fields (SMFs) have been used in dental prostheses and osseointegrated implants, their biological effects on osteoblastic and cementoblastic differentiation in cells involved in periodontal regeneration remain unknown. This study was undertaken to investigate the effects of SMFs (15 mT) on the osteoblastic and cementoblastic differentiation of human osteoblasts, periodontal ligament cells (PDLCs), and cementoblasts, and to explore the possible mechanisms underlying these effects. Methods Differentiation was evaluated by measuring alkaline phosphatase (ALP) activity, mineralized nodule formation based on Alizarin red staining, calcium content, and the expression of marker mRNAs assessed by reverse transcription polymerase chain reaction (RT-PCR). Signaling pathways were analyzed by western blotting and immunocytochemistry. Results The activities of the early marker ALP and the late markers matrix mineralization and calcium content, as well as osteoblast- and cementoblast-specific gene expression in osteoblasts, PDLCs, and cementoblasts were enhanced. SMFs upregulated the expression of Wnt proteins, and increased the phosphorylation of glycogen synthase kinase-3β (GSK-3β) and total β-catenin protein expression. Furthermore, p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK), and nuclear factor-κB (NF-κB) pathways were activated. Conclusions SMF treatment enhanced osteoblastic and/or cementoblastic differentiation in osteoblasts, cementoblasts, and PDLCs. These findings provide a molecular basis for the beneficial osteogenic and/or cementogenic effect of SMFs, which could have potential in stimulating bone or cementum formation during bone regeneration and in patients with periodontal disease. PMID:29093986

  1. Canonical Wnt signaling in differentiated osteoblasts controls osteoclast differentiation.

    PubMed

    Glass, Donald A; Bialek, Peter; Ahn, Jong Deok; Starbuck, Michael; Patel, Millan S; Clevers, Hans; Taketo, Mark M; Long, Fanxin; McMahon, Andrew P; Lang, Richard A; Karsenty, Gerard

    2005-05-01

    Inactivation of beta-catenin in mesenchymal progenitors prevents osteoblast differentiation; inactivation of Lrp5, a gene encoding a likely Wnt coreceptor, results in low bone mass (osteopenia) by decreasing bone formation. These observations indicate that Wnt signaling controls osteoblast differentiation and suggest that it may regulate bone formation in differentiated osteoblasts. Here, we study later events and find that stabilization of beta-catenin in differentiated osteoblasts results in high bone mass, while its deletion from differentiated osteoblasts leads to osteopenia. Surprisingly, histological analysis showed that these mutations primarily affect bone resorption rather than bone formation. Cellular and molecular studies showed that beta-catenin together with TCF proteins regulates osteoblast expression of Osteoprotegerin, a major inhibitor of osteoclast differentiation. These findings demonstrate that beta-catenin, and presumably Wnt signaling, promote the ability of differentiated osteoblasts to inhibit osteoclast differentiation; thus, they broaden our knowledge of the functions Wnt proteins have at various stages of skeletogenesis.

  2. Alpinia officinarum Stimulates Osteoblast Mineralization and Inhibits Osteoclast Differentiation.

    PubMed

    Shim, Ki-Shuk; Lee, Chung-Jo; Yim, Nam-Hui; Gu, Min Jung; Ma, Jin Yeul

    2016-01-01

    Alpinia officinarum rhizome has been used as a traditional herbal remedy to treat inflammatory and internal diseases. Based on the previously observed inhibitory effect of A. officinarum rhizome in an arthritis model, we evaluated whether a water extract of A. officinarum rhizome (WEAO) would enhance in vitro osteoblast mineralization using calvarial osteoblast precursor cells or would inhibit in vitro osteoclast differentiation and bone resorption using bone marrow derived macrophages. In osteoblasts, WEAO enhanced the mRNA levels of transcription factor (runt-related transcription factor 2, smad1, smad5, and junB) and marker (bone morphogenetic protein-2, collagen type 1alpha1, and osteocalcin) genes related to osteoblast mineralization, consistent with increased alizarin red S staining intensity. WEAO markedly inhibited osteoclast differentiation by suppressing the receptor activator for nuclear factor-[Formula: see text]B ligand-induced downregulation of inhibitor of DNA binding 2 and V-maf musculoaponeurotic fibrosarcoma oncogene homolog B and the phosphorylation of c-Jun N-terminal kinase, p38, nuclear factor-[Formula: see text]B, c-Src, and Bruton's tyrosine kinase to induce nuclear factor of activated T cells cytoplasmic 1 expression. WEAO also suppressed the resorbing activity of mature osteoclasts by altering actin ring formation. Therefore, the results of this study demonstrate that WEAO stimulates osteoblast mineralization and inhibits osteoclast differentiation. Thus, WEAO may be a promising herbal candidate to treat or prevent pathological bone diseases by regulating the balance between osteoclast and osteoblast activity.

  3. The protective effect of platelet released growth factors and bone augmentation (Bio-Oss®) on ethanol impaired osteoblasts.

    PubMed

    Sönmez, Tolga Taha; Bayer, Andreas; Cremer, Tillman; Hock, Jennifer Vanessa Phi; Lethaus, Bernd; Kweider, Nisreen; Wruck, Christoph Jan; Drescher, Wolf; Jahr, Holger; Lippross, Sebastian; Pufe, Thomas; Tohidnezhad, Mersedeh

    2017-11-01

    Chronic alcohol consumption is a known limiting factor for bone healing. One promising strategy to improve bone augmentation techniques with Bio-Oss ® in oral and maxillofacial surgery might be the supportive application of platelet-concentrated biomaterials as platelet-released growth factor (PRGF). To address this matter, we performed an in vitro study investigating the protective effects of PRGF and Bio-Oss ® in ethanol (EtOH) treated osteoblasts. The SAOS-2 osteosarcoma cell line, with and without EtOH pretreatment was used. The cell viability, proliferation and alkali phosphatase activity (ALP) after application of 0%, 5% and 10% PRGF and Bio-Oss ® were assessed. The application of PRGF and Bio-Oss ® in EtOH impaired osteoblasts showed a significant beneficial influence increasing the viability of the osteoblasts in cell culture. The synergistic effect of Bio-Oss ® and 5% PRGF on the proliferation of osteoblasts was also demonstrated. Bio-Oss ® only in combination with PRGF increases the alkaline phosphatase (ALP) activity in EtOH pretreated cells. These results indicate that the simultaneous application of PRGF and Bio-Oss ® inhibits EtOH induced bone healing impairment. Furthermore, in the cells, PRGF induced a protective mechanism which might promote bone regeneration. Copyright © 2017 Elsevier GmbH. All rights reserved.

  4. Osteoblast and osteocyte: games without frontiers.

    PubMed

    Capulli, Mattia; Paone, Riccardo; Rucci, Nadia

    2014-11-01

    The portrait of osteoblasts and osteocytes has been subjected to a revision, since a large body of evidence is attributing these cells amazing roles both inside and outside the bone. The osteoblast, long confined to its bone building function, is actually a very eclectic cell, actively regulating osteoclast formation and function as well as hematopoietic stem cells homeostasis. It is also an endocrine cell, affecting energy metabolism, male fertility and cognition through the release of osteocalcin, a perfect definition-fitting hormone in its uncarboxylated state. As for the osteocytes, many evidence shows that they do not merely represent the final destination of the osteoblasts, but they are instead very active cells that, besides a mechanosensorial function, actively contribute to the bone remodelling by regulating bone formation and resorption. The regulation is exerted by the production of sclerostin (SOST), which in turn inhibits osteoblast differentiation by blocking Wnt/beta-catenin pathway. At the same time, osteocytes influence bone resorption both indirectly, by producing RANKL, which stimulates osteoclastogenesis, and directly by means of a local osteolysis, which is observed especially under pathological conditions. The great versatility of both these cells reflects the complexity of the bone tissue, which has not only a structural role, but influences and is influenced by different organs, taking part in homeostatic and adaptive responses affecting the whole organism. Copyright © 2014. Published by Elsevier Inc.

  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.

  6. Protein Kinase Inhibitor γ reciprocally regulates osteoblast and adipocyte differentiation by downregulating Leukemia Inhibitory Factor

    PubMed Central

    Chen, Xin; Hausman, Bryan S.; Luo, Guangbin; Zhou, Guang; Murakami, Shunichi; Rubin, Janet; Greenfield, Edward M.

    2013-01-01

    The Protein Kinase Inhibitor (Pki) gene family inactivates nuclear PKA and terminates PKA-induced gene expression. We previously showed that Pkig is the primary family member expressed in osteoblasts and that Pkig knockdown increases the effects of parathyroid hormone and isoproterenol on PKA activation, gene expression, and inhibition of apoptosis. Here, we determined whether endogenous levels of Pkig regulate osteoblast differentiation. Pkig is the primary family member in MEFs, murine marrow-derived mesenchymal stem cells, and human mesenchymal stem cells. Pkig deletion increased forskolin-dependent nuclear PKA activation and gene expression and Pkig deletion or knockdown increased osteoblast differentiation. PKA signaling is known to stimulate adipogenesis; however, adipogenesis and osteogenesis are often reciprocally regulated. We found that the reciprocal regulation predominates over the direct effects of PKA since adipogenesis was decreased by Pkig deletion or knockdown. Pkig deletion or knockdown simultaneously increased osteogenesis and decreased adipogenesis in mixed osteogenic/adipogenic medium. Pkig deletion increased PKA-induced expression of Leukemia Inhibitory Factor (Lif) mRNA and LIF protein. LIF neutralizing antibodies inhibited the effects on osteogenesis and adipogenesis of either Pkig deletion in MEFs or PKIγ knockdown in both murine and human mesenchymal stem cells. Collectively, our results show that endogenous levels of Pkig reciprocally regulate osteoblast and adipocyte differentiation and that this reciprocal regulation is mediated in part by LIF. PMID:23963683

  7. Stimulation of fibroblast growth factor 23 by metabolic acidosis requires osteoblastic intracellular calcium signaling and prostaglandin synthesis.

    PubMed

    Krieger, Nancy S; Bushinsky, David A

    2017-10-01

    Serum fibroblast growth factor 23 (FGF23) increases progressively in chronic kidney disease (CKD) and is associated with increased mortality. FGF23 is synthesized in osteoblasts and osteocytes; however, the factors regulating its production are not clear. Patients with CKD have decreased renal acid excretion leading to metabolic acidosis (MET). During MET, acid is buffered by bone with release of mineral calcium (Ca) and phosphate (P). MET increases intracellular Ca signaling and cyclooxygenase 2 (COX2)-induced prostaglandin production in the osteoblast, leading to decreased bone formation and increased bone resorption. We found that MET directly stimulates FGF23 in mouse bone organ cultures and primary osteoblasts. We hypothesized that MET increases FGF23 through similar pathways that lead to bone resorption. Neonatal mouse calvariae were incubated in neutral (NTL, pH = 7.44, Pco 2 = 38 mmHg, [HCO 3 - ] = 27 mM) or acid (MET, pH = 7.18, Pco 2 = 37 mmHg, [HCO 3 - ] = 13 mM) medium without or with 2-APB (50 μM), an inhibitor of intracellular Ca signaling or NS-398 (1 μM), an inhibitor of COX2. Each agent significantly inhibited MET stimulation of medium FGF23 protein and calvarial FGF23 RNA as well as bone resorption at 48 h. To exclude the potential contribution of MET-induced bone P release, we utilized primary calvarial osteoblasts. In these cells each agent inhibited MET stimulation of FGF23 RNA expression at 6 h. Thus stimulation of FGF23 by MET in mouse osteoblasts utilizes the same initial signaling pathways as MET-induced bone resorption. Therapeutic interventions directed toward correction of MET, especially in CKD, have the potential to not only prevent bone resorption but also lower FGF23 and perhaps decrease mortality. Copyright © 2017 the American Physiological Society.

  8. Slight changes in the mechanical stimulation affects osteoblast- and osteoclast-like cells in co-culture.

    PubMed

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

    2013-12-01

    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. 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. 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. 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.

  9. Cloning the promoter for transforming growth factor-beta type III receptor. Basal and conditional expression in fetal rat osteoblasts

    NASA Technical Reports Server (NTRS)

    Ji, C.; Chen, Y.; McCarthy, T. L.; Centrella, M.

    1999-01-01

    Transforming growth factor-beta binds to three high affinity cell surface molecules that directly or indirectly regulate its biological effects. The type III receptor (TRIII) is a proteoglycan that lacks significant intracellular signaling or enzymatic motifs but may facilitate transforming growth factor-beta binding to other receptors, stabilize multimeric receptor complexes, or segregate growth factor from activating receptors. Because various agents or events that regulate osteoblast function rapidly modulate TRIII expression, we cloned the 5' region of the rat TRIII gene to assess possible control elements. DNA fragments from this region directed high reporter gene expression in osteoblasts. Sequencing showed no consensus TATA or CCAAT boxes, whereas several nuclear factors binding sequences within the 3' region of the promoter co-mapped with multiple transcription initiation sites, DNase I footprints, gel mobility shift analysis, or loss of activity by deletion or mutation. An upstream enhancer was evident 5' proximal to nucleotide -979, and a silencer region occurred between nucleotides -2014 and -2194. Glucocorticoid sensitivity mapped between nucleotides -687 and -253, whereas bone morphogenetic protein 2 sensitivity co-mapped within the silencer region. Thus, the TRIII promoter contains cooperative basal elements and dispersed growth factor- and hormone-sensitive regulatory regions that can control TRIII expression by osteoblasts.

  10. Role of fibroblast growth factor receptors (FGFR) and FGFR like-1 (FGFRL1) in mesenchymal stromal cell differentiation to osteoblasts and adipocytes.

    PubMed

    Kähkönen, T E; Ivaska, K K; Jiang, M; Büki, K G; Väänänen, H K; Härkönen, P L

    2018-02-05

    Fibroblast growth factors (FGF) and their receptors (FGFRs) regulate many developmental processes including differentiation of mesenchymal stromal cells (MSC). We developed two MSC lines capable of differentiating to osteoblasts and adipocytes and studied the role of FGFRs in this process. We identified FGFR2 and fibroblast growth factor receptor like-1 (FGFRL1) as possible actors in MSC differentiation with gene microarray and qRT-PCR. FGFR2 and FGFRL1 mRNA expression strongly increased during MSC differentiation to osteoblasts. FGF2 treatment, resulting in downregulation of FGFR2, or silencing FGFR2 expression with siRNAs inhibited osteoblast differentiation. During adipocyte differentiation expression of FGFR1 and FGFRL1 increased and was down-regulated by FGF2. FGFR1 knockdown inhibited adipocyte differentiation. Silencing FGFR2 and FGFR1 in MSCs was associated with decreased FGFRL1 expression in osteoblasts and adipocytes, respectively. Our results suggest that FGFR1 and FGFR2 regulate FGFRL1 expression. FGFRL1 may mediate or modulate FGFR regulation of MSC differentiation together with FGFR2 in osteoblastic and FGFR1 in adipocytic lineage. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Osteoblast gene expression is differentially regulated by TGF-beta isoforms.

    PubMed

    Fagenholz, P J; Warren, S M; Greenwald, J A; Bouletreau, P J; Spector, J A; Crisera, F E; Longaker, M T

    2001-03-01

    The transforming growth factor beta (TGF-beta) superfamily encompasses a number of important growth factors including several TGF-beta isoforms, the bone morphogenetic proteins, activins, inhibins, and growth and differentiation factors. TGF-beta 1, -beta 2, and -beta 3 are three closely related isoforms that are widely expressed during skeletal morphogenesis and bone repair. Numerous studies suggest that each isoform has unique in vivo functions; however, the effects of these TGF-beta isoforms on osteoblast gene expression and maturation have never been directly compared. In the current study, we treated undifferentiated neonatal rat calvaria osteoblast-enriched cell cultures with 2.5 ng/ml of each TGF-beta isoform and analyzed gene expression at 0, 3, 6, and 24 hours. We demonstrated unique isoform-specific regulation of endogenous TGF-beta 1 and type I collagen mRNA transcription. To assess the effects of extended TGF-beta treatment on osteoblast maturation, we differentiated osteoblast cultures in the presence of 2.5 ng/ml of each TGF-beta isoform. Analysis of collagen I, alkaline phosphatase, and osteocalcin demonstrated that each TGF-beta isoform uniquely suppressed the transcription of these osteoblast differentiation markers. Interestingly, TGF-beta isoform treatment increased osteopontin expression in primary osteoblasts after 4 and 10 days of differentiation. To our knowledge, these data provide the first direct comparison of the effects of the TGF-beta isoforms on osteoblast gene expression in vitro. Furthermore, these data suggest that TGF-beta isoforms may exert their unique in vivo effects by differentially regulating osteoblast cytokine secretion, extracellular matrix production, and the rate of cellular maturation.

  12. Building strong bones: molecular regulation of the osteoblast lineage.

    PubMed

    Long, Fanxin

    2011-12-22

    The past 15 years have witnessed tremendous progress in the molecular understanding of osteoblasts, the main bone-forming cells in the vertebrate skeleton. In particular, all of the major developmental signals (including WNT and Notch signalling), along with an increasing number of transcription factors (such as RUNX2 and osterix), have been shown to regulate the differentiation and/or function of osteoblasts. As evidence indicates that osteoblasts may also regulate the behaviour of other cell types, a clear understanding of the molecular identity and regulation of osteoblasts is important beyond the field of bone biology.

  13. Osteoblast Menin Regulates Bone Mass in Vivo*

    PubMed Central

    Kanazawa, Ippei; Canaff, Lucie; Abi Rafeh, Jad; Angrula, Aarti; Li, Jingjing; Riddle, Ryan C.; Boraschi-Diaz, Iris; Komarova, Svetlana V.; Clemens, Thomas L.; Murshed, Monzur; Hendy, Geoffrey N.

    2015-01-01

    Menin, the product of the multiple endocrine neoplasia type 1 (Men1) tumor suppressor gene, mediates the cell proliferation and differentiation actions of transforming growth factor-β (TGF-β) ligand family members. In vitro, menin modulates osteoblastogenesis and osteoblast differentiation promoted and sustained by bone morphogenetic protein-2 (BMP-2) and TGF-β, respectively. To examine the in vivo function of menin in bone, we conditionally inactivated Men1 in mature osteoblasts by crossing osteocalcin (OC)-Cre mice with floxed Men1 (Men1f/f) mice to generate mice lacking menin in differentiating osteoblasts (OC-Cre;Men1f/f mice). These mice displayed significant reduction in bone mineral density, trabecular bone volume, and cortical bone thickness compared with control littermates. Osteoblast and osteoclast number as well as mineral apposition rate were significantly reduced, whereas osteocyte number was increased. Primary calvarial osteoblasts proliferated more quickly but had deficient mineral apposition and alkaline phosphatase activity. Although the mRNA expression of osteoblast marker and cyclin-dependent kinase inhibitor genes were all reduced, that of cyclin-dependent kinase, osteocyte marker, and pro-apoptotic genes were increased in isolated Men1 knock-out osteoblasts compared with controls. In contrast to the knock-out mice, transgenic mice overexpressing a human menin cDNA in osteoblasts driven by the 2.3-kb Col1a1 promoter, showed a gain of bone mass relative to control littermates. Osteoblast number and mineral apposition rate were significantly increased in the Col1a1-Menin-Tg mice. Therefore, osteoblast menin plays a key role in bone development, remodeling, and maintenance. PMID:25538250

  14. Improving effects of chitosan nanofiber scaffolds on osteoblast proliferation and maturation

    PubMed Central

    Ho, Ming-Hua; Liao, Mei-Hsiu; Lin, Yi-Ling; Lai, Chien-Hao; Lin, Pei-I; Chen, Ruei-Ming

    2014-01-01

    Osteoblast maturation plays a key role in regulating osteogenesis. Electrospun nanofibrous products were reported to possess a high surface area and porosity. In this study, we developed chitosan nanofibers and examined the effects of nanofibrous scaffolds on osteoblast maturation and the possible mechanisms. Macro- and micro observations of the chitosan nanofibers revealed that these nanoproducts had a flat surface and well-distributed fibers with nanoscale diameters. Mouse osteoblasts were able to attach onto the chitosan nanofiber scaffolds, and the scaffolds degraded in a time-dependent manner. Analysis by scanning electron microscopy further showed mouse osteoblasts adhered onto the scaffolds along the nanofibers, and cell–cell communication was also detected. Mouse osteoblasts grew much better on chitosan nanofiber scaffolds than on chitosan films. In addition, human osteoblasts were able to adhere and grow on the chitosan nanofiber scaffolds. Interestingly, culturing human osteoblasts on chitosan nanofiber scaffolds time-dependently increased DNA replication and cell proliferation. In parallel, administration of human osteoblasts onto chitosan nanofibers significantly induced osteopontin, osteocalcin, and alkaline phosphatase (ALP) messenger (m)RNA expression. As to the mechanism, chitosan nanofibers triggered runt-related transcription factor 2 mRNA and protein syntheses. Consequently, results of ALP-, alizarin red-, and von Kossa-staining analyses showed that chitosan nanofibers improved osteoblast mineralization. Taken together, results of this study demonstrate that chitosan nanofibers can stimulate osteoblast proliferation and maturation via runt-related transcription factor 2-mediated regulation of osteoblast-associated osteopontin, osteocalcin, and ALP gene expression. PMID:25246786

  15. Resveratrol Inhibits the Epidermal Growth Factor-Induced Migration of Osteoblasts: the Suppression of SAPK/JNK and Akt.

    PubMed

    Kawabata, Tetsu; Tokuda, Haruhiko; Fujita, Kazuhiko; Kainuma, Shingo; Sakai, Go; Matsushima-Nishiwaki, Rie; Kozawa, Osamu; Otsuka, Takanobu

    2017-01-01

    Resveratrol is a polyphenol enriched in the skins of grapes and berries, that shows various beneficial effects for human health. In the present study, we investigated the mechanism behind the epidermal growth factor (EGF)-induced migration of osteoblast-like MC3T3-E1 cells, and the effect of resveratrol on this cell migration. The cell migration was examined using Boyden chamber, and phosphorylation of each kinase was analyzed by Western blotting. The EGF-induced migration was suppressed by PD98059, an inhibitor of MEK1/2, as well as SB203580, an inhibitor of p38 MAP kinase, SP600125, an inhibitor of SAPK/JNK, and deguelin, an inhibitor of Akt. In contrast, rapamycin, an inhibitor of upstream kinase of p70 S6 kinase, and fasudil, an inhibitor of Rho-kinase, hardly affected the migration. Resveratrol significantly reduced the EGF-induced migration in a dose-dependent manner. SRT1720, an SIRT1 activator, suppressed the migration by EGF. In addition, resveratrol markedly attenuated the EGF-induced phosphorylation of SAPK/JNK and Akt without affecting the phosphorylation of p44/p42 MAP kinase or p38 MAP kinase. The phosphorylation of SAPK/JNK and Akt induced by EGF was down-regulated by SRT1720. Our results strongly suggest that resveratrol reduces the EGF-stimulated migration of osteoblasts via suppression of SAPK and Akt, and that the inhibitory effect of resveratrol is mediated in part via SIRT1. © 2017 The Author(s). Published by S. Karger AG, Basel.

  16. 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.

  17. 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

  18. Expression of adipokines in osteoarthritis osteophytes and their effect on osteoblasts.

    PubMed

    Junker, Susann; Frommer, Klaus W; Krumbholz, Grit; Tsiklauri, Lali; Gerstberger, Rüdiger; Rehart, Stefan; Steinmeyer, Jürgen; Rickert, Markus; Wenisch, Sabine; Schett, Georg; Müller-Ladner, Ulf; Neumann, Elena

    2017-10-01

    Osteophyte formation in osteoarthritis (OA) is mediated by increased osteoblast activity, which is -in turn- regulated by the Wnt signaling pathway. Obesity is regarded a risk factor in OA, yet little is known about the interaction between adipose tissue-derived factors, the adipokines, and bone formation, although adipokines are associated with the pathogenesis of OA. Therefore, the effect of adipokines on bone and cartilage forming cells and osteophyte development was analyzed. Human OA osteophytes were histologically characterized and adipokine expression was evaluated by immunohistochemistry. Osteoblasts and chondrocytes were isolated from OA tissue and stimulated with adiponectin, resistin, or visfatin. Cytokine and osteoblast/chondrocyte markers were quantified and activation of Wnt and p38 MAPK signaling was analyzed. Adiponectin, resistin, and visfatin were expressed in OA osteophytes by various articular cell types. Stimulation of OA osteoblasts with adiponectin and of OA chondrocytes with visfatin led to an increased release of proinflammatory mediators but not to osteoblast differentiation or activation. Additionally, visfatin increased matrix degrading factors in chondrocytes. Wnt signaling was not altered by adipokines, but adiponectin induced p38 MAPK signaling in osteoblasts. Adipokines are present in OA osteophytes, and adiponectin and visfatin increase the release of proinflammatory mediators by osteoblasts and chondrocytes. The effects of adiponectin were mediated by p38 MAPK but not Wnt signaling in osteoblasts. Therefore, the results support the idea that adipokines do not directly influence osteophyte development but the proinflammatory conditions in OA. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. [Biocompatibility of HA/TCP biphasic ceramics with co-cultured human osteoblasts in vitro].

    PubMed

    Lu, X; Li, S; Zhang, J; Zhang, Z; Lu, B; Bu, H; Li, Y; Cheng, J

    2001-12-01

    The biocompatibility of HA/TCP ceramic was evaluated by investigation of attachment and growth of osteoblasts on biomaterial, as well as monitoring the effects of biomaterial on expression of functional phenotypes of co-cultured osteoblasts in vitro. When co-cultured with HA/TCP ceramics, osteoblasts firstly attached to the surface of HA/TCP disk, then attached to notches and grew into the micropores of biomaterial during further culture period. At last, the ceramics were almost packed with osteoblasts. Additionally, osteoblasts co-cultured with HA/TCP were similar to osteoblasts cultured under normal condition in osteoblastic phenotypes; the secreted lots of collagen type I, possess strong activity of Alkaline Phosphatase and mineralized extracellular matrix. The fact that osteoblasts could grow well on HA/TCP ceramics and the biomaterial did not affect their physiological function suggest that HA/TCP ceramic is biocompatible with human osteoblasts.

  20. Response of human rheumatoid arthritis osteoblasts and osteoclasts to adiponectin.

    PubMed

    Krumbholz, Grit; Junker, Susann; Meier, Florian M P; Rickert, Markus; Steinmeyer, Jürgen; Rehart, Stefan; Lange, Uwe; Frommer, Klaus W; Schett, Georg; Müller-Ladner, Ulf; Neumann, Elena

    2017-01-01

    Adiponectin is an effector molecule in the pathophysiology of rheumatoid arthritis, e.g. by inducing cytokines and matrix degrading enzymes in synovial fibroblasts. There is growing evidence that adiponectin affects osteoblasts and osteoclasts although the contribution to the aberrant bone metabolism in rheumatoid arthritis is unclear. Therefore, the adiponectin effects on rheumatoid arthritis-derived osteoblasts and osteoclasts were evaluated. Adiponectin and its receptors were examined in bone tissue. Primary human osteoblasts and osteoclasts were stimulated with adiponectin and analysed using realtime polymerase chain-reaction and immunoassays. Effects on matrix-production by osteoblasts and differentiation and resorptive activity of osteoclasts were examined. Immunohistochemistry of rheumatoid arthritis bone tissue showed adiponectin expression in key cells of bone remodelling. Adiponectin altered gene expression and cytokine release in osteoblasts and increased IL-8 secretion by osteoclasts. Adiponectin inhibited osterix and induced osteoprotegerin mRNA in osteoblasts. In osteoclasts, MMP-9 and tartrate resistant acid phosphatase expression was increased. Accordingly, mineralisation capacity of osteoblasts decreased whereas resorptive activity of osteoclasts increased. The results confirm the proinflammatory potential of adiponectin and support the idea that adiponectin influences rheumatoid arthritis bone remodelling through alterations in osteoblast and osteoclast.

  1. Mechanisms of palmitate-induced cell death in human osteoblasts

    PubMed Central

    Gunaratnam, Krishanthi; Vidal, Christopher; Boadle, Ross; Thekkedam, Chris; Duque, Gustavo

    2013-01-01

    Summary Lipotoxicity is an overload of lipids in non-adipose tissues that affects function and induces cell death. Lipotoxicity has been demonstrated in bone cells in vitro using osteoblasts and adipocytes in coculture. In this condition, lipotoxicity was induced by high levels of saturated fatty acids (mostly palmitate) secreted by cultured adipocytes acting in a paracrine manner. In the present study, we aimed to identify the underlying mechanisms of lipotoxicity in human osteoblasts. Palmitate induced autophagy in cultured osteoblasts, which was preceded by the activation of autophagosomes that surround palmitate droplets. Palmitate also induced apoptosis though the activation of the Fas/Jun kinase (JNK) apoptotic pathway. In addition, osteoblasts could be protected from lipotoxicity by inhibiting autophagy with the phosphoinositide kinase inhibitor 3-methyladenine or by inhibiting apoptosis with the JNK inhibitor SP600125. In summary, we have identified two major molecular mechanisms of lipotoxicity in osteoblasts and in doing so we have identified a new potential therapeutic approach to prevent osteoblast dysfunction and death, which are common features of age-related bone loss and osteoporosis. PMID:24285710

  2. Bone marrow osteoblast vulnerability to chemotherapy

    PubMed Central

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

    2013-01-01

    Osteoblasts are a major component of the bone marrow microenvironment which provide support for hematopoietic cell development. Functional disruption of any element of the bone marrow niche, including osteoblasts, can potentially impair hematopoiesis. We have studied the effect of two widely used drugs with different mechanisms of action, etoposide (VP16) and melphalan, on murine osteoblasts at distinct stages of maturation. VP16 and melphalan delayed maturation of preosteoblasts and altered CXCL12 protein levels, a key regulator of hematopoietic cell homing to the bone marrow. Sublethal concentrations of VP16 and melphalan also decreased the levels of several transcripts which contribute to the composition of the extracellular matrix (ECM) including osteopontin (OPN), osteocalcin (OCN) and collagen 1A1 (Col1a1). The impact of chemotherapy on message and protein levels for some targets was not always aligned, suggesting differential responses at the transcription and translation or protein stability levels. Since one of the main functions of a mature osteoblast is to synthesize ECM of a defined composition, disruption of the ratio of its components may be one mechanism by which chemotherapy affects the ability of osteoblasts to support hematopoietic recovery coincident with altered marrow architecture. Collectively, these observations suggest that the osteoblast compartment of the marrow hematopoietic niche is vulnerable to functional dysregulation by damage imposed by agents frequently used in clinical settings. Understanding the mechanistic underpinning of chemotherapy-induced changes on the hematopoietic support capacity of the marrow microenvironment may contribute to improved strategies to optimize patient recovery post-transplantation. PMID:23551534

  3. Gravity, an Regulation Factor in BMSCs Differentiation to osteoblasts

    NASA Astrophysics Data System (ADS)

    Yan, Huang; Yinghui, Li; Fen, Yang; Zhongquan, Dai

    PURPOSE Most studies of regulatory mechanisms of adult stem cell differentiation are concentrated in chemical factors but few efforts are put into physical factors Recent space life science studies indicate mechanical factors participate in the differentiation of cells The aim of this study is to investigate the effects of simulated microgravity or hypergravity on the osteogenic differentiation of rat bone marrow mesenchymal stem cells BMSCs METHODOLOGY The BMSCs at day 7 were added osteogenic inducer 10nM dexamethasone 10mM beta -glycerophosphate and 50 mu M asorbic acid-2-phosphate for 7 days and cultured under simulated microgravity or hypergravity 2g for 1 day 3 days 5 days or 7 days RESULTS After treating BMSCs with osteogenic inducer and hypergravity the cells expressed more ColIA1 Cbfa1 and ALP than in single steogenic inducer treatment Reversely the cells treated with osteogenic inducer and simulated microgravity expressed less ColIA1 Cbfa1 and ALP CONCLUSIONS Our study suggests that hypergravity promotes the osteogenic differentiation of BMSCs and simulated microgravity inhibits this process Gravity is an important regulation factor in BMSCs differentiation to osteoblasts

  4. The Sox2 high mobility group transcription factor inhibits mature osteoblast function in transgenic mice

    PubMed Central

    Holmes, Greg; Bromage, Timothy G.; Basilico, Claudio

    2011-01-01

    We have previously shown that in osteoblasts Sox2 expression can be induced by Fgfs, and can inhibit Wnt signaling and differentiation. Furthermore, in mice in which Sox2 is conditionally deleted in the osteoblastic lineage, bones are osteopenic, and Sox2 inactivation in cultured osteoblasts leads to a loss of proliferative ability with a senescent phenotype. To help understand the role of Sox2 in osteoblast development we have specifically expressed Sox2 in bone from a Col1α1 promoter, which extended Sox2 expression into more mature osteoblasts. In long bones, trabecular cartilage remodeling was delayed and the transition from endochondral to cortical bone was disrupted, resulting in porous and undermineralized cortical bone. Collagen deposition was disorganized, and patterns of osteoclast activity were altered. Calvarial bones were thinner and parietal bones failed to develop the diploic space. Microarray analysis showed significant up- or downregulation of a variety of genes coding for non-collagenous extracellular matrix proteins, with a number of genes typical of mature osteoblasts being downregulated. Our results position Sox2 as a negative regulator of osteoblast maturation in vivo. PMID:21703370

  5. Effect of human vascular endothelial growth factor gene transfer on endogenous vascular endothelial growth factor mRNA expression in a rat fibroblast and osteoblast culture model.

    PubMed

    Li, Ru; Li, Claire H; Nauth, Aaron; McKee, Michael D; Schemitsch, Emil H

    2010-09-01

    Vascular endothelial growth factor (VEGF) plays an important role in promoting angiogenesis and osteogenesis during fracture repair. Our previous studies have shown that cell-based VEGF gene therapy enhances bone healing of a rabbit tibia segmental bone defect in vivo. The aim of this project was to examine the effect of exogenous human VEGF on the endogenous rat VEGF messenger RNA (mRNA) expression in a cell-based gene transfer model. Rat fibroblasts and osteoblasts were harvested from the dermal tissue and periosteum, respectively, of Fisher 344 rats. The cells were then cultured and transfected with pcDNA-human VEGF using Superfect reagent (Qiagen). Four experimental groups were created: 1) fibroblast-VEGF; 2) osteoblast-VEGF; 3) nontransfected fibroblast controls; and 4) nontransfected osteoblast controls. The cultured cells were harvested at 1, 3, and 7 days after the gene transfection. The total mRNA was extracted (Trizol; Invitrogen); both human VEGF and rat VEGF mRNA were measured by reverse transcriptase-polymerase chain reaction and quantified by VisionWorksLS. The human VEGF165 mRNA was detected by reverse transcriptase-polymerase chain reaction from transfected fibroblasts and osteoblasts at 1, 3, and 7 days after gene transfection. The human VEGF165 levels peaked at Day 1 and then gradually reduced expression in both transfected fibroblasts and osteoblasts. Two endogenous rat VEGF isoforms were detected in this cell culture model: rat VEGF120 and rat VEGF164. We compared the rat VEGF120 and rat VEGF164 expression level of the fibroblasts or osteoblasts that were transfected with human VEGF165, with nontransfected control cells. Both the transfected fibroblasts and osteoblasts showed greater expression of rat VEGF164 than nontransfected controls at Day 1 (peak level) and Day 3, but not at Day 7. The expression of rat VEGF120 was lower in transfected fibroblasts, but higher in transfected osteoblasts, than the relevant control groups at any time point

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

    PubMed

    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

    2011-05-01

    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(TM)) affects bone mass and remodeling in vivo. Male C57BL/6J mice received daily SAHA (100mg/kg) or vehicle injections for 3 to 4weeks. 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 4weeks after 4weeks 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 clinical use of

  7. Knockdown of microtubule actin crosslinking factor 1 inhibits cell proliferation in MC3T3-E1 osteoblastic cells

    PubMed Central

    Hu, Lifang; Su, Peihong; Li, Runzhi; Yan, Kun; Chen, Zhihao; Shang, Peng; Qian, Airong

    2015-01-01

    Microtubule actin crosslinking factor 1 (MACF1), a widely expressed cytoskeletal linker, plays important roles in various cells by regulating cytoskeleton dynamics. However, its role in osteoblastic cells is not well understood. Based on our previous findings that the association of MACF1 with F-actin and microtubules in osteoblast-like cells was altered under magnetic force conditions, here, by adopting a stable MACF1-knockdown MC3T3-E1 osteoblastic cell line, we found that MACF1 knockdown induced large cells with a binuclear/multinuclear structure. Further, immunofluorescence staining showed disorganization of F-actin and microtubules in MACF1-knockdown cells. Cell counting revealed significant decrease of cell proliferation and cell cycle analysis showed an S phase cell cycle arrest in MACF1-knockdown cells. Moreover and interestingly, MACF1 knockdown showed a potential effect on cellular MTT reduction activity and mitochondrial content, suggesting an impact on cellular metabolic activity. These results together indicate an important role of MACF1 in regulating osteoblastic cell morphology and function. [BMB Reports 2015; 48(10): 583-588] PMID:26277981

  8. Knockdown of microtubule actin crosslinking factor 1 inhibits cell proliferation in MC3T3-E1 osteoblastic cells.

    PubMed

    Hu, Lifang; Su, Peihong; Li, Runzhi; Yan, Kun; Chen, Zhihao; Shang, Peng; Qian, Airong

    2015-10-01

    Microtubule actin crosslinking factor 1 (MACF1), a widely expressed cytoskeletal linker, plays important roles in various cells by regulating cytoskeleton dynamics. However, its role in osteoblastic cells is not well understood. Based on our previous findings that the association of MACF1 with F-actin and microtubules in osteoblast-like cells was altered under magnetic force conditions, here, by adopting a stable MACF1-knockdown MC3T3-E1 osteoblastic cell line, we found that MACF1 knockdown induced large cells with a binuclear/multinuclear structure. Further, immunofluorescence staining showed disorganization of F-actin and microtubules in MACF1-knockdown cells. Cell counting revealed significant decrease of cell proliferation and cell cycle analysis showed an S phase cell cycle arrest in MACF1-knockdown cells. Moreover and interestingly, MACF1 knockdown showed a potential effect on cellular MTT reduction activity and mitochondrial content, suggesting an impact on cellular metabolic activity. These results together indicate an important role of MACF1 in regulating osteoblastic cell morphology and function.

  9. High Glucose Concentrations Suppress the Proliferation of Human Periodontal Ligament Stem Cells and Their Differentiation Into Osteoblasts.

    PubMed

    Kato, Hirohito; Taguchi, Yoichiro; Tominaga, Kazuya; Kimura, Daisuke; Yamawaki, Isao; Noguchi, Masahiro; Yamauchi, Nobuhiro; Tamura, Isao; Tanaka, Akio; Umeda, Makoto

    2016-04-01

    Diabetes mellitus (DM) is a major risk factor for periodontal disease and affects various cellular functions. Periodontal ligament stem cells (PDLSCs) play an important role in periodontal tissue regeneration; however, the effect of hyperglycemia on PDLSCs is unclear. The aim of this study is to investigate whether hyperglycemia affects periodontal tissue regeneration, using human PDLSCs and high-glucose medium as a model of DM. PDLSCs were obtained from healthy adult human mandibular third molars. Cell proliferation, osteoblastic differentiation, and proinflammatory cytokine expression were investigated by culturing PDLSCs in media supplemented with four different glucose concentrations representative of control patients (5.5 mM), patients with postprandial or controlled DM (8.0 mM), and patients with uncontrolled DM (12.0 and 24.0 mM). The molecular effects of hyperglycemia on PDLSC physiology were examined with a focus on the nuclear factor (NF)-(κB signaling pathway. The involvement of NF-κB was investigated with a specific NF-κB inhibitor in PDLSCs under hyperglycemic conditions. High glucose levels inhibited PDLSC proliferation and differentiation into osteoblasts but induced NF-κB activation and subsequent interleukin (IL)-6 and IL-8 expression. Treatment with an NF-κB inhibitor rescued the defects in cell proliferation and osteoblastic differentiation and inhibited the IL-6 expression caused by the high-glucose environment. The results of this study demonstrate that hyperglycemia inhibits human PDLSC proliferation and osteoblastic differentiation.

  10. Down-Regulation by Resveratrol of Basic Fibroblast Growth Factor-Stimulated Osteoprotegerin Synthesis through Suppression of Akt in Osteoblasts

    PubMed Central

    Kuroyanagi, Gen; Otsuka, Takanobu; Yamamoto, Naohiro; Matsushima-Nishiwaki, Rie; Nakakami, Akira; Mizutani, Jun; Kozawa, Osamu; Tokuda, Haruhiko

    2014-01-01

    It is firmly established that resveratrol, a natural food compound abundantly found in grape skins and red wine, has beneficial properties for human health. In the present study, we investigated the effect of basic fibroblast growth factor (FGF-2) on osteoprotegerin (OPG) synthesis in osteoblast-like MC3T3-E1 cells and whether resveratrol affects the OPG synthesis. FGF-2 stimulated both the OPG release and the expression of OPG mRNA. Resveratrol significantly suppressed the FGF-2-stimulated OPG release and the mRNA levels of OPG. SRT1720, an activator of SIRT1, reduced the FGF-2-induced OPG release and the OPG mRNA expression. PD98059, an inhibitor of upstream kinase activating p44/p42 mitogen-activated protein (MAP) kinase, had little effect on the FGF-2-stimulated OPG release. On the other hand, SB203580, an inhibitor of p38 MAP kinase, SP600125, an inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and Akt inhibitor suppressed the OPG release induced by FGF-2. Resveratrol failed to affect the FGF-2-induced phosphorylation of p44/p42 MAP kinase, p38 MAP kinase or SAPK/JNK. The phosphorylation of Akt induced by FGF-2 was significantly suppressed by resveratrol or SRT1720. These findings strongly suggest that resveratrol down-regulates FGF-2-stimulated OPG synthesis through the suppression of the Akt pathway in osteoblasts and that the inhibitory effect of resveratrol is mediated at least in part by SIRT1 activation. PMID:25290095

  11. Role of diabetes- and obesity-related protein in the regulation of osteoblast differentiation

    PubMed Central

    Linares, Gabriel R.; Xing, Weirong; Burghardt, Hans; Baumgartner, Bernhard; Chen, Shin-Tai; Ricart, Wifredo; Fernández-Real, José Manuel; Zorzano, Antonio

    2011-01-01

    Although thyroid hormone (TH) is known to exert important effects on the skeleton, the nuclear factors constituting the TH receptor coactivator complex and the molecular pathways by which TH mediates its effects on target gene expression in osteoblasts remain poorly understood. A recent study demonstrated that the actions of TH on myoblast differentiation are dependent on diabetes- and obesity-related protein (DOR). However, the role of DOR in osteoblast differentiation is unknown. We found DOR expression increased during in vitro differentiation of bone marrow stromal cells into osteoblasts and also in MC3T3-E1 cells treated with TH. However, DOR expression decreased during cellular proliferation. To determine whether DOR acts as a modulator of TH action during osteoblast differentiation, we examined whether overexpression or knockdown of DOR in MC3T3-E1 cells affects the ability of TH to induce osteoblast differentiation by evaluating alkaline phosphatase (ALP) activity. ALP activity was markedly increased in DOR-overexpressing cells treated with TH. In contrast, loss of DOR dramatically reduced TH stimulation of ALP activity in MC3T3-E1 cells and primary calvaria osteoblasts transduced with lentiviral DOR shRNA. Consistent with reduced ALP activity, mRNA levels of osteocalcin, ALP, and Runx2 were decreased significantly in DOR shRNA cells. In addition, a common single nucleotide polymorphism (SNP), DOR1 found on the promoter of human DOR gene, was associated with circulating osteocalcin levels in nondiabetic subjects. Based on these data, we conclude that DOR plays an important role in TH-mediated osteoblast differentiation, and a DOR SNP associates with plasma osteocalcin in men. PMID:21467300

  12. The Transient Receptor Potential Ion Channel TRPV6 Is Expressed at Low Levels in Osteoblasts and Has Little Role in Osteoblast Calcium Uptake

    PubMed Central

    Little, Robert; Muimo, Richmond; Robson, Louise; Harris, Kate; Grabowski, Peter S.

    2011-01-01

    Background TRPV6 ion channels are key mediators of regulated transepithelial absorption of Ca2+ within the small intestine. Trpv6 -/- mice were reported to have lower bone density than wild-type littermates and significant disturbances in calcium homeostasis that suggested a role for TRPV6 in osteoblasts during bone formation and mineralization. TRPV6 and molecules related to transepithelial Ca2+ transport have been reported to be expressed at high levels in human and mouse osteoblasts. Results Transmembrane ion currents in whole cell patch clamped SaOS-2 osteoblasts did not show sensitivity to ruthenium red, an inhibitor of TRPV5/6 ion channels, and 45Ca uptake was not significantly affected by ruthenium red in either SaOS-2 (P = 0.77) or TE-85 (P = 0.69) osteoblastic cells. In contrast, ion currents and 45Ca uptake were both significantly affected in a human bronchial epithelial cell line known to express TRPV6. TRPV6 was expressed at lower levels in osteoblastic cells than has been reported in some literature. In SaOS-2 TRPV6 mRNA was below the assay detection limit; in TE-85 TRPV6 mRNA was detected at 6.90±1.9 × 10−5 relative to B2M. In contrast, TRPV6 was detected at 7.7±3.0 × 10−2 and 2.38±0.28 × 10−4 the level of B2M in human carcinoma-derived cell lines LNCaP and CaCO-2 respectively. In murine primary calvarial osteoblasts TRPV6 was detected at 3.80±0.24 × 10−5 relative to GAPDH, in contrast with 4.3±1.5 × 10−2 relative to GAPDH in murine duodenum. By immunohistochemistry, TRPV6 was expressed mainly in myleocytic cells of the murine bone marrow and was observed only at low levels in murine osteoblasts, osteocytes or growth plate cartilage. Conclusions TRPV6 is expressed only at low levels in osteoblasts and plays little functional role in osteoblastic calcium uptake. PMID:22163264

  13. MicroRNA-194 promotes osteoblast differentiation via downregulating STAT1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Li, Jun; He, Xijing; Wei, Wenzhi

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

  14. The LIM protein LIMD1 influences osteoblast differentiation and function

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Luderer, Hilary F.; Bai Shuting; Longmore, Gregory D.

    2008-09-10

    The balance between bone resorption and bone formation involves the coordinated activities of osteoblasts and osteoclasts. Communication between these two cell types is essential for maintenance of normal bone homeostasis; however, the mechanisms regulating this cross talk are not completely understood. Many factors that mediate differentiation and function of both osteoblasts and osteoclasts have been identified. The LIM protein Limd1 has been implicated in the regulation of stress osteoclastogenesis through an interaction with the p62/sequestosome protein. Here we show that Limd1 also influences osteoblast progenitor numbers, differentiation, and function. Limd1{sup -/-} calvarial osteoblasts display increased mineralization and accelerated differentiation. Whilemore » no significant differences in osteoblast number or function were detected in vivo, bone marrow stromal cells isolated from Limd1{sup -/-} mice contain significantly more osteoblast progenitors compared to wild type controls when cultured ex vivo. Furthermore, we observed a significant increase in nuclear {beta}-catenin staining in differentiating Limd1{sup -/-} calvarial osteoblasts suggesting that Limd1 is a negative regulator of canonical Wnt signaling in osteoblasts. These results demonstrate that Limd1 influences not only stress osteoclastogenesis but also osteoblast function and osteoblast progenitor commitment. Together, these data identify Limd1 as a novel regulator of both bone osetoclast and bone osteoblast development and function.« less

  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. Large gradient high magnetic fields affect osteoblast ultrastructure and function by disrupting collagen I or fibronectin/αβ1 integrin.

    PubMed

    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.

  17. Endothelial-to-Osteoblast Conversion Generates Osteoblastic Metastasis of Prostate Cancer.

    PubMed

    Lin, Song-Chang; Lee, Yu-Chen; Yu, Guoyu; Cheng, Chien-Jui; Zhou, Xin; Chu, Khoi; Murshed, Monzur; Le, Nhat-Tu; Baseler, Laura; Abe, Jun-Ichi; Fujiwara, Keigi; deCrombrugghe, Benoit; Logothetis, Christopher J; Gallick, Gary E; Yu-Lee, Li-Yuan; Maity, Sankar N; Lin, Sue-Hwa

    2017-06-05

    Prostate cancer (PCa) bone metastasis is frequently associated with bone-forming lesions, but the source of the osteoblastic lesions remains unclear. We show that the tumor-induced bone derives partly from tumor-associated endothelial cells that have undergone endothelial-to-osteoblast (EC-to-OSB) conversion. The tumor-associated osteoblasts in PCa bone metastasis specimens and patient-derived xenografts (PDXs) were found to co-express endothelial marker Tie-2. BMP4, identified in PDX-conditioned medium, promoted EC-to-OSB conversion of 2H11 endothelial cells. BMP4 overexpression in non-osteogenic C4-2b PCa cells led to ectopic bone formation under subcutaneous implantation. Tumor-induced bone was reduced in trigenic mice (Tie2 cre /Osx f/f /SCID) with endothelial-specific deletion of osteoblast cell-fate determinant OSX compared with bigenic mice (Osx f/f /SCID). Thus, tumor-induced EC-to-OSB conversion is one mechanism that leads to osteoblastic bone metastasis of PCa. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Time- and dose-related interactions between glucocorticoid and cyclic adenosine 3',5'-monophosphate on CCAAT/enhancer-binding protein-dependent insulin-like growth factor I expression by osteoblasts

    NASA Technical Reports Server (NTRS)

    McCarthy, T. L.; Ji, C.; Chen, Y.; Kim, K.; Centrella, M.

    2000-01-01

    Glucocorticoid has complex effects on osteoblasts. Several of these changes appear to be related to steroid concentration, duration of exposure, or specific effects on growth factor expression or activity within bone. One important bone growth factor, insulin-like growth factor I (IGF-I), is induced in osteoblasts by hormones such as PGE2 that increase intracellular cAMP levels. In this way, PGE2 activates transcription factor CCAAT/enhancer-binding protein-delta (C/EBPdelta) and enhances its binding to a specific control element found in exon 1 in the IGF-I gene. Our current studies show that preexposure to glucocorticoid enhanced C/EBPdelta and C/EBPbeta expression by osteoblasts and thereby potentiated IGF-I gene promoter activation in response to PGE2. Importantly, this directly contrasts with inhibitory effects on IGF-I expression that result from sustained or pharmacologically high levels of glucocorticoid exposure. Consistent with the stimulatory effect of IGF-I on bone protein synthesis, pretreatment with glucocorticoid sensitized osteoblasts to PGE2, and in this context significantly enhanced new collagen and noncollagen protein synthesis. Therefore, pharmacological levels of glucocorticoid may reduce IGF-I expression by osteoblasts and cause osteopenic disease, whereas physiological transient increases in glucocorticoid may permit or amplify the effectiveness of hormones that regulate skeletal tissue integrity. These events appear to converge on the important role of C/EBPdelta and C/EBPbeta on IGF-I expression by osteoblasts.

  19. Constitutive β-catenin activation in osteoblasts impairs terminal osteoblast differentiation and bone quality

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bao, Quanwei; Chen, Sixu; Qin, Hao

    Accumulating evidence suggests that Wnt/β-catenin signaling plays a central role in controlling bone mass. We previously reported that constitutive activation of β-catenin (CA-β-catenin) in osteoblasts potentially has side effects on the bone growth and bone remodeling process, although it could increase bone mass. The present study aimed to observe the effects of osteoblastic CA-β-catenin on bone quality and to investigate possible mechanisms of these effects. It was found that CA-β-catenin mice exhibited lower mineralization levels and disorganized collagen in long bones as confirmed by von Kossa staining and sirius red staining, respectively. Also, bone strength decreased significantly in CA-β-catenin mice.more » Then the effect of CA-β-catenin on biological functions of osteoblasts were investigated and it was found that the expression levels of osteocalcin, a marker for the late differentiation of osteoblasts, decreased in CA-β-catenin mice, while the expression levels of osterix and alkaline phosphatase, two markers for the early differentiation of osteoblasts, increased in CA-β-catenin mice. Furthermore, higher proliferation rate were revealed in osteoblasts that were isolated from CA-β-catenin mice. The Real-time PCR and western blot examination found that the expression level of c-myc and cyclin D1, two G1 progression-related molecules, increased in osteoblasts that were isolated from the CA-β-catenin mice, and the expression levels of CDK14 and cyclin Y, two mitotic-related molecules that can accelerate cells entering into S and G2/M phases, increased in osteoblasts that were isolated from the CA-β-catenin mice. In summary, osteoblastic CA-β-catenin kept osteoblasts in high proliferative state and impaired the terminal osteoblast differentiation, and this led to changed bone structure and decreased bone strength. - Highlights: • Wnt/β-catenin signaling plays a central role in controlling bone mass. • CA-β-catenin has side effects on

  20. ATF3 mediates the inhibitory action of TNF-α on osteoblast differentiation through the JNK signaling pathway.

    PubMed

    Jeong, Byung-Chul

    2018-05-15

    Tumor necrosis factor (TNF)-α, which is a proinflammatory cytokine, inhibits osteoblast differentiation under diverse inflammatory conditions. Activating transcription factor 3 (ATF3), which is a member of the ATF/cAMP response element-binding protein family of transcription factors, has been implicated in the regulation of cell proliferation and differentiation. However, the precise interactions between ATF3 and the TNF-α signaling pathway in the regulation of osteoblast differentiation remain unclear. In this study, we examined the role of ATF3 in the TNF-α-mediated inhibition of osteoblast differentiation and investigated the signaling pathways involved. The treatment of cells with TNF-α downregulated osteogenic markers, but significantly upregulated the expression of Atf3. The inhibition of Atf3 by small interfering RNAs rescued osteogenesis, which was inhibited by TNF-α. Conversely, the enforced expression of Atf3 enhanced the TNF-α-mediated inhibition of osteoblast differentiation, as revealed by the measurement of osteogenic markers and alkaline phosphatase staining. Mechanistically, TNF-α-induced Atf3 expression was significantly suppressed by the inhibition of the c-Jun N-terminal kinase (JNK) pathway. Furthermore, the overexpression of Atf3 did not affect the rescue effect that inhibiting TNF-α expression using a JNK inhibitor had on alkaline phosphatase activity and mineralization. Taken together, these results indicate that ATF3 mediates the inhibitory action of TNF-α on osteoblast differentiation and that the TNF-α-activated JNK pathway is responsible for the induction of Atf3 expression. Copyright © 2018 Elsevier Inc. All rights reserved.

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

    PubMed Central

    Hempel, Ute; Preissler, Carolin; Möller, Stephanie; Becher, Jana; Rauner, Martina; Hofbauer, Lorenz C.; Dieter, Peter

    2014-01-01

    Sulfated glycosaminoglycans (GAG) are components of the bone marrow stem cell niche and to a minor extent of mature bone tissue with important functions in regulating stem cell lineage commitment and differentiation. We anticipated that artificial extracellular matrices (aECM) composed of collagen I and synthetically oversulfated GAG derivatives affect preferentially the differentiation of osteoblast-precursor cells and early osteoblasts. A set of gradually sulfated chondroitin sulfate and hyaluronan derivatives was used for the preparation of aECM. All these matrices were analysed with human bone marrow stromal cells to identify the most potent aECM and to determine the influence of the degree and position of sulfate groups and the kind of disaccharide units on the osteogenic differentiation. Oversulfated GAG derivatives with a sulfate group at the C-6 position of the N-acetylglycosamine revealed the most pronounced proosteogenic effect as determined by tissue nonspecific alkaline phosphatase activity and calcium deposition. A subset of the aECM was further analysed with different primary osteoblasts and cell lines reflecting different maturation stages to test whether the effect of sulfated GAG derivatives depends on the maturation status of the cells. It was shown that the proosteogenic effect of aECM was most prominent in early osteoblasts. PMID:24864267

  2. 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

  3. 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.

  4. Bioenergetics during calvarial osteoblast differentiation reflect strain differences in bone mass.

    PubMed

    Guntur, Anyonya R; Le, Phuong T; Farber, Charles R; Rosen, Clifford J

    2014-05-01

    Osteoblastogenesis is the process by which mesenchymal stem cells differentiate into osteoblasts that synthesize collagen and mineralize matrix. The pace and magnitude of this process are determined by multiple genetic and environmental factors. Two inbred strains of mice, C3H/HeJ and C57BL/6J, exhibit differences in peak bone mass and bone formation. Although all the heritable factors that differ between these strains have not been elucidated, a recent F1 hybrid expression panel (C3H × B6) revealed major genotypic differences in osteoblastic genes related to cellular respiration and oxidative phosphorylation. Thus, we hypothesized that the metabolic rate of energy utilization by osteoblasts differed by strain and would ultimately contribute to differences in bone formation. In order to study the bioenergetic profile of osteoblasts, we measured oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) first in a preosteoblastic cell line MC3T3-E1C4 and subsequently in primary calvarial osteoblasts from C3H and B6 mice at days 7, 14, and 21 of differentiation. During osteoblast differentiation in media containing ascorbic acid and β-glycerophosphate, all 3 cell types increased their oxygen consumption and extracellular acidification rates compared with the same cells grown in regular media. These increases are sustained throughout differentiation. Importantly, C3H calvarial osteoblasts had greater oxygen consumption rates than B6 consistent with their in vivo phenotype of higher bone formation. Interestingly, osteoblasts utilized both oxidative phosphorylation and glycolysis during the differentiation process although mature osteoblasts were more dependent on glycolysis at the 21-day time point than oxidative phosphorylation. Thus, determinants of oxygen consumption reflect strain differences in bone mass and provide the first evidence that during collagen synthesis osteoblasts use both glycolysis and oxidative phosphorylation to synthesize and

  5. Bioenergetics During Calvarial Osteoblast Differentiation Reflect Strain Differences in Bone Mass

    PubMed Central

    Le, Phuong T.; Farber, Charles R.; Rosen, Clifford J.

    2014-01-01

    Osteoblastogenesis is the process by which mesenchymal stem cells differentiate into osteoblasts that synthesize collagen and mineralize matrix. The pace and magnitude of this process are determined by multiple genetic and environmental factors. Two inbred strains of mice, C3H/HeJ and C57BL/6J, exhibit differences in peak bone mass and bone formation. Although all the heritable factors that differ between these strains have not been elucidated, a recent F1 hybrid expression panel (C3H × B6) revealed major genotypic differences in osteoblastic genes related to cellular respiration and oxidative phosphorylation. Thus, we hypothesized that the metabolic rate of energy utilization by osteoblasts differed by strain and would ultimately contribute to differences in bone formation. In order to study the bioenergetic profile of osteoblasts, we measured oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) first in a preosteoblastic cell line MC3T3-E1C4 and subsequently in primary calvarial osteoblasts from C3H and B6 mice at days 7, 14, and 21 of differentiation. During osteoblast differentiation in media containing ascorbic acid and β-glycerophosphate, all 3 cell types increased their oxygen consumption and extracellular acidification rates compared with the same cells grown in regular media. These increases are sustained throughout differentiation. Importantly, C3H calvarial osteoblasts had greater oxygen consumption rates than B6 consistent with their in vivo phenotype of higher bone formation. Interestingly, osteoblasts utilized both oxidative phosphorylation and glycolysis during the differentiation process although mature osteoblasts were more dependent on glycolysis at the 21-day time point than oxidative phosphorylation. Thus, determinants of oxygen consumption reflect strain differences in bone mass and provide the first evidence that during collagen synthesis osteoblasts use both glycolysis and oxidative phosphorylation to synthesize and

  6. A serum factor promotes collagenase synthesis by an osteoblastic cell line

    NASA Technical Reports Server (NTRS)

    Puccinelli, J. M.; Omura, T. H.; Strege, D. W.; Jeffrey, J. J.; Partridge, N. C.

    1991-01-01

    Regulation of the synthesis of collagenase was investigated in the osteoblastic cell line, UMR 106-01. The cells were stained by the avidin-biotin-complex technique for the presence of the enzyme. By this method, it was possible to identify cells producing collagenase. Synthesis, but not secretion, was found to be constitutive in these cells with the enzyme located intracellularly in cytoplasmic vesicles and the Golgi apparatus. The amount of collagenase contained within UMR cells and the number of cells synthesizing the enzyme were proportional to the concentration of fetal bovine serum in the incubating medium. When serum was withdrawn from the osteosarcoma cells, the content of collagenase decreased with time and the enzyme became undetectable by 48 h of serum depletion. The decrease in collagenase content could be completely reversed by resupplying serum to the cells. The collagenase promoting activity of serum could not be eliminated by adsorption on activated charcoal but was retained by a dialysis membrane with a 12,000 mol wt cutoff. A range of bone-seeking hormones or agents known to affect collagenase secretion was added to the medium in an attempt to mimic the effect of serum on collagenase accumulation. None of these agonists, including parathyroid hormone, could reproduce the effect of serum on these cells, although parathyroid hormone could act as a collagenase secretagogue in the presence or absence of serum. It is concluded that fetal bovine serum contains a yet unidentified factor or factors greater than 12,000 mol wt responsible for the continued synthesis of collagenase by UMR 106-01 cells.

  7. Role of platelet-released growth factors in detoxification of reactive oxygen species in osteoblasts.

    PubMed

    Tohidnezhad, Mersedeh; Wruck, Christoph-Jan; Slowik, Alexander; Kweider, Nisreen; Beckmann, Rainer; Bayer, Andreas; Houben, Astrid; Brandenburg, Lars-Ove; Varoga, Deike; Sönmez, Tolga-Taha; Stoffel, Marcus; Jahr, Holger; Lippross, Sebastian; Pufe, Thomas

    2014-08-01

    Oxidative stress can impair fracture healing. To protect against oxidative damage, a system of detoxifying and antioxidative enzymes works to reduce the cellular stress. The transcription of these enzymes is regulated by antioxidant response element (ARE). The nuclear factor (erythroid-derived 2)-like2 (Nrf2) plays a major role in transcriptional activation of ARE-driven genes. Recently it has been shown that vascular endothelial growth factor (VEGF) prevents oxidative damage via activation of the Nrf2 pathway in vitro. Platelet-released growth factor (PRGF) is a mixture of autologous proteins and growth factors, prepared from a determined volume of platelet-rich plasma (PRP). It has already used to enhance fracture healing in vitro. The aim of the present study was to elucidate if platelets can lead to upregulation of VEGF and if platelets can regulate the activity of Nrf2-ARE system in primary human osteoblast (hOB) and in osteoblast-like cell line (SAOS-2). Platelets and PRGF were obtained from healthy human donors. HOB and SAOS-2 osteosarcoma cell line were used. The ARE activity was analysed using a dual luciferase reporter assay system. We used Western blot to detect the nuclear accumulation of Nrf2 and the amount of cytosolic antioxidant Thioredoxin Reductase-1 (TXNRD-1), Heme Oxygenase-1 (HO-1) and NAD(P)H quinine oxidoreductase-1 (NQO1). Gene expression analysis was performed by real-time RT PCR. ELISA was used for the quantification of growth factors. The activity of ARE was increased in the presence of PRGF up to 50%. Western blotting demonstrated enhanced nuclear accumulation of Nrf2. This was followed by an increase in the protein expression of the aforementioned downstream targets of Nrf2. Real-time RT PCR data showed an upregulation in the gene expression of the VEGF after PRGF treatment. This was confirmed by ELISA, where the treatment with PRGF induced the protein level of VEGF in both cells. These results provide a new insight into PRGF's mode of

  8. 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

  9. Fibroblast growth factor 2 inhibits up-regulation of bone morphogenic proteins and their receptors during osteoblastic differentiation of human mesenchymal stem cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Biver, Emmanuel, E-mail: ebiver@yahoo.fr; Department of Rheumatology, Lille University Hospital, Roger Salengro Hospital, 59037 Lille cedex; Service of Bone Diseases, Department of Internal Medicine Specialties, University Hospital of Geneva, CH-1211 Geneva 14

    2012-11-02

    Highlights: Black-Right-Pointing-Pointer FGF modulates BMPs pathway in HMSCs by down-regulating BMP/BMPR expression. Black-Right-Pointing-Pointer This effect is mediated by ERK and JNK MAPKs pathways. Black-Right-Pointing-Pointer Crosstalk between FGF and BMPs must be taken into account in skeletal bioengineering. Black-Right-Pointing-Pointer It must also be considered in the use of recombinant BMPs in orthopedic and spine surgeries. -- Abstract: Understanding the interactions between growth factors and bone morphogenic proteins (BMPs) signaling remains a crucial issue to optimize the use of human mesenchymal stem cells (HMSCs) and BMPs in therapeutic perspectives and bone tissue engineering. BMPs are potent inducers of osteoblastic differentiation. They exertmore » their actions via BMP receptors (BMPR), including BMPR1A, BMPR1B and BMPR2. Fibroblast growth factor 2 (FGF2) is expressed by cells of the osteoblastic lineage, increases their proliferation and is secreted during the healing process of fractures or in surgery bone sites. We hypothesized that FGF2 might influence HMSC osteoblastic differentiation by modulating expressions of BMPs and their receptors. BMP2, BMP4, BMPR1A and mainly BMPR1B expressions were up-regulated during this differentiation. FGF2 inhibited HMSCs osteoblastic differentiation and the up-regulation of BMPs and BMPR. This effect was prevented by inhibiting the ERK or JNK mitogen-activated protein kinases which are known to be activated by FGF2. These data provide a mechanism explaining the inhibitory effect of FGF2 on osteoblastic differentiation of HMSCs. These crosstalks between growth and osteogenic factors should be considered in the use of recombinant BMPs in therapeutic purpose of fracture repair or skeletal bioengineering.« less

  10. Mitotic inheritance of mRNA facilitates translational activation of the osteogenic-lineage commitment factor Runx2 in progeny of osteoblastic cells

    PubMed Central

    Varela, Nelson; Aranguiz, Alejandra; Lizama, Carlos; Sepulveda, Hugo; Antonelli, Marcelo; Thaler, Roman; Moreno, Ricardo D.; Montecino, Martin; Stein, Gary S.; van Wijnen, Andre J.; Galindo, Mario

    2017-01-01

    Epigenetic mechanisms mediate the acquisition of specialized cellular phenotypes during tissue development, maintenance and repair. When phenotype-committed cells transit through mitosis, chromosomal condensation counteracts epigenetic activation of gene expression. Subsequent post-mitotic re-activation of transcription depends on epigenetic DNA and histone modifications, as well as other architecturally bound proteins that ‘bookmark’ the genome. Osteogenic lineage commitment, differentiation and progenitor proliferation require the bone-related runt-related transcription factor Runx2. Here, we characterized a non-genomic mRNA mediated mechanism by which osteoblast precursors retain their phenotype during self-renewal. We show that osteoblasts produce maximal levels of Runx2 mRNA, but not protein, prior to mitotic cell division. Runx2 mRNA partitions symmetrically between daughter cells in a non-chromosomal tubulin-containing compartment. Subsequently, transcription-independent de novo synthesis of Runx2 protein in early G1 phase results in increased functional interactions of Runx2 with a representative osteoblast-specific target gene (osteocalcin/BGLAP2) in chromatin. Somatic transmission of Runx2 mRNAs in osteoblasts and osteosarcoma cells represents a versatile mechanism for translational rather than transcriptional induction of this principal gene regulator to maintain osteoblast phenotype identity after mitosis. PMID:26381402

  11. 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. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. 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

  13. Osteoblastic molecular scaffold Gab1 is required for maintaining bone homeostasis.

    PubMed

    Weng, Tujun; Mao, Fengfeng; Wang, Youliang; Sun, Qiang; Li, Ruixin; Yang, Guan; Zhang, Xizheng; Luo, Jincai; Feng, Gen-Sheng; Yang, Xiao

    2010-03-01

    The Grb2-associated binder 1 (Gab1), which serves as a scaffolding adaptor protein, plays a crucial role in transmitting key signals that control cell growth, differentiation and function from multiple receptors. However, its biological role in osteoblast activity and postnatal bone metabolism remains unclear. To elucidate the in vivo function of Gab1 in postnatal bone remodeling, we generated osteoblast-specific Gab1 knockout mice. Disruption of Gab1 expression in osteoblasts led to decreased trabecular bone mass with a reduced bone formation rate and a decreased bone resorption. Bones from Gab1 mutants also exhibited inferior mechanical properties. Moreover, primary osteoblasts from Gab1 mutant mice demonstrated markedly suppressed osteoblast mineralization, increased susceptibility to apoptosis and decreased expression of receptor activator of NF-kappaB ligand (RANKL). Activation of serine-threonine Akt kinase and extracellular signal-regulated kinase in response to insulin and insulin-like growth factor 1 was attenuated in Gab1 mutant osteoblasts. Our results show that Gab1-mediated signals in osteoblasts are crucial for normal postnatal bone homeostasis.

  14. MEK5 suppresses osteoblastic differentiation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kaneshiro, Shoichi; Department of Orthopaedic Surgery, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871; Otsuki, Dai

    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, osteocalcinmore » (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.« less

  15. [Effects of laser solid forming of porous titanium on proliferation of osteoblast and RANKL/OPG expression].

    PubMed

    Chen, Hui; Du, Shanshan; Zhen, Ping; Li, Xusheng; Liang, Xiaoyan; Fan, Lijuan; Jiang, Jie; Yang, Haiou; Liu, Jun

    2016-12-28

    To evaluate the effect of laser solid forming (LSF) of porous titanium on receptor activator of NF-κB ligand (RANKL)/osteoprorotegerin (OPG) expression and osteoblast cells growth.
 Methods: The DMEM and sterile saline were used for porous titanium extract. The osteoblast cells were cultured in the extract while equal amount of  DMEM and sterile saline were added to the control group. The growth of the cells were observed under an inverted phase contrast microscope. MTT was used to detect the growth inhibitory rates. The adhesion capacity of osteoblasts were measured. The growth in the material surface was examined by the electron microscope, and the expressions of RANKL and OPG were determined by Westen blot.
 Results: At the first day, the osteoblast proliferation rate was significantly different (P<0.05), at the fourth and seventh day, the osteoblast proliferation rate was not significantly affected in the LSF group (P>0.05); at each time point, the osteoblast proliferation rate were significantly different between the two groups (P<0.05). Compared with the control group, RANKL and OPG protein expression were not significantly different (P>0.05). The laser solid forming of porous titanium showed well bone compatibility.
 Conclusion: The porous titanium did not affect osteoblast proliferation due to its well bone compatibility. It did not affect the OPG/RANKL/RANK-axis system of bone metabolism, exibiting a wide applicable prospect for tissue engineering.

  16. Osteopontin inhibits osteoblast responsiveness through the down-regulation of focal adhesion kinase mediated by the induction of low-molecular weight protein tyrosine phosphatase.

    PubMed

    Kusuyama, Joji; Bandow, Kenjiro; Ohnishi, Tomokazu; Hisadome, Mitsuhiro; Shima, Kaori; Semba, Ichiro; Matsuguchi, Tetsuya

    2017-05-15

    Osteopontin (OPN) is an osteogenic marker protein. Osteoblast functions are affected by inflammatory cytokines and pathological conditions. OPN is highly expressed in bone lesions such as those in rheumatoid arthritis. However, local regulatory effects of OPN on osteoblasts remain ambiguous. Here we examined how OPN influences osteoblast responses to mechanical stress and growth factors. Expression of NO synthase 1 ( Nos1 ) and Nos2 was increased by low-intensity pulsed ultrasound (LIPUS) in MC3T3-E1 cells and primary osteoblasts. The increase of Nos1/2 expression was abrogated by both exogenous OPN overexpression and recombinant OPN treatment, whereas it was promoted by OPN-specific siRNA and OPN antibody. Moreover, LIPUS-induced phosphorylation of focal adhesion kinase (FAK), a crucial regulator of mechanoresponses, was down-regulated by OPN treatments. OPN also attenuated hepatocyte growth factor-induced vitamin D receptor ( Vdr ) expression and platelet-derived growth factor-induced cell mobility through the repression of FAK activity. Of note, the expression of low-molecular weight protein tyrosine phosphatase (LMW-PTP), a FAK phosphatase, was increased in both OPN-treated and differentiated osteoblasts. CD44 was a specific OPN receptor for LWW-PTP induction. Consistently, the suppressive influence of OPN on osteoblast responsiveness was abrogated by LMW-PTP knockdown. Taken together, these results reveal novel functions of OPN in osteoblast physiology. © 2017 Kusuyama et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  17. 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. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    PubMed Central

    Mantripragada, Venkata P.; Jayasuriya, Ambalangodage C.

    2016-01-01

    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 100 μg/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 1000 μg/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 21.5 μg/ml could inhibit ~85% growth of S. epidermidis, whereas vancomycin at a concentration of 30 μg/ml was found to inhibit ~80% bacterial growth. PMID:27287137

  19. Beneficial effects of sulfonamide-based gallates on osteoblasts in vitro

    PubMed Central

    Huang, Li; Jin, Pan; Lin, Xiao; Lin, Cuiwu; Zheng, Li; Zhao, Jinmin

    2017-01-01

    Effective treatments for osteoporosis remain fairly elusive; however, studies have reported that antioxidants may aid in the maintenance of reactive oxygen species at a favorable level, in order to prevent osteoporosis. Gallic acid (GA) and its derivatives are potent antioxidative and anti-inflammatory agents that affect several biochemical and pharmacological pathways; however, GA is slightly cytotoxic and suppresses cell proliferation. The present study modified GA by the introduction of sulfonamide, in order to obtain a novel compound known as JEZ-C, and investigated its effects on osteoblasts by measuring cell proliferation, viability, morphology, alkaline phosphatase (ALP) activity, and the expression of relevant osteoblast markers. Results indicated that JEZ-C may effectively promote osteoblast growth. JEZ-C increased ALP activity, upregulated the expression of osteogenic-related genes, including runt-related transcription factor 2, bone sialoprotein, osteocalcin and alpha-1 type I collagen, thus indicating that JEZ-C enhances bone matrix production and mineralization. The recommended range of JEZ-C concentration is between 6.25×10−3 and 6.25×10−1 µg/ml, within which cell growth was promoted compared with the control. Specifically, treatment with 6.25×10−2 µg/ml JEZ-C is ideal. These findings may represent a novel approach to cell-based therapy for the treatment of osteoporosis. PMID:28138702

  20. Rebamipide delivered by brushite cement enhances osteoblast and macrophage proliferation.

    PubMed

    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 occurs 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 0.4 uM 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 1 uM), and quenches ROS by 40-60%. This is the first investigation of Rebamipide in osteoblasts.

  1. 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. © 2016 Japanese Society of Developmental Biologists.

  2. Eluted zinc ions stimulate osteoblast differentiation and mineralization in human dental pulp stem cells for bone tissue engineering.

    PubMed

    Yusa, Kazuyuki; Yamamoto, Osamu; Iino, Mitsuyoshi; Takano, Hiroshi; Fukuda, Masayuki; Qiao, Zhiwei; Sugiyama, Toshihiro

    2016-11-01

    Zinc is an essential element for proliferation, differentiation and survival in various cell types. In a previous study, we found that zinc ions released from zinc-modified titanium surfaces (eluted zinc ions; EZ) stimulate cell viability, osteoblast marker gene expression and calcium deposition in human bone marrow-derived mesenchymal cells (hBMCs). The aim of the present study was to investigate the effects of EZ on osteoblast differentiation among dental pulp stem cells (DPSCs) in vitro. In this study, we evaluated the effects of EZ on osteogenesis in DPSCs. Osteoblast and osteoclast marker gene expression was evaluated by real-time PCR. We also evaluated alkaline phosphatase (ALP) staining and calcium deposition. We found that EZ stimulated osteoblast marker gene (type I collagen, alkaline phosphatase (ALP), osteocalcin (OCN) and Runx2) expression, vascular endothelial growth factor A (VEGF-A), and TGF-beta signaling pathway-related gene expression after 7days of incubation. Osteoclastogenesis occurs in a receptor for activated nuclear-factor kappa B ligand (RANKL)/osteoprotegerin (OPG)-independent manner. Real-time PCR analysis revealed that EZ did not affect RANKL or OPG mRNA expression. It was also revealed that EZ induced alkaline phosphatase (ALP) staining and calcium deposition in DPSCs. Collectively, these results demonstrate the potential for clinical application to prospective treatment of bone diseases. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Asporin and transforming growth factor-beta gene expression in osteoblasts from subchondral bone and osteophytes in osteoarthritis.

    PubMed

    Sakao, Kei; Takahashi, Kenji A; Arai, Yuji; Saito, Masazumi; Honjyo, Kuniaki; Hiraoka, Nobuyuki; Kishida, Tsunao; Mazda, Osam; Imanishi, Jiro; Kubo, Toshikazu

    2009-11-01

    To clarify the significance of subchondral bone and osteophytes in the pathology of osteoarthritis (OA), we investigated the expression of asporin (ASPN), transforming growth factor-beta1 (TGF-beta1), TGF-beta2, TGF-beta3, and runt-related transcription factor-2 (Runx2) genes involved in bone metabolism. Osteoblasts were isolated from 19 patients diagnosed with knee OA and from 4 patients diagnosed with femoral neck fracture. Osteoblast expression of mRNA encoding ASPN, TGF-beta1, TGF-beta2, TGF-beta3, and Runx2 was analyzed using real-time RT-PCR. Expression of ASPN, TGF-beta1, and TGF-beta3 mRNA in the subchondral bone and osteophytes of OA patients increased compared with that of non-OA patients. The ratio of ASPN to TGF-beta1 mRNA in patients with severe cartilage damage was higher than that in patients with mild cartilage damage. The increased ratio of ASPN mRNA to TGF-beta1 mRNA in patients with severe relative to mild cartilage damage indicates that increased ASPN mRNA expression was significantly associated with the severity of cartilage degeneration. This finding suggests that ASPN may regulate TGF-beta1-mediated factors in the development of OA, which may provide clues as to the underlying pathology of OA.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tsukasaki, Masayuki; Yamada, Atsushi, E-mail: yamadaa@dent.showa-u.ac.jp; Suzuki, Dai

    2011-07-15

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

  5. Transdifferentiation of adipocytes to osteoblasts: potential for orthopaedic treatment.

    PubMed

    Lin, Daphne P L; Dass, Crispin R

    2018-03-01

    As both adipocytes and osteoblasts originate from the same pool of mesenchymal stem cells, increasing clinical evidence has emerged of the plasticity between the two lineages. For instance, the downregulation of osteoblast differentiation and upregulation of adipogenesis are common features of conditions such as multiple myeloma, obesity and drug-induced bone loss in diabetes mellitus. However, despite in-vitro and in-vivo observations of adipocyte transdifferentiation into osteoblasts, little is known of the underlying mechanisms. This review summarises the current knowledge of this particular transdifferentiation process whereby the Wnt/β-catenin signalling pathway and Runx2 overexpression have been postulated to play a critical role. Furthermore, due to the possibility of a novel therapy in the treatment of bone conditions, a number of agents with the potential to induce adipo-to-osteoblast transdifferentiation have been investigated such as all-trans retinoic acid, bone morphogenetic protein-9 and vascular endothelial growth factor. © 2018 Royal Pharmaceutical Society.

  6. Identification and proteomic analysis of osteoblast-derived exosomes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ge, Min; Ke, Ronghu; Cai, Tianyi

    Exosomes are nanometer-sized vesicles with the function of intercellular communication, and they are released by various cell types. To reveal the knowledge about the exosomes from osteoblast, and explore the potential functions of osteogenesis, we isolated microvesicles from supernatants of mouse Mc3t3 by ultracentrifugation, characterized exosomes by electron microscopy and immunoblotting and presented the protein profile by proteomic analysis. The result demonstrated that microvesicles were between 30 and 100 nm in diameter, round shape with cup-like concavity and expressed exosomal marker tumor susceptibility gene (TSG) 101 and flotillin (Flot) 1. We identified a total number of 1069 proteins among which 786more » proteins overlap with ExoCarta database. Gene Oncology analysis indicated that exosomes mostly derived from plasma membrane and mainly involved in protein localization and intracellular signaling. The Ingenuity Pathway Analysis showed pathways are mostly involved in exosome biogenesis, formation, uptake and osteogenesis. Among the pathways, eukaryotic initiation factor 2 pathways played an important role in osteogenesis. Our study identified osteoblast-derived exosomes, unveiled the content of them, presented potential osteogenesis-related proteins and pathways and provided a rich proteomics data resource that will be valuable for further studies of the functions of individual proteins in bone diseases. - Highlights: • We for the first time identified exosomes from mouse osteoblast. • Osteoblasts-derived exosomes contain osteoblast peculiar proteins. • Proteins from osteoblasts-derived exosomes are intently involved in EIF2 pathway. • EIF2α from the EIF2 pathway plays an important role in osteogenesis.« less

  7. 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

  8. Pro416Arg cherubism mutation in Sh3bp2 knock-in mice affects osteoblasts and alters bone mineral and matrix properties

    PubMed Central

    Wang, Chiachien J.; Chen, I-Ping; Koczon-Jaremko, Boguslawa; Boskey, Adele L.; Ueki, Yasuyoshi; Kuhn, Liisa; Reichenberger, Ernst J.

    2010-01-01

    Cherubism is an autosomal dominant disorder in children characterized by unwarranted symmetrical bone resorption of the jaws with fibrous tissue deposition. Mutations causing cherubism have been identified in the adaptor protein SH3BP2. Knock-in mice with a Pro416Arg mutation in Sh3bp2 exhibit a generalized osteoporotic bone phenotype. In this study, we examined the effects of this “cherubism” mutation on spectroscopic indices of “bone quality” and on osteoblast differentiation. Fourier-transform infrared imaging (FTIRI) analysis of femurs from wild-type and Sh3bp2 knock-in mice showed decreased mineral content, decreased mineral crystallinity/crystal size, and increased collagen maturity in homozygous mutants. To assess osteoblast maturation in vivo, knock-in mice were crossed with transgenic mice over-expressing GFP driven by 3.6-kb or 2.3-kb Col1a1 promoter fragments. Reduced numbers of mature osteoblasts were observed in homozygous mice. Neonatal calvarial cultures, which were enriched for osteoblasts by depletion of hematopoietic cells (negative selection for Ter119- and CD45-positive cells) were investigated for osteoblast-specific gene expression and differentiation, which demonstrated that differentiation and mineralization in homozygous osteoblast cultures was impaired. Co-cultures with calvarial osteoblasts and bone marrow macrophages showed that mutant osteoblasts appear to increase osteoclastogenesis resulting in increased bone resorption on bone chips. In summary, the Sh3bp2 mutation in cherubism mice alters bone quality, reduces osteoblast function, and may contribute to excessive bone resorption by osteoclasts. Our data, together with previous osteoclast studies, demonstrate a critical role of Sh3bp2 in bone remodeling and osteoblast differentiation. PMID:20117257

  9. 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

  10. Spatial distribution of osteoblast-specific transcription factor Cbfa1 and bone formation in atherosclerotic arteries.

    PubMed

    Bobryshev, Yuri V; Killingsworth, Murray C; Lord, Reginald S A

    2008-08-01

    The mechanisms of ectopic bone formation in arteries are poorly understood. Osteoblasts might originate either from stem cells that penetrate atherosclerotic plaques from the blood stream or from pluripotent mesenchymal cells that have remained in the arterial wall from embryonic stages of the development. We have examined the frequency of the expression and spatial distribution of osteoblast-specific factor-2/core binding factor-1 (Osf2/Cbfa1) in carotid and coronary arteries. Cbfa1-expressing cells were rarely observed but were found in all tissue specimens in the deep portions of atherosclerotic plaques under the necrotic cores. The deep portions of atherosclerotic plaques under the necrotic cores were characterized by the lack of capillaries of neovascularization. In contrast, plaque shoulders, which were enriched by plexuses of neovascularization, lacked Cbfa1-expressing cells. No bone formation was found in any of the 21 carotid plaques examined and ectopic bone was observed in only two of 12 coronary plaques. We speculate that the sparse invasion of sprouts of neovascularization into areas underlying the necrotic cores, where Cbfa1-expressing cells reside, might explain the rarity of events of ectopic bone formation in the arterial wall. This study has also revealed that Cbfa1-expressing cells contain alpha-smooth muscle actin and myofilaments, indicating their relationship with arterial smooth muscle cells.

  11. Therapeutic touch affects DNA synthesis and mineralization of human osteoblasts in culture.

    PubMed

    Jhaveri, Ankur; Walsh, Stephen J; Wang, Yatzen; McCarthy, MaryBeth; Gronowicz, Gloria

    2008-11-01

    Complementary and alternative medicine (CAM) techniques are commonly used in hospitals and private medical facilities; however, the effectiveness of many of these practices has not been thoroughly studied in a scientific manner. Developed by Dr. Dolores Krieger and Dora Kunz, Therapeutic Touch is one of these CAM practices and is a highly disciplined five-step process by which a practitioner can generate energy through their hands to promote healing. There are numerous clinical studies on the effects of TT but few in vitro studies. Our purpose was to determine if Therapeutic Touch had any effect on osteoblast proliferation, differentiation, and mineralization in vitro. TT was performed twice a week for 10 min each on human osteoblasts (HOBs) and on an osteosarcoma-derived cell line, SaOs-2. No significant differences were found in DNA synthesis, assayed by [(3)H]-thymidine incorporation at 1 or 2 weeks for SaOs-2 or 1 week for HOBs. However, after four TT treatments in 2 weeks, TT significantly (p = 0.03) increased HOB DNA synthesis compared to controls. Immunocytochemistry for Proliferating Cell Nuclear Antigen (PCNA) confirmed these data. At 2 weeks in differentiation medium, TT significantly increased mineralization in HOBs (p = 0.016) and decreased mineralization in SaOs-2 (p = 0.0007), compared to controls. Additionally, Northern blot analysis indicated a TT-induced increase in mRNA expression for Type I collagen, bone sialoprotein, and alkaline phosphatase in HOBs and a decrease of these bone markers in SaOs-2 cells. In conclusion, Therapeutic Touch appears to increase human osteoblast DNA synthesis, differentiation and mineralization, and decrease differentiation and mineralization in a human osteosarcoma-derived cell line. (c) 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hong, Dun; Orthopedic Department, Taizhou Hospital, Wenzhou Medical College, Linhai, Zhejiang 317000; Chen, Hai-Xiao, E-mail: Hxchen-1@163.net

    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 formmore » 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.« less

  13. The effect of Platelet Lysate on osteoblast proliferation associated with a transient increase of the inflammatory response in bone regeneration.

    PubMed

    Ruggiu, Alessandra; Ulivi, Valentina; Sanguineti, Francesca; Cancedda, Ranieri; Descalzi, Fiorella

    2013-12-01

    Platelet Lysate (PL) contains a cocktail of growth factors and cytokines, which actively participates in tissue repair and its clinical application has been broadly described. The aim of this study was to assess the regenerative potential of PL for bone repair. We demonstrated that PL stimulation induces a transient increase of the inflammatory response in quiescent human osteoblasts, via NF-kB activation, COX-2 induction, PGE2 production and secretion of pro-inflammatory cytokines. Furthermore, we showed that long-term PL stimulation enhances proliferation of actively replicating osteoblasts, without affecting their differentiation potential, along with changes of cell morphology, resulting in increased cell density at confluence. In confluent resting osteoblasts, PL treatment induced resumption of proliferation, change in cell morphology and increase of cell density at confluence. A burst of PL treatment (24-h) was sufficient to trigger such processes in both conditions. These results correlated with up-regulation of the proliferative and survival pathways ERKs and Akt and with cell cycle re-activation via induction of CyclinD1 and phosphorylation of Rb, following PL stimulation. Our findings demonstrate that PL treatment results in activation and expansion of resting osteoblasts, without affecting their differentiation potential. Therefore PL represents a good therapeutic candidate in regenerative medicine for bone repair. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. The role of selenium in insulin-like growth factor I receptor (IGF-IR) expression and regulation of apoptosis in mouse osteoblasts.

    PubMed

    Ren, Gaixian; Ali, Tariq; Chen, Wei; Han, Dandan; Zhang, Limei; Gu, Xiaolong; Zhang, Shiyao; Ding, Laidi; Fanning, Séamus; Han, Bo

    2016-02-01

    Selenium (Se) is an essential component for animals and human beings. The chemoprotective role of Se, via the regulation of the cell cycle, stimulation of apoptosis and activation of some cytokines among others, is well known; however, the comprehensive effects of Se on the expression of IGF-IR and its regulation of apoptosis have not been investigated. Thus the aim of this study was to report on the effects that different concentrations of Se extert on body weight, blood serum IGF-IR levels and histopathology in mice; and on IGF-IR expression, proliferation and apoptosis in mouse osteoblasts. In vivo experiments showed a significant decrease in body weight, serum levels of IGF-IR and prominent toxicant effects on the liver, kidney, heart and spleen following the administration of defined concentrations of Se for 30 d. However, moderate levels (0.1 mg/kg) of Se gradually improved weight and serum IGF-IR. In vitro osteoblast experiments revealed that at concentrations of 5 × 10(-6) and 10(-5) mol/L Se, MTT activity decreased in comparison with control cells. Cell cycle, TEM and caspase-3 activity supported these observations including an increase in the sub-G1 phase and notable apoptosis in osteoblasts, along with a decrease in the expression of mRNA and protein levels of IGF-IR. Moreover, the MTT activity, mRNA and protein levels of IGF-IR in osteoblasts were decreased and caspase-3 activity was increased in siRNA groups as compared with non-siRNA groups. These data suggest that Se significantly affects IGF-IR expression, and that it contributes to the proliferation and regulation of apoptosis in osteoblasts. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Nuclear chromatin-concentrated osteoblasts in renal bone diseases.

    PubMed

    Kazama, Junichiro James; Yamamoto, Suguru; Narita, Ichiei; Kurihara, Satoshi

    2011-06-01

    The morphological appearance of an osteoblast largely alters with its differentiation and maturation, along with the change of cell function. We quantitatively observed the osteoblast morphology and compared it with bone metabolism. Biopsied iliac bone samples obtained from 77 dialysis patients (14 mild change, 37 osteitis fibrosa, 2 osteomalacia, 8 mixed, and 16 adynamic bone) were included in the study. Osteoblast appearances were classified into three groups: (i) type II and III osteoblasts, namely, active osteoblasts characterized by cuboidal or columnar shapes with or without a nuclear clear zone; (ii) type IV osteoblasts, lining osteoblasts characterized by extremely thin cytoplasm; and (iii) type V osteoblasts, apoptotic osteoblasts characterized by nuclear chromatin concentration. The results were quantitatively expressed as the length of bone surface covered by each type of osteoblasts. The type II and III osteoblasts were predominant in osteitis fibrosa, mixed, and mild change. The type IV osteoblasts were overwhelmingly predominant in adynamic bone. The type V osteoblasts appeared most frequently in osteitis fibrosa, followed by mixed and mild change. Both absolute and relative lengths of bone surface covered by the type V osteoblasts were significantly higher in the high-turnover bone group (osteitis fibrosa and mixed) than the low-turnover bone group (adynamic bone and osteomalacia). The type V osteoblasts were slightly correlated with serum intact parathyroid hormone levels. In conclusion, a high bone-turnover condition seems to be associated with the promotion of osteoblastic apoptosis in dialysis patients. This finding may explain the fact that osteopenia develops faster in CKD patients with high turnover of bone. © 2011 The Authors. Therapeutic Apheresis and Dialysis © 2011 International Society for Apheresis.

  16. 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

  17. Electrical Polarization of Titanium Surfaces for the Enhancement of Osteoblast Differentiation

    PubMed Central

    Gittens, Rolando A.; Olivares-Navarrete, Rene; Rettew, Robert; Butera, Robert J.; Alamgir, Faisal M.; Boyan, Barbara D.; Schwartz, Zvi

    2014-01-01

    Electrical stimulation has been used clinically to promote bone regeneration in cases of fractures with delayed union or nonunion, with several in vitro and in vivo reports suggesting its beneficial effects on bone formation. However, the use of electrical stimulation of titanium (Ti) implants to enhance osseointegration is less understood, in part because of the few in vitro models that attempt to represent the in vivo environment. In this article, the design of a new in vitro system that allows direct electrical stimulation of osteoblasts through their Ti substrates without the flow of exogenous currents through the media is presented, and the effect of applied electrical polarization on osteoblast differentiation and local factor production was evaluated. A custom-made polycarbonate tissue culture plate was designed to allow electrical connections directly underneath Ti disks placed inside the wells, which were supplied with electrical polarization ranging from 100 to 500 mV to stimulate MG63 osteoblasts. Our results show that electrical polarization applied directly through Ti substrates on which the cells are growing in the absence of applied electrical currents may increase osteoblast differentiation and local factor production in a voltage-dependent manner. PMID:23996899

  18. Dual targeting c-met and VEGFR2 in osteoblasts suppresses growth and osteolysis of prostate cancer bone metastasis.

    PubMed

    Lee, Changki; Whang, Young Mi; Campbell, Preston; Mulcrone, Patrick L; Elefteriou, Florent; Cho, Sun Wook; Park, Serk In

    2018-02-01

    Prostate cancer characteristically induces osteoblastic bone metastasis, for which no therapies are available. A dual kinase inhibitor of c-Met and VEGFR-2 (cabozantinib) was shown to reduce prostate cancer growth in bone, with evidence for suppressing osteoblastic activity. However, c-Met and VEGFR2 signaling in osteoblasts in the context of bone metastasis remain unclear. Here we show using cultured osteoblasts that hepatocyte growth factor (HGF) and VEGF-A increased receptor activator of NFκB ligand (RANKL) and M-CSF, two essential factors for osteoclastogenesis. Insulin-like growth factor-1 (IGF1) also increased RANKL and M-CSF via c-Met transactivation. The conditioned media from IGF1-, HGF-, or VEGFA-treated osteoblasts promoted osteoclastogenesis that was reversed by inhibiting c-Met and/or VEGFR2 in osteoblasts. In vivo experiments used cabozantinib-resistant prostate cancer cells (PC-3 and C4-2B) to test the effects of c-Met/VEGFR2 inhibition specifically in osteoblasts. Cabozantinib (60 mg/kg, 3 weeks) suppressed tumor growth in bone and reduced expression of RANKL and M-CSF and subsequent tumor-induced osteolysis. Collectively, inhibition of c-Met and VEGFR2 in osteoblasts reduced RANKL and M-CSF expression, and associated with reduction of tumor-induced osteolysis, suggesting that c-Met and VEGFR2 are promising therapeutic targets in bone metastasis. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. 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

    detected in any of the samples. There were no significant differences in the expression of actin mRNA between the 0-g and 1-g samples. These data indicate that quiescent osteoblasts are slower to enter the cell cycle in microgravity, suggesting that the force of gravity itself may be a significant factor in bone loss in spaceflight. Preliminary data from our STS 76 flight experiment support our hypothesis that a basic biological response occurs at the tissue, cellular, and molecular level in 0-g. Here we examine ground-based and space flown data on osteoblast growth in ground-based experiments mimicking space flight conditions and in microgravity to simulate lack of gravity stress to help us understand the mechanism of bone loss by experiments.

  20. Knockdown of Indian hedgehog protein induces an inhibition of cell growth and differentiation in osteoblast MC3T3‑E1 cells.

    PubMed

    Deng, Ang; Zhang, Hongqi; Hu, Minyu; Liu, Shaohua; Gao, Qile; Wang, Yuxiang; Guo, Chaofeng

    2017-12-01

    Indian hedgehog protein (Ihh) is evolutionarily conserved and serves important roles in controlling the differentiation of progenitor cells into osteoblasts. Ihh null mutant mice exhibit a failure of osteoblast development in endochondral bone. Although studies have demonstrated that Ihh signaling is a potent local factor that regulates osteoblast differentiation, the specific transcription factors that determine osteoblast differentiation remain unclear. Further studies are required to determine the precise mechanism through which Ihh regulates osteoblast differentiation. In the present study, Ihh was knocked down in osteoblast MC3T3‑E1 cells using short hairpin RNA, to investigate the function of Ihh in osteoblast proliferation and differentiation and to examine the potential mechanism through which Ihh induces osteoblast apoptosis and cell cycle arrest. It was observed that the knockdown of Ihh induced a marked inhibition of cell growth and increased the apoptosis rate compared with the negative control osteoblasts. Downregulation of Ihh resulted in a cell cycle arrest at the G1 to S phase boundary in osteoblasts. In addition, the knockdown of Ihh decreased the alkaline phosphatase activity and mineral deposition of osteoblasts. The inhibitory roles of Ihh downregulation in osteoblast growth and differentiation may be associated with the transforming growth factor‑β/mothers against decapentaplegic homolog and tumor necrosis factor receptor superfamily member 11B/tumor necrosis factor ligand superfamily member 11 signaling pathways. Manipulating either Ihh expression or its signaling components may be of benefit for the treatment of skeletal diseases.

  1. CCAAT/enhancer-binding protein delta activates insulin-like growth factor-I gene transcription in osteoblasts. Identification of a novel cyclic AMP signaling pathway in bone

    NASA Technical Reports Server (NTRS)

    Umayahara, Y.; Ji, C.; Centrella, M.; Rotwein, P.; McCarthy, T. L.

    1997-01-01

    Insulin-like growth factor-I (IGF-I) plays a key role in skeletal growth by stimulating bone cell replication and differentiation. We previously showed that prostaglandin E2 (PGE2) and other cAMP-activating agents enhanced IGF-I gene transcription in cultured primary rat osteoblasts through promoter 1, the major IGF-I promoter, and identified a short segment of the promoter, termed HS3D, that was essential for hormonal regulation of IGF-I gene expression. We now demonstrate that CCAAT/enhancer-binding protein (C/EBP) delta is a major component of a PGE2-stimulated DNA-protein complex involving HS3D and find that C/EBPdelta transactivates IGF-I promoter 1 through this site. Competition gel shift studies first indicated that a core C/EBP half-site (GCAAT) was required for binding of a labeled HS3D oligomer to osteoblast nuclear proteins. Southwestern blotting and UV-cross-linking studies showed that the HS3D probe recognized a approximately 35-kDa nuclear protein, and antibody supershift assays indicated that C/EBPdelta comprised most of the PGE2-activated gel-shifted complex. C/EBPdelta was detected by Western immunoblotting in osteoblast nuclear extracts after treatment of cells with PGE2. An HS3D oligonucleotide competed effectively with a high affinity C/EBP site from the rat albumin gene for binding to osteoblast nuclear proteins. Co-transfection of osteoblast cell cultures with a C/EBPdelta expression plasmid enhanced basal and PGE2-activated IGF-I promoter 1-luciferase activity but did not stimulate a reporter gene lacking an HS3D site. By contrast, an expression plasmid for the related protein, C/EBPbeta, did not alter basal IGF-I gene activity but did increase the response to PGE2. In osteoblasts and in COS-7 cells, C/EBPdelta, but not C/EBPbeta, transactivated a reporter gene containing four tandem copies of HS3D fused to a minimal promoter; neither transcription factor stimulated a gene with four copies of an HS3D mutant that was unable to bind osteoblast

  2. PEG attachment to osteoblasts enhances mechanosensitivity.

    PubMed

    Hamamura, Kazunori; Weng, Yiming; Zhao, Jun; Yokota, Hiroki; Xie, Dong

    2008-06-01

    Fluid flow induces proliferation and differentiation of osteoblasts, and fibrous structure like a primary cilium on a cell surface contributes to flow sensing and flow-driven gene regulation. We address a question: Does attachment of synthetic polymers on a cell surface enhance mechanosensitivity of osteoblasts? Using MC3T3 osteoblast cells (C4 clone) and a PEG polymer, one of whose termini was covalently linked to a succinimidyl succinate group (functionalized PEG-PEGSS), we examined attachment of PEGSS to osteoblasts and evaluated its effects on the mRNA expression of stress-responsive genes. AFM images exhibited globular PEGSS conformation of approximately 100 nm in size, and SEM images confirmed the attachment of a cluster of pancake-like PEGSS molecules on the osteoblast surface. Compared to control cells incubated with unfunctionalized PEG, real-time PCR revealed that RNA upregulation of c-fos, egr1, ATF3 and Cox2 genes was magnified in the cells incubated with PEGSS. These results support a PEG-induced increase in mechanosensitivity of osteoblasts and indicate that the described approach would be useful to accelerate growth and development of osteoblasts for bone repair and tissue engineering.

  3. Particulate wear debris activates protein tyrosine kinases and nuclear factor kappaB, which down-regulates type I collagen synthesis in human osteoblasts.

    PubMed

    Vermes, C; Roebuck, K A; Chandrasekaran, R; Dobai, J G; Jacobs, J J; Glant, T T

    2000-09-01

    Particulate wear debris generated mechanically from prosthetic materials is phagocytosed by a variety of cell types within the periprosthetic space including osteoblasts, which cells with an altered function may contribute to periprosthetic osteolysis. Exposure of osteoblast-like osteosarcoma cells or bone marrow-derived primary osteoblasts to either metallic or polymeric particles of phagocytosable sizes resulted in a marked decrease in the steady-state messenger RNA (mRNA) levels of procollagen alpha1[I] and procollagen alpha1[III]. In contrast, no significant effect was observed for the osteoblast-specific genes, such as osteonectin and osteocalcin (OC). In kinetic studies, particles once phagocytosed, maintained a significant suppressive effect on collagen gene expression and type I collagen synthesis for up to five passages. Large particles of a size that cannot be phagocytosed also down-regulated collagen gene expression suggesting that an initial contact between cells and particles can generate gene responsive signals independently of the phagocytosis process. Concerning such signaling, titanium particles rapidly increased protein tyrosine phosphorylation and nuclear transcription factor kappaB (NF-kappaB) binding activity before the phagocytosis of particles. Protein tyrosine kinase (PTK) inhibitors such as genistein and the NF-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC) significantly reduced the suppressive effect of titanium on collagen gene expression suggesting particles suppress collagen gene expression through the NF-kappaB signaling pathway. These results provide a mechanism by which particulate wear debris can antagonize the transcription of the procollagen alpha1[I] gene in osteoblasts, which may contribute to reduced bone formation and progressive periprosthetic osteolysis.

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

    NASA Technical Reports Server (NTRS)

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

    1996-01-01

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

  5. Ligand-Mediated Activation of an Engineered Gs G Protein-Coupled Receptor in Osteoblasts Increases Trabecular Bone Formation

    PubMed Central

    Hsiao, Edward C.; Millard, Susan M.; Louie, Alyssa; Huang, Yong; Conklin, Bruce R.; Nissenson, Robert A.

    2010-01-01

    Age-dependent changes in skeletal growth play important roles in regulating skeletal expansion and in the course of many diseases affecting bone. How G protein-coupled receptor (GPCR) signaling affects these changes is poorly understood. Previously, we described a mouse model expressing Rs1, an engineered receptor with constitutive Gs activity. Rs1 expression in osteoblasts from gestation induced a dramatic age-dependent increase in trabecular bone with features resembling fibrous dysplasia; however, these changes were greatly minimized if Rs1 expression was delayed until after puberty. To further investigate whether ligand-induced activation of the Gs-GPCR pathway affects bone formation in adult mice, we activated Rs1 in adult mice with the synthetic ligand RS67333 delivered continuously via an osmotic pump or intermittently by daily injections. We found that osteoblasts from adult animals can be stimulated to form large amounts of bone, indicating that adult mice are sensitive to the dramatic bone- forming actions of Gs signaling in osteoblasts. In addition, our results show that intermittent and continuous activation of Rs1 led to structurally similar but quantitatively different degrees of trabecular bone formation. These results indicate that activation of a Gs-coupled receptor in osteoblasts of adult animals by either intermittent or continuous ligand administration can increase trabecular bone formation. In addition, osteoblasts located at the bone epiphyses may be more responsive to Gs signaling than osteoblasts at the bone diaphysis. This model provides a powerful tool for investigating the effects of ligand-activated Gs-GPCR signaling on dynamic bone growth and remodeling. PMID:20150184

  6. 17beta-estradiol potently suppresses cAMP-induced insulin-like growth factor-I gene activation in primary rat osteoblast cultures

    NASA Technical Reports Server (NTRS)

    McCarthy, T. L.; Ji, C.; Shu, H.; Casinghino, S.; Crothers, K.; Rotwein, P.; Centrella, M.

    1997-01-01

    Insulin-like growth factor-I (IGF-I) is a key factor in bone remodeling. In osteoblasts, IGF-I synthesis is enhanced by parathyroid hormone and prostaglandin E2 (PGE2) through cAMP-activated protein kinase. In rats, estrogen loss after ovariectomy leads to a rise in serum IGF-I and an increase in bone remodeling, both of which are reversed by estrogen treatment. To examine estrogen-dependent regulation of IGF-I expression at the molecular level, primary fetal rat osteoblasts were co-transfected with the estrogen receptor (hER, to ensure active ER expression), and luciferase reporter plasmids controlled by promoter 1 of the rat IGF-I gene (IGF-I P1), used exclusively in these cells. As reported, 1 microM PGE2 increased IGF-I P1 activity by 5-fold. 17beta-Estradiol alone had no effect, but dose-dependently suppressed the stimulatory effect of PGE2 by up to 90% (ED50 approximately 0.1 nM). This occurred within 3 h, persisted for at least 16 h, required ER, and appeared specific, since 17alpha-estradiol was 100-300-fold less effective. By contrast, 17beta-estradiol stimulated estrogen response element (ERE)-dependent reporter expression by up to 10-fold. 17beta-Estradiol also suppressed an IGF-I P1 construct retaining only minimal promoter sequence required for cAMP-dependent gene activation, but did not affect the 60-fold increase in cAMP induced by PGE2. There is no consensus ERE in rat IGF-I P1, suggesting novel downstream interactions in the cAMP pathway that normally enhances IGF-I expression in skeletal cells. To explore this, nuclear extract from osteoblasts expressing hER were examined by electrophoretic mobility shift assay using the atypical cAMP response element in IGF-I P1. Estrogen alone did not cause DNA-protein binding, while PGE2 induced a characteristic gel shift complex. Co-treatment with both hormones caused a gel shift greatly diminished in intensity, consistent with their combined effects on IGF-I promoter activity. Nonetheless, hER did not bind

  7. Smad4 controls bone homeostasis through regulation of osteoblast/osteocyte viability.

    PubMed

    Moon, Young Jae; Yun, Chi-Young; Choi, Hwajung; Ka, Sun-O; Kim, Jung Ryul; Park, Byung-Hyun; Cho, Eui-Sic

    2016-09-02

    Regulation of osteoblast and osteocyte viability is essential for bone homeostasis. Smad4, a major transducer of bone morphogenetic protein and transforming growth factor-β signaling pathways, regulates apoptosis in various cell types through a mitochondrial pathway. However, it remains poorly understood whether Smad4 is necessary for the regulation of osteoblast and osteocyte viability. In this study, we analyzed Smad4Δ(Os) mice, in which Smad4 was subjected to tissue-specific disruption under the control of the 2.3-kb Col1a1 promoter, to understand the functional significance of Smad4 in regulating osteoblast/osteocyte viability during bone formation and remodeling. Smad4Δ(Os) mice showed a significant increase in osteoblast number and osteocyte density in the trabecular and cortical regions of the femur, whereas osteoclast activity was significantly decreased. The proliferation of osteoblasts/osteocytes did not alter, as shown by measuring 5'-bromo-2'deoxyuridine incorporation. By contrast, the percentage of TUNEL-positive cells decreased, together with a decrease in the Bax/Bcl-2 ratio and in the proteolytic cleavage of caspase 3, in Smad4Δ(Os) mice. Apoptosis in isolated calvaria cells from Smad4Δ(Os) mice decreased after differentiation, which was consistent with the results of the TUNEL assay and western blotting in Smad4Δ(Os) mice. Conversely, osteoblast cells overexpressing Smad4 showed increased apoptosis. In an apoptosis induction model of Smad4Δ(Os) mice, osteoblasts/osteocytes were more resistant to apoptosis than were control cells, and, consequently, bone remodeling was attenuated. These findings indicate that Smad4 has a significant role in regulating osteoblast/osteocyte viability and therefore controls bone homeostasis.

  8. 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

  9. Extracellular Vesicles from Adipose-Derived Mesenchymal Stem Cells Downregulate Senescence Features in Osteoarthritic Osteoblasts

    PubMed Central

    Tofiño-Vian, Miguel; Pérez del Caz, María Dolores; Castejón, Miguel Angel

    2017-01-01

    Osteoarthritis (OA) affects all articular tissues leading to pain and disability. The dysregulation of bone metabolism may contribute to the progression of this condition. Adipose-derived mesenchymal stem cells (ASC) are attractive candidates in the search of novel strategies for OA treatment and exert anti-inflammatory and cytoprotective effects on cartilage. Chronic inflammation in OA is a relevant factor in the development of cellular senescence and joint degradation. In this study, we extend our previous observations of ASC paracrine effects to study the influence of conditioned medium and extracellular vesicles from ASC on senescence induced by inflammatory stress in OA osteoblasts. Our results in cells stimulated with interleukin- (IL-) 1β indicate that conditioned medium, microvesicles, and exosomes from ASC downregulate senescence-associated β-galactosidase activity and the accumulation of γH2AX foci. In addition, they reduced the production of inflammatory mediators, with the highest effect on IL-6 and prostaglandin E2. The control of mitochondrial membrane alterations and oxidative stress may provide a mechanism for the protective effects of ASC in OA osteoblasts. We have also shown that microvesicles and exosomes mediate the paracrine effects of ASC. Our study suggests that correction of abnormal osteoblast metabolism by ASC products may contribute to their protective effects. PMID:29230269

  10. Extracellular Vesicles from Adipose-Derived Mesenchymal Stem Cells Downregulate Senescence Features in Osteoarthritic Osteoblasts.

    PubMed

    Tofiño-Vian, Miguel; Guillén, Maria Isabel; Pérez Del Caz, María Dolores; Castejón, Miguel Angel; Alcaraz, Maria José

    2017-01-01

    Osteoarthritis (OA) affects all articular tissues leading to pain and disability. The dysregulation of bone metabolism may contribute to the progression of this condition. Adipose-derived mesenchymal stem cells (ASC) are attractive candidates in the search of novel strategies for OA treatment and exert anti-inflammatory and cytoprotective effects on cartilage. Chronic inflammation in OA is a relevant factor in the development of cellular senescence and joint degradation. In this study, we extend our previous observations of ASC paracrine effects to study the influence of conditioned medium and extracellular vesicles from ASC on senescence induced by inflammatory stress in OA osteoblasts. Our results in cells stimulated with interleukin- (IL-) 1 β indicate that conditioned medium, microvesicles, and exosomes from ASC downregulate senescence-associated β -galactosidase activity and the accumulation of γ H2AX foci. In addition, they reduced the production of inflammatory mediators, with the highest effect on IL-6 and prostaglandin E 2 . The control of mitochondrial membrane alterations and oxidative stress may provide a mechanism for the protective effects of ASC in OA osteoblasts. We have also shown that microvesicles and exosomes mediate the paracrine effects of ASC. Our study suggests that correction of abnormal osteoblast metabolism by ASC products may contribute to their protective effects.

  11. Curcumin induces osteoblast differentiation through mild-endoplasmic reticulum stress-mediated such as BMP2 on osteoblast cells.

    PubMed

    Son, Hyo-Eun; Kim, Eun-Jung; Jang, Won-Gu

    2018-01-15

    Curcumin (diferuloylmethane or [1E,6E]-1,7-bis[4-hydroxy-3-methoxyphenyl]-1,6heptadiene-3,5-dione) is a phenolic natural product derived from the rhizomes of the turmeric plant, Curcuma longa. It is reported to have various biological actions such as anti-oxidative, anti-inflammatory, and anti-cancer effects. However, the molecular mechanism of osteoblast differentiation by curcumin has not yet been reported. The cytotoxicity of curcumin was identified using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Expression of osteogenic markers and endoplasmic reticulum (ER) stress markers in C3H1-T1/2 cells were measured using reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blotting. Alkaline phosphatase (ALP) staining was performed to assess ALP activity in C3H10T1/2 cells. Transcriptional activity was detected using a luciferase reporter assay. Curcumin increased the expression of genes such as distal-less homeobox 5 (Dlx5), runt-related transcription factor 2 (Runx2), ALP, and osteocalcin (OC), which subsequently induced osteoblast differentiation in C3H10T1/2 cells. In addition, ALP activity and mineralization was found to be increased by curcumin treatment. Curcumin also induced mild ER stress similar to bone morphogenetic protein 2 (BMP2) function in osteoblast cells. Next, we confirmed that curcumin increased mild ER stress and osteoblast differentiation similar to BMP2 in C3H10T1/2 mesenchymal stem cells. Transient transfection studies also showed that curcumin increased ATF6-Luc activity, while decreasing the activities of CREBH-Luc and SMILE-Luc. In addition, similar to BMP2, curcumin induced the phosphorylation of Smad 1/5/9. Overall, these results demonstrate that curcumin-induced mild ER stress increases osteoblast differentiation via ATF6 expression in C3H10T1/2 cells. Copyright © 2017. Published by Elsevier Inc.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wan, Qiaoqiao; Cho, Eunhye; Yokota, Hiroki

    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.more » 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

  13. TRIB3 mediates the expression of Wnt5a and activation of nuclear factor-κB in Porphyromonas endodontalis lipopolysaccharide-treated osteoblasts.

    PubMed

    Yu, Y; Qiu, L; Guo, J; Yang, D; Qu, L; Yu, J; Zhan, F; Xue, M; Zhong, M

    2015-08-01

    Porphyromonas endodontalis lipopolysaccharide (LPS) is considered to be correlated with the progression of bone resorption in periodontal and periapical diseases. Wnt5a has recently been implicated in inflammatory processes, but its role is unclear as a P. endodontalis LPS-induced mediator in osteoblasts. Tribbles homolog 3 (TRIB3) encodes a pseudokinase and has been linked to inflammation in certain situations. Here, we found that P. endodontalis LPS induced Wnt5a expression in a dose- and time-dependent manner and it also upregulated translocation, phosphorylation and transcriptional activity of nuclear factor-κB (NF-κB) in MC3T3-E1 cells. Bay 11-7082 blocked the translocation of NF-κB and Wnt5a expression induced by P. endodontalis LPS. Chromatin immunoprecipitation assay further established that induction of Wnt5a by P. endodontalis LPS was mediated through the NF-κB p65 subunit. Additionally, P. endodontalis LPS increased expression of TRIB3 in osteoblasts after 10 h simulated time. Overexpression of TRIB3 enhanced NF-κB phosphorylation and Wnt5a induction, whereas knockdown of TRIB3 inhibited NF-κB phosphorylation and Wnt5a expression in P. endodontalis LPS-stimulated osteoblasts. These results suggest that P. endodontalis LPS has the ability to promote the expression of Wnt5a in mouse osteoblasts, and this induction is mainly mediated by NF-κB pathway. TRIB3 seems to modulate the sustained expression of Wnt5a in osteoblasts stimulated by P. endodontalis LPS, as well as regulating NF-κB phosphorylation. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  14. 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.

  15. Effects of Frequency and Acceleration Amplitude on Osteoblast Mechanical Vibration Responses: A Finite Element Study

    PubMed Central

    Hsu, Hung-Yao

    2016-01-01

    Bone cells are deformed according to mechanical stimulation they receive and their mechanical characteristics. However, how osteoblasts are affected by mechanical vibration frequency and acceleration amplitude remains unclear. By developing 3D osteoblast finite element (FE) models, this study investigated the effect of cell shapes on vibration characteristics and effect of acceleration (vibration intensity) on vibrational responses of cultured osteoblasts. Firstly, the developed FE models predicted natural frequencies of osteoblasts within 6.85–48.69 Hz. Then, three different levels of acceleration of base excitation were selected (0.5, 1, and 2 g) to simulate vibrational responses, and acceleration of base excitation was found to have no influence on natural frequencies of osteoblasts. However, vibration response values of displacement, stress, and strain increased with the increase of acceleration. Finally, stress and stress distributions of osteoblast models under 0.5 g acceleration in Z-direction were investigated further. It was revealed that resonance frequencies can be a monotonic function of cell height or bottom area when cell volumes and material properties were assumed as constants. These findings will be useful in understanding how forces are transferred and influence osteoblast mechanical responses during vibrations and in providing guidance for cell culture and external vibration loading in experimental and clinical osteogenesis studies. PMID:28074178

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sato, Chieri; Iwasaki, Tsuyoshi, E-mail: tsuyo-i@huhs.ac.jp; Kitano, Sachie

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

  17. CCAAT/enhancer-binding protein beta promotes osteoblast differentiation by enhancing Runx2 activity with ATF4.

    PubMed

    Tominaga, Hiroyuki; Maeda, Shingo; Hayashi, Makoto; Takeda, Shu; Akira, Shizuo; Komiya, Setsuro; Nakamura, Takashi; Akiyama, Haruhiko; Imamura, Takeshi

    2008-12-01

    Although CCAAT/enhancer-binding protein beta (C/EBPbeta) is involved in osteocalcin gene expression in osteoblast in vitro, the physiological importance of and molecular mechanisms governing C/EBPbeta in bone formation remain to be elucidated. In particular, it remains unclear whether C/EBPbeta acts as a homodimer or a heterodimer with other proteins during osteoblast differentiation. Here, deletion of the C/EBPbeta gene from mice resulted in delayed bone formation with concurrent suppression of chondrocyte maturation and osteoblast differentiation. The expression of type X collagen as well as chondrocyte hypertrophy were suppressed in mutant bone, providing new insight into the possible roles of C/EBPbeta in chondrocyte maturation. In osteoblasts, luciferase reporter, gel shift, DNAP, and ChIP assays demonstrated that C/EBPbeta heterodimerized with activating transcription factor 4 (ATF4), another basic leucine zipper transcription factor crucial for osteoblast maturation. This complex interacted and transactivated osteocalcin-specific element 1 (OSE1) of the osteocalcin promoter. C/EBPbeta also enhanced the synergistic effect of ATF4 and Runx2 on osteocalcin promoter transactivation by enhancing their interaction. Thus, our results provide evidence that C/EBPbeta is a crucial cofactor in the promotion of osteoblast maturation by Runx2 and ATF4.

  18. Effects of combined mechanical stimulation on the proliferation and differentiation of pre-osteoblasts

    PubMed Central

    Kang, Kyung Shin; Lee, Seung-Jae; Lee, Haksue; Moon, Wonkyu

    2011-01-01

    We observed how combined mechanical stimuli affect the proliferation and differentiation of pre-osteoblasts. For this research, a bioreactor system was developed that can simultaneously stimulate cells with cyclic strain and ultrasound, each of which is known to effectively stimulate bone tissue regeneration. MC3T3-E1 pre-osteoblasts were chosen for bone tissue engineering due to their osteoblast-like characteristics. 3-D scaffolds were fabricated with polycaprolactone and poly-L-lactic acid using the salt leaching method. The cells were stimulated by the bioreactor with cyclic strain and ultrasound. The bioreactor was set at a frequency of 1.0 Hz and 10% strain for cyclic strain and 1.0 MHz and 30 mW/cm2 for ultrasound. Three experimental groups (ultrasound, cyclic strain, and combined stimulation) and a control group were examined. Each group was stimulated for 20 min/day. Mechanical stimuli did not affect MC3T3-E1 cell proliferation significantly up to 10 days when measured with the cell counting kit-8. However, gene expression analysis of collagen type-I, osteocalcin, RUNX2, and osterix revealed that the combined mechanical stimulation accelerated the matrix maturation of MC3T3-E1 cells. These results indicate that the combined mechanical stimulation can enhance the differentiation of pre-osteoblasts more efficiently than simple stimuli, in spite of no effect on cell proliferation. PMID:21532314

  19. 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

  20. Whole body vibration improves osseointegration by up-regulating osteoblastic activity but down-regulating osteoblast-mediated osteoclastogenesis via ERK1/2 pathway.

    PubMed

    Zhou, Yi; Guan, Xiaoxu; Liu, Tie; Wang, Xinhua; Yu, Mengfei; Yang, Guoli; Wang, Huiming

    2015-02-01

    Due to the reduction in bone mass and deterioration in bone microarchitecture, osteoporosis is an important risk factor for impairing implant osseointegration. Recently, low-magnitude, high-frequency (LMHF) vibration (LM: <1×g; HF: 20-90Hz) has been shown to exhibit anabolic, but anti-resorptive effects on skeletal homeostasis. Therefore, we hypothesized that LMHF loading, in terms of whole body vibration (WBV), may improve implant fixation under osteoporotic status. In the in vivo study, WBV treatment (magnitude: 0.3g, frequency: 40Hz, time: 30min/12h, 5days/week) was applied after hydroxyapatite-coated titanium implants were inserted in the bilateral tibiae of ovariectomized rats. The bone mass and the osteospecific gene expressions were measured at 12weeks post implantation. In the in vitro study, the cellular and molecular mechanisms underlying osteoblastic and osteoclastic activities were fully investigated using various experimental assays. Micro-CT examination showed that WBV could enhance osseointegration by improving microstructure parameters surrounding implants. WBV-regulated gene levels in favor of bone formation over resorption may be the reason for the favorable adaptive bone remolding on bone-implant surface. The in vitro study showed that vibration (magnitude: 0.3g, frequency: 40Hz, time: 30min/12h) up-regulated osteoblast differentiation, matrix synthesis and mineralization. However, mechanically regulated osteoclastic activity was mainly through the effect on osteoblastic cells producing osteoclastogenesis-associated key soluble factors, including RANKL and M-CSF. Osteoblasts were therefore the direct target cells during the mechanotransduction process. The ERK1/2 pathway was demonstrated to play an essential role in vibration-induced enhancement of bone formation and decreased bone resorption. Our data suggests that WBV was a helpful non-pharmacological intervention for improving osseointegration under osteoporosis. Copyright © 2014 Elsevier

  1. The flavonoid fisetin promotes osteoblasts differentiation through Runx2 transcriptional activity.

    PubMed

    Léotoing, Laurent; Davicco, Marie-Jeanne; Lebecque, Patrice; Wittrant, Yohann; Coxam, Véronique

    2014-06-01

    Flavonoids represent a group of polyphenolic compounds commonly found in daily nutrition with proven health benefits. Among this group, the flavonol fisetin has been previously shown to protect bone by repressing osteoclast differentiation. In the present study, we investigated the role of fisetin in regulating osteoblasts physiology. In vivo mice treated with LPSs exhibited osteoporosis features associated with a dramatic repression of osteoblast marker expression. In this model, inhibition of osteocalcin and type I collagen alpha 1 transcription was partially countered by a daily consumption of fisetin. Interestingly, in vitro, fisetin promoted both osteoblast alkaline phosphatase activity and mineralization process. To decipher how fisetin may exert its positive effect on osteoblastogenesis, we analyzed its ability to control the runt-related transcription factor 2 (Runx2), a key organizer in developing and maturing osteoblasts. While fisetin did not impact Runx2 mRNA and protein levels, it upregulated its transcriptional activity. Actually, fisetin stimulated the luciferase activity of a reporter plasmid driven by the osteocalcin gene promoter that contains Runx2 binding sites and promoted the mRNA expression of osteocalcin and type I collagen alpha 1 targets. Bone sparing properties of fisetin also rely on its positive influence on osteoblast differentiation and activity. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. EGFR trans-activation mediates pleiotrophin-induced activation of Akt and Erk in cultured osteoblasts.

    PubMed

    Fan, Jian-Bo; Liu, Wei; Yuan, Kun; Zhu, Xin-Hui; Xu, Da-Wei; Chen, Jia-Jia; Cui, Zhi-Ming

    2014-05-09

    Pleiotrophin (Ptn) plays an important role in bone growth through regulating osteoblasts' functions. The underlying signaling mechanisms are not fully understood. In the current study, we found that Ptn induced heparin-binding epidermal growth factor (HB-EGF) release to trans-activate EGF-receptor (EGFR) in both primary osteoblasts and osteoblast-like MC3T3-E1 cells. Meanwhile, Ptn activated Akt and Erk signalings in cultured osteoblasts. The EGFR inhibitor AG1478 as well as the monoclonal antibody against HB-EGF (anti-HB-EGF) significantly inhibited Ptn-induced EGFR activation and Akt and Erk phosphorylations in MC3T3-E1 cells and primary osteoblasts. Further, EGFR siRNA depletion or dominant negative mutation suppressed also Akt and Erk activation in MC3T3-E1 cells. Finally, we observed that Ptn increased alkaline phosphatase (ALP) activity and inhibited dexamethasone (Dex)-induced cell death in both MC3T3-E1 cells and primary osteoblasts, such effects were alleviated by AG1478 or anti-HB-EGF. Together, these results suggest that Ptn-induced Akt/Erk activation and some of its pleiotropic functions are mediated by EGFR trans-activation in cultured osteoblasts. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Calcineurin/NFAT signaling in osteoblasts regulates bone mass.

    PubMed

    Winslow, Monte M; Pan, Minggui; Starbuck, Michael; Gallo, Elena M; Deng, Lei; Karsenty, Gerard; Crabtree, Gerald R

    2006-06-01

    Development and repair of the vertebrate skeleton requires the precise coordination of bone-forming osteoblasts and bone-resorbing osteoclasts. In diseases such as osteoporosis, bone resorption dominates over bone formation, suggesting a failure to harmonize osteoclast and osteoblast function. Here, we show that mice expressing a constitutively nuclear NFATc1 variant (NFATc1(nuc)) in osteoblasts develop high bone mass. NFATc1(nuc) mice have massive osteoblast overgrowth, enhanced osteoblast proliferation, and coordinated changes in the expression of Wnt signaling components. In contrast, viable NFATc1-deficient mice have defects in skull bone formation in addition to impaired osteoclast development. NFATc1(nuc) mice have increased osteoclastogenesis despite normal levels of RANKL and OPG, indicating that an additional NFAT-regulated mechanism influences osteoclastogenesis in vivo. Calcineurin/NFATc signaling in osteoblasts controls the expression of chemoattractants that attract monocytic osteoclast precursors, thereby coupling bone formation and bone resorption. Our results indicate that NFATc1 regulates bone mass by functioning in both osteoblasts and osteoclasts.

  4. Transdifferentiation of myoblasts into osteoblasts - possible use for bone therapy.

    PubMed

    Lin, Daphne P L; Carnagarin, Revathy; Dharmarajan, Arun; Dass, Crispin R

    2017-12-01

    Transdifferentiation is defined as the conversion of one cell type to another and is an ever-expanding field with a growing number of cells found to be capable of such a process. To date, the fact remains that there are limited treatment options for fracture healing, osteoporosis and bone repair post-destruction by bone tumours. Hence, this review focuses on the transdifferentiation of myoblast to osteoblast as a means to further understand the transdifferentiation process and to investigate a potential therapeutic option if successful. The potent osteoinductive effects of the bone morphogenetic protein-2 are largely implicated in the transdifferentiation of myoblast to osteoblast. Bone morphogenetic protein-2-induced activation of the Smad1 protein ultimately results in JunB synthesis, the first transcriptional step in myoblast dedifferentiation. The upregulation of the activating protein-1 binding activity triggers the transcription of the runt-related transcription factor 2 gene, a transcription factor that plays a major role in osteoblast differentiation. This potential transdifferentiation treatment may be utilised for dental implants, fracture healing, osteoporosis and bone repair post-destruction by bone tumours. © 2017 Royal Pharmaceutical Society.

  5. Abrogation of Cbl-PI3K interaction increases bone formation and osteoblast proliferation.

    PubMed

    Brennan, Tracy; Adapala, Naga Suresh; Barbe, Mary F; Yingling, Vanessa; Sanjay, Archana

    2011-11-01

    Cbl is an adaptor protein and E3 ligase that plays both positive and negative roles in several signaling pathways that affect various cellular functions. Tyrosine 737 is unique to Cbl and phosphorylated by Src family kinases. Phosphorylated CblY737 creates a binding site for the p85 regulatory subunit of phosphatidylinositol 3 kinase (PI3K) that also plays an important role in the regulation of bone homeostasis. To investigate the role of Cbl-PI3K interaction in bone homeostasis, we examined knock-in mice in which the PI3K binding site on Cbl was ablated due to the substitution of tyrosine 737 to phenylalanine (Cbl(YF/YF), YF mice). We previously reported that bone volume in these mice is increased due to decreased osteoclast function (Adapala et al., J Biol Chem 285:36745-36758, 19). Here, we report that YF mice also have increased bone formation and osteoblast numbers. In ex vivo cultures bone marrow-derived YF osteoblasts showed increased Col1A expression and their proliferation was also significantly augmented. Moreover, proliferation of MC3T3-E1 cells was increased after treatment with conditioned medium generated by culturing YF bone marrow stromal cells. Expression of stromal derived factor-1 (SDF-1) was increased in YF bone marrow stromal cells compared to wild type. Increased immunostaining of SDF-1 and CXCR4 was observed in YF bone marrow stromal cells compared to wild type. Treatment of YF condition medium with neutralizing anti-SDF-1 and anti-CXCR4 antibodies attenuated MC3T3-E1 cell proliferation. Cumulatively, these results show that abrogation of Cbl-PI3K interaction perturbs bone homeostasis, affecting both osteoclast function and osteoblast proliferation.

  6. A trans-acting enhancer modulates estrogen-mediated transcription of reporter genes in osteoblasts.

    PubMed

    Sasaki-Iwaoka, H; Maruyama, K; Endoh, H; Komori, T; Kato, S; Kawashima, H

    1999-02-01

    The presence of bone-specific estrogen agonists and discovery of the osteoblast-specific transcription factor (TF), Cbfa1, together with the discovery of synergism between a TF Pit-1 and estrogen receptor alpha (ERalpha) on rat prolactin gene, led to investigation of Cbfa1 in the modulation of osteoblast-specific actions of estrogen. Reverse transcribed-polymerase chain reaction demonstrated expression of Cbfa1 in the osteoblastic cell lines, MG63, ROS17/2.8, and MC3T3E1, but not in nonosteoblastic cell lines, MCF7, C3H10T1/2, and HeLa. An ER expression vector and a series of luciferase (Luc) reporter plasmids harboring the Cbfa1 binding site OSE2 (the osteoblast-specific cis element in the osteocalcin promoter) and palindromic estrogen response elements (EREs) were cotransfected into both osteoblastic and nonosteoblastic cells. OSE2 worked as a cis- acting element in osteoblastic cells but not nonosteoblastic cells, whereas EREs were cis- acting in all cell lines. Synergistic transactivation was observed in osteoblastic cells only when both ERE and OSE2 were placed in juxtaposition to the promoter. Forced expression of Cbfa1 in C3H10T1/2 cells also induced synergism. Tamoxifen, a partial agonist/antagonist of estrogen, acted as an osteoblast-specific agonist in cells transfected with a promoter containing ERE and acted synergistically with a promoter containing the ERE-OSE2 enhancer combination. These results support the idea that bone-specific TFs modulate the actions of estrogen in a tissue-specific manner.

  7. Salmon DNA Accelerates Bone Regeneration by Inducing Osteoblast Migration

    PubMed Central

    Sato, Ayako; Kajiya, Hiroshi; Mori, Nana; Sato, Hironobu; Fukushima, Tadao; Kido, Hirofumi

    2017-01-01

    The initial step of bone regeneration requires the migration of osteogenic cells to defective sites. Our previous studies suggest that a salmon DNA-based scaffold can promote the bone regeneration of calvarial defects in rats. We speculate that the salmon DNA may possess osteoinductive properties, including the homing of migrating osteogenic cells. In the present study, we investigated the influence of the salmon DNA on osteoblastic differentiation and induction of osteoblast migration using MG63 cells (human preosteoblasts) in vitro. Moreover, we analyzed the bone regeneration of a critical-sized in vivo calvarial bone defect (CSD) model in rats. The salmon DNA enhanced both mRNA and protein expression of the osteogenesis-related factors, runt-related transcription factor 2 (Runx2), alkaline phosphatase, and osterix (OSX) in the MG63 cells, compared with the cultivation using osteogenic induction medium alone. From the histochemical and immunohistochemical assays using frozen sections of the bone defects from animals that were implanted with DNA disks, many cells were found to express aldehyde dehydrogenase 1, one of the markers for mesenchymal stem cells. In addition, OSX was observed in the replaced connective tissue of the bone defects. These findings indicate that the DNA induced the migration and accumulation of osteogenic cells to the regenerative tissue. Furthermore, an in vitro transwell migration assay showed that the addition of DNA enhanced an induction of osteoblast migration, compared with the medium alone. The implantation of the DNA disks promoted bone regeneration in the CSD of rats, compared with that of collagen disks. These results indicate that the salmon DNA enhanced osteoblastic differentiation and induction of migration, resulting in the facilitation of bone regeneration. PMID:28060874

  8. Orphan nuclear receptor chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) protein negatively regulates bone morphogenetic protein 2-induced osteoblast differentiation through suppressing runt-related gene 2 (Runx2) activity.

    PubMed

    Lee, Kkot-Nim; Jang, Won-Gu; Kim, Eun-Jung; Oh, Sin-Hye; Son, Hye-Ju; Kim, Sun-Hun; Franceschi, Renny; Zhang, Xiao-Kun; Lee, Shee-Eun; Koh, Jeong-Tae

    2012-06-01

    Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) is an orphan nuclear receptor of the steroid-thyroid hormone receptor superfamily. COUP-TFII is widely expressed in multiple tissues and organs throughout embryonic development and has been shown to regulate cellular growth, differentiation, and organ development. However, the role of COUP-TFII in osteoblast differentiation has not been systematically evaluated. In the present study, COUP-TFII was strongly expressed in multipotential mesenchymal cells, and the endogenous expression level decreased during osteoblast differentiation. Overexpression of COUP-TFII inhibited bone morphogenetic protein 2 (BMP2)-induced osteoblastic gene expression. The results of alkaline phosphatase, Alizarin Red staining, and osteocalcin production assay showed that COUP-TFII overexpression blocks BMP2-induced osteoblast differentiation. In contrast, the down-regulation of COUP-TFII synergistically induced the expression of BMP2-induced osteoblastic genes and osteoblast differentiation. Furthermore, the immunoprecipitation assay showed that COUP-TFII and Runx2 physically interacted and COUP-TFII significantly impaired the Runx2-dependent activation of the osteocalcin promoter. From the ChIP assay, we found that COUP-TFII repressed DNA binding of Runx2 to the osteocalcin gene, whereas Runx2 inhibited COUP-TFII expression via direct binding to the COUP-TFII promoter. Taken together, these findings demonstrate that COUP-TFII negatively regulates osteoblast differentiation via interaction with Runx2, and during the differentiation state, BMP2-induced Runx2 represses COUP-TFII expression and promotes osteoblast differentiation.

  9. Does size difference in allogeneic cancellous bone granules loaded with differentiated autologous cultured osteoblasts affect osteogenic potential?

    PubMed

    Lee, Sang-Uk; Chung, Yang-Guk; Kim, Seok-Jung; Oh, Il-Hoan; Kim, Yong-Sik; Ju, Sung-Hun

    2014-02-01

    We study the efficacy of bone regeneration by using two differently sized allogeneic cancellous bone granules loaded with autologous cultured osteoblasts in a rabbit model. Critical-sized bone defects of the radial shaft were made in 40 New Zealand White rabbits. Small allogeneic bone granules (150-300 μm in diameter) loaded with cultured differentiated autologous osteoblasts were implanted into one forearm (SBG group) and large bone granules (500-710 μm) loaded with osteoblasts were implanted into the forearm of the other side (LBG group). Radiographic evaluations were performed at 3, 6, 9 and 12 weeks and histology and micro-CT image analysis were carried out at 6 and 12 weeks post-implantation. On radiographic evaluation, the LBG group showed a higher bone quantity index at 3 and 6 weeks post-implantation (P < 0.05) but statistical significance was lost at 9 and 12 weeks. The progression of biological processes of the SBG group was faster than that of the LBG group. On micro-CT image analysis, the LBG group revealed a higher total bone volume and surface area than the SBG group at 6 weeks (P < 0.05) but the difference decreased at 12 weeks and was without statistical significance. Histological evaluation also revealed faster progression of new bone formation and maturation in the SBG group. Thus, the two differently sized allogeneic bone granules loaded with co-cultured autologous osteoblasts show no differences in the amount of bone regeneration, although the SBG group exhibits faster progression of bone regeneration and remodeling. This method might therefore provide benefits, such as a short healing time and easy application in an injectable form, in a clinical setting.

  10. Constitutively expressed COX-2 in osteoblasts positively regulates Akt signal transduction via suppression of PTEN activity.

    PubMed

    Li, Ching-Ju; Chang, Je-Ken; Wang, Gwo-Jaw; Ho, Mei-Ling

    2011-02-01

    Cyclooxygenase-2 (COX-2) is thought to be an inducible enzyme, but increasing reports indicate that COX-2 is constitutively expressed in several organs. The status of COX-2 expression in bone and its physiological role remains undefined. Non-selective non-steroidal anti-inflammatory drugs (NSAIDs) and selective COX-2 inhibitors, which commonly suppress COX-2 activity, were reported to suppress osteoblast proliferation via Akt/FOXO3a/p27(Kip1) signaling, suggesting that COX-2 may be the key factor of the suppressive effects of NSAIDs on proliferation. Although Akt activation correlates with PTEN deficiency and cell viability, the role of COX-2 on PTEN/Akt regulation remains unclear. In this study, we hypothesized that COX-2 may be constitutively expressed in osteoblasts and regulate PTEN/Akt-related proliferation. We examined the localization and co-expression of COX-2 and p-Akt in normal mouse femurs and in cultured mouse (mOBs) and human osteoblasts (hOBs). Our results showed that osteoblasts adjacent to the trabeculae, periosteum and endosteum in mouse femurs constitutively expressed COX-2, while COX-2 co-expressed with p-Akt in osteoblasts sitting adjacent to trabeculae in vivo, and in mOBs and hOBs in vitro. We further used COX-2 siRNA to test the role of COX-2 in Akt signaling in hOBs; COX-2 silencing significantly inhibited PTEN phosphorylation, enhanced PTEN activity, and suppressed p-Akt level and proliferation. However, replenishment of the COX-2 enzymatic product, PGE2, failed to reverse COX-2-dependent Akt phosphorylation. Furthermore, transfection with recombinant human COX-2 (rhCOX-2) significantly reversed COX-2 siRNA-suppressed PTEN phosphorylation, but this effect was reduced when the enzymatic activity of rhCOX-2 was blocked. This finding indicated that the effect of COX-2 on PTEN/Akt signaling is not related to PGE2 but still dependent on COX-2 enzymatic activity. Conversely, COX-1 silencing did not affect PTEN/Akt signaling. Our findings provide

  11. 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

  12. Activity of the rat osteocalcin basal promoter in osteoblastic cells is dependent upon homeodomain and CP1 binding motifs.

    PubMed

    Towler, D A; Bennett, C D; Rodan, G A

    1994-05-01

    A detailed analysis of the transcriptional machinery responsible for osteoblast-specific gene expression should provide tools useful for understanding osteoblast commitment and differentiation. We have defined three cis-elements important for basal activity of the rat osteocalcin (OC) promoter, located at about -200 to -180, -170 to -138, and -121 to -64 relative to the transcription initiation site. A motif (TCTGATTGTGT) present in the region between -200 and -170 that binds a multisubunit CP1/NFY/CBF-like CAAT factor complex contributes significantly to high level basal activity and presumably functions as the CAAT box for the rat OC promoter. We show that the region -121 to 32 is sufficient to confer osteoblastic cell type specificity in transient transfection assays of cultured cell lines using luciferase as a reporter. The basal promoter is active in rodent osteoblastic cell lines, but not in rodent fibroblastic or muscle cell lines. Although the rat OC box (-100 to -74) contains a CAAT motif, we could not detect CP1-like CAAT factor binding to this region. In fact, we demonstrate that a Msx-1 (Hox 7.1) homeodomain binding motif (ACTAATTG; bottom strand) in the 3'-end of the rat OC box is necessary for high level activity of the rat OC basal promoter in osteoblastic cells. A nuclear factor that recognizes this motif appears to be present in osteoblastic ROS 17/2.8 cells, which produce OC, but not in fibroblastic ROS 25/1 cells, which fail to express OC. This ROS 17/2.8 nuclear factor also recognizes the A/T-rich DNA cognates of the homeodomain-containing POU family of transcription factors. Taken together, these data suggest that a ubiquitous CP1-like CAAT factor and a cell type-restricted homeodomain containing (Msx or POU family) transcription factor interact with the proximal rat OC promoter to direct appropriate basal OC transcription in osteoblastic cells.

  13. NURR1 Downregulation Favors Osteoblastic Differentiation of MSCs

    PubMed Central

    Di Benedetto, Adriana; Cantore, Stefania; Centonze, Matteo; Grano, Maria; Cavalcanti-Adam, Elisabetta A.

    2017-01-01

    Mesenchymal stem cells (MSCs) have been identified in human dental tissues. Dental pulp stem cells (DPSCs) were classified within MSC family, are multipotent, can be isolated from adult teeth, and have been shown to differentiate, under particular conditions, into various cell types including osteoblasts. In this work, we investigated how the differentiation process of DPSCs toward osteoblasts is controlled. Recent literature data attributed to the nuclear receptor related 1 (NURR1), a still unclarified role in osteoblast differentiation, while NURR1 is primarily involved in dopaminergic neuron differentiation and activity. Thus, in order to verify if NURR1 had a role in DPSC osteoblastic differentiation, we silenced it during all the processes and compared the expression of the main osteoblastic markers with control cultures. Our results showed that the inhibition of NURR1 significantly increased the expression of osteoblast markers collagen I and alkaline phosphatase. Further, in long time cultures, the mineral matrix deposition was strongly enhanced in NURR1-silenced cultures. These results suggest that NURR1 plays a key role in switching DPSC differentiation toward osteoblasts rather than neuronal or even other cell lines. In conclusion, DPSCs represent a source of osteoblast-like cells and downregulation of NURR1 strongly prompted their differentiation toward the osteoblastogenesis process. PMID:28769982

  14. Additively manufactured 3D porous Ti-6Al-4V constructs mimic trabecular bone structure and regulate osteoblast proliferation, differentiation and local factor production in a porosity and surface roughness dependent manner.

    PubMed

    Cheng, Alice; Humayun, Aiza; Cohen, David J; Boyan, Barbara D; Schwartz, Zvi

    2014-10-07

    Additive manufacturing by laser sintering is able to produce high resolution metal constructs for orthopedic and dental implants. In this study, we used a human trabecular bone template to design and manufacture Ti-6Al-4V constructs with varying porosity via laser sintering. Characterization of constructs revealed interconnected porosities ranging from 15-70% with compressive moduli of 2579-3693 MPa. These constructs with macro porosity were further surface-treated to create a desirable multi-scale micro-/nano-roughness, which has been shown to enhance the osseointegration process. Osteoblasts (MG63 cells) exhibited high viability when grown on the constructs. Proliferation (DNA) and alkaline phosphatase specific activity, an early differentiation marker, decreased as porosity increased, while osteocalcin, a late differentiation marker, as well as osteoprotegerin, vascular endothelial growth factor and bone morphogenetic proteins 2 and 4 increased with increasing porosity. Three-dimensional (3D) constructs with the highest porosity and surface modification supported the greatest osteoblast differentiation and local factor production. These results indicate that additively manufactured 3D porous constructs mimicking human trabecular bone and produced with additional surface treatment can be customized for increased osteoblast response. Increased factors for osteoblast maturation and differentiation on high porosity constructs suggest the enhanced performance of these surfaces for increasing osseointegration in vivo.

  15. Osthole Enhances Osteogenesis in Osteoblasts by Elevating Transcription Factor Osterix via cAMP/CREB Signaling In Vitro and In Vivo.

    PubMed

    Zhang, Zhong-Rong; Leung, Wing Nang; Li, Gang; Kong, Siu Kai; Lu, Xiong; Wong, Yin Mei; Chan, Chun Wai

    2017-06-08

    Anabolic anti-osteoporotic agents are desirable for treatment and prevention of osteoporosis and fragility fractures. Osthole is a coumarin derivative extracted from the medicinal herbs Cnidium monnieri (L.) Cusson and Angelica pubescens Maxim.f. Osthole has been reported with osteogenic and anti-osteoporotic properties, whereas the underlying mechanism of its benefit still remains unclear. The objective of the present study was to investigate the osteopromotive action of osthole on mouse osteoblastic MC3T3-E1 cells and on mouse femoral fracture repair, and to explore the interaction between osthole-induced osteopromotive effect and cyclic adenosine monophosphate (cAMP) elevating effect. Osthole treatment promoted osteogenesis in osteoblasts by enhancing alkaline phosphatase (ALP) activity and mineralization. Oral gavage of osthole enhanced fracture repair and increased bone strength. Mechanistic study showed osthole triggered the cAMP/CREB pathway through the elevation of the intracellular cAMP level and activation of the phosphorylation of the cAMP response element-binding protein (CREB). Blockage of cAMP/CREB downstream signals with protein kinase A (PKA) inhibitor KT5720 partially suppressed osthole-mediated osteogenesis by inhibiting the elevation of transcription factor, osterix. In conclusion, osthole shows osteopromotive effect on osteoblasts in vitro and in vivo. Osthole-mediated osteogenesis is related to activation of the cAMP/CREB signaling pathway and downstream osterix expression.

  16. Osthole Enhances Osteogenesis in Osteoblasts by Elevating Transcription Factor Osterix via cAMP/CREB Signaling In Vitro and In Vivo

    PubMed Central

    Zhang, Zhong-Rong; Leung, Wing Nang; Li, Gang; Kong, Siu Kai; Lu, Xiong; Wong, Yin Mei; Chan, Chun Wai

    2017-01-01

    Anabolic anti-osteoporotic agents are desirable for treatment and prevention of osteoporosis and fragility fractures. Osthole is a coumarin derivative extracted from the medicinal herbs Cnidium monnieri (L.) Cusson and Angelica pubescens Maxim.f. Osthole has been reported with osteogenic and anti-osteoporotic properties, whereas the underlying mechanism of its benefit still remains unclear. The objective of the present study was to investigate the osteopromotive action of osthole on mouse osteoblastic MC3T3-E1 cells and on mouse femoral fracture repair, and to explore the interaction between osthole-induced osteopromotive effect and cyclic adenosine monophosphate (cAMP) elevating effect. Osthole treatment promoted osteogenesis in osteoblasts by enhancing alkaline phosphatase (ALP) activity and mineralization. Oral gavage of osthole enhanced fracture repair and increased bone strength. Mechanistic study showed osthole triggered the cAMP/CREB pathway through the elevation of the intracellular cAMP level and activation of the phosphorylation of the cAMP response element-binding protein (CREB). Blockage of cAMP/CREB downstream signals with protein kinase A (PKA) inhibitor KT5720 partially suppressed osthole-mediated osteogenesis by inhibiting the elevation of transcription factor, osterix. In conclusion, osthole shows osteopromotive effect on osteoblasts in vitro and in vivo. Osthole-mediated osteogenesis is related to activation of the cAMP/CREB signaling pathway and downstream osterix expression. PMID:28629115

  17. 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.

  18. 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

  19. Leptin promotes ossification through multiple ways of bone metabolism in osteoblast: a pilot study.

    PubMed

    Zhang, Jing; Li, Tingting; Xu, Liangzhi; Li, Wenjuan; Cheng, Meng; Zhuang, Jing; Chen, Yan; Xu, Wenming

    2013-08-01

    Leptin may be a potential option in preventing osteoporosis for menopausal women. The objective of this study is to explore the molecular mechanism of leptin on bone metabolism in osteoblast. Primary osteoblasts were isolated from parietal bone of adult female rats. mRNA level of OB-Rb in osteoblasts was inhibited by siRNA to block leptin signal transmission. The whole genome expression was tested by using gene chip to preliminarily explore the molecular mechanism of leptin in regulating osteoblast activity. The optimal concentration of siRNA was 25 nM, resulting in a maximal inhibition of OB-Rb mRNA. Ossification (p < 0.05) and bone mineralization (p = 0.0001) were downregulated by inhibiting leptin signal transmission, while bone resorption (p = 0.007), osteoblast differentiation (p = 0.026) and negative regulation of bone remodeling (p = 0.004) were upregulated. The expressions of some genes were regulated by OB-Rb siRNA. The expressions of alkaline phosphatase (p = 0.014) and osteocalcin (p = 0.002) were reduced, while that of vascular endothelial growth factor A (p = 0.0076) and IL-6 (p = 0.021) were increased. In a model of osteoblast, leptin positively promotes ossification through multiple ways including bone mineralization, remodeling, resorption and osteoblast differentiation, but which way plays the most critical role is not discussed in this study and needs to be clarified in future.

  20. Identification of the cAMP response element that controls transcriptional activation of the insulin-like growth factor-I gene by prostaglandin E2 in osteoblasts

    NASA Technical Reports Server (NTRS)

    Thomas, M. J.; Umayahara, Y.; Shu, H.; Centrella, M.; Rotwein, P.; McCarthy, T. L.

    1996-01-01

    Insulin-like growth factor-I (IGF-I), a multifunctional growth factor, plays a key role in skeletal growth and can enhance bone cell replication and differentiation. We previously showed that prostaglandin E2 (PGE2) and other agents that increase cAMP activated IGF-I gene transcription in primary rat osteoblast cultures through promoter 1 (P1), the major IGF-I promoter, and found that transcriptional induction was mediated by protein kinase A. We now have identified a short segment of P1 that is essential for full hormonal regulation and have characterized inducible DNA-protein interactions involving this site. Transient transfections of IGF-I P1 reporter genes into primary rat osteoblasts showed that the 328-base pair untranslated region of exon 1 was required for a full 5.3-fold response to PGE2; mutation in a previously footprinted site, HS3D (base pairs +193 to +215), reduced induction by 65%. PGE2 stimulated nuclear protein binding to HS3D. Binding, as determined by gel mobility shift assay, was not seen in nuclear extracts from untreated osteoblast cultures, was detected within 2 h of PGE2 treatment, and was maximal by 4 h. This DNA-protein interaction was not observed in cytoplasmic extracts from PGE2-treated cultures, indicating nuclear localization of the protein kinase A-activated factor(s). Activation of this factor was not blocked by cycloheximide (Chx), and Chx did not impair stimulation of IGF-I gene expression by PGE2. In contrast, binding to a consensus cAMP response element (CRE; 5'-TGACGTCA-3') from the rat somatostatin gene was not modulated by PGE2 or Chx. Competition gel mobility shift analysis using mutated DNA probes identified 5'-CGCAATCG-3' as the minimal sequence needed for inducible binding. All modified IGF-I P1 promoterreporter genes with mutations within this CRE sequence also showed a diminished functional response to PGE2. These results identify the CRE within the 5'-untranslated region of IGF-I exon 1 that is required for hormonal

  1. A new role for HERPUD1 and ERAD activation in osteoblast differentiation and mineralization.

    PubMed

    Américo-Da-Silva, Luan; Diaz, Jheimmy; Bustamante, Mario; Mancilla, Georthan; Oyarzún, Ingrid; Verdejo, Hugo E; Quiroga, Clara

    2018-03-23

    Bone integrity depends on a finely tuned balance between bone synthesis by osteoblasts and resorption by osteoclasts. The secretion capacity of mature osteoblasts requires strict control of proteostasis. Endoplasmic reticulum-associated degradation (ERAD) prevents the accumulation of unfolded ER proteins via dislocation to the cytosol and degradation by the proteasome. The ER membrane protein, homocysteine-inducible endoplasmic reticulum protein with ubiquitin-like domain 1 (HERPUD1), is a key component of the ERAD multiprotein complex which helps to stabilize the complex and facilitate the efficient degradation of unfolded proteins. HERPUD1 expression is strongly up-regulated by the unfolded protein response and cellular stress. The aim of the current study was to establish whether HERPUD1 and ERAD play roles in osteoblast differentiation and maturation. We evaluated preosteoblastic MC3T3-E1 cell and primary rat osteoblast differentiation by measuring calcium deposit levels, alkaline phosphatase activity, and runt-related transcription factor 2 and osterix expression. We found that ERAD and proteasomal degradation were activated and that HERPUD1 expression was increased as osteoblast differentiation progressed. The absence of HERPUD1 blocked osteoblast mineralization in vitro and significantly reduced alkaline phosphatase activity. In contrast, HERPUD1 overexpression activated the osteoblast differentiation program. Our results demonstrate that HERPUD1 and ERAD are important for the activation of the osteoblast maturation program and may be useful new targets for elucidating bone physiology.-Américo-Da-Silva, L., Diaz, J., Bustamante, M., Mancilla, G., Oyarzún, I., Verdejo, H. E., Quiroga, C. A new role for HERPUD1 and ERAD activation in osteoblast differentiation and mineralization.

  2. 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

  3. Conditional ablation of osteoblasts in medaka.

    PubMed

    Willems, Bernd; Büttner, Anita; Huysseune, Ann; Renn, Joerg; Witten, P Eckhard; Winkler, Christoph

    2012-04-15

    Different from tetrapods, teleost vertebral centra form without prior establishment of a cartilaginous scaffold, in two steps: First, mineralization of the notochord sheath establishes the vertebral centra. Second, sclerotome derived mesenchymal cells migrate around the notochord sheath. These cells differentiate into osteoblasts and deposit bone onto the mineralized notochord sheath in a process of intramembranous bone formation. In contrast, most skeletal elements of the cranial skeleton arise by chondral bone formation, with remarkably similar mechanisms in fish and tetrapods. To further investigate the role of osteoblasts during formation of the cranial and axial skeleton, we generated a transgenic osx:CFP-NTR medaka line which enables conditional ablation of osterix expressing osteoblasts. By expressing a bacterial nitroreductase (NTR) fused to Cyan Fluorescent Protein (CFP) under control of the osterix promoter these cells become sensitive towards Metronidazole (Mtz). Mtz treatment of stable osx:CFP-NTR transgenic medaka for several consecutive days led to significant loss of osteoblasts by apoptosis. Live staining of mineralized bone matrix revealed reduced ossification in head skeletal elements such as cleithrum and operculum, as well as in the vertebral arches. Interestingly in Mtz treated larvae, intervertebral spaces were missing and the notochord sheath was often continuously mineralized resulting in the fusion of centra. We therefore propose a dual role for osx-positive osteoblasts in fish. Besides a role in bone deposition, we suggest an additional border function during mineralization of the chordal centra. After termination of Mtz treatment, osteoblasts gradually reappeared, indicating regenerative properties in this cell lineage. Taken together, the osx:CFP-NTR medaka line represents a valuable tool to study osteoblast function and regeneration at different stages of development in whole vertebrate specimens in vivo. Copyright © 2012 Elsevier

  4. Vitamin A Is a Negative Regulator of Osteoblast Mineralization

    PubMed Central

    Hu, Lijuan; Pejler, Gunnar; Andersson, Göran; Jacobson, Annica; Melhus, Håkan

    2013-01-01

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

  5. Communication-dependent mineralization of osteoblasts via gap junctions.

    PubMed

    Hashida, Yukihiko; Nakahama, Ken-ichi; Shimizu, Kaori; Akiyama, Masako; Harada, Kiyoshi; Morita, Ikuo

    2014-04-01

    Connexin43 (Cx43) is a major gap junction (GJ) protein in bone and plays a critical role in osteoblast differentiation. Several studies show that osteoblast differentiation is delayed by Cx43 ablation. However, the precise mechanism underlying the role of Cx43 in osteoblast differentiation is not fully understood. Firstly, we analyzed the phenotype of a conditional knockout mouse, which was generated by mating of an osterix promoter-driven Cre expressing mouse with a Cx43-floxed mouse. As expected, delayed ossification was observed. Secondly, we demonstrated that the cell communication via gap junctions played an important role in osteoblast differentiation using a tamoxifen-inducible knockout system in vitro. Genetic ablation of Cx43 resulted in both the disruption of cell-communications and the attenuation of osteoblast mineralization induced by BMP-2, but not by ascorbic acid. Moreover, restoring full-length Cx43 (382aa) expression rescued the impairment of osteoblast cell-communication and osteoblast mineralization; however, the expression of the Cx43 N-terminal mutant (382aaG2V) did not rescue either of them. Comparing the gene expression profiles, the genes directly regulated by BMP-2 were attenuated by Cx43 gene ablation. These results suggested that the cell-communication mediated by gap junctions was indispensable for normal differentiation of osteoblast induced by BMP-2. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. SMURF2 regulates bone homeostasis by disrupting SMAD3 interaction with vitamin D receptor in osteoblasts

    PubMed Central

    Xu, Zhan; Greenblatt, Matthew B.; Yan, Guang; Feng, Heng; Sun, Jun; Lotinun, Sutada; Brady, Nicholas; Baron, Roland; Glimcher, Laurie H.; Zou, Weiguo

    2017-01-01

    Coordination between osteoblasts and osteoclasts is required for bone health and homeostasis. Here we show that mice deficient in SMURF2 have severe osteoporosis in vivo. This low bone mass phenotype is accompanied by a pronounced increase in osteoclast numbers, although Smurf2-deficient osteoclasts have no intrinsic alterations in activity. Smurf2-deficient osteoblasts display increased expression of RANKL, the central osteoclastogenic cytokine. Mechanistically, SMURF2 regulates RANKL expression by disrupting the interaction between SMAD3 and vitamin D receptor by altering SMAD3 ubiquitination. Selective deletion of Smurf2 in the osteoblast lineage recapitulates the phenotype of germline Smurf2-deficient mice, indicating that SMURF2 regulates osteoblast-dependent osteoclast activity rather than directly affecting the osteoclast. Our results reveal SMURF2 as an important regulator of the critical communication between osteoblasts and osteoclasts. Furthermore, the bone mass phenotype in Smurf2- and Smurf1-deficient mice is opposite, indicating that SMURF2 has a non-overlapping and, in some respects, opposite function to SMURF1. PMID:28216630

  7. Effect of Magnolol on the Function of Osteoblastic MC3T3-E1 Cells

    PubMed Central

    Kwak, Eun Jung; Lee, Young Soon; Choi, Eun Mi

    2012-01-01

    Objectives. In the present study, the ability of magnolol, a hydroxylated biphenyl compound isolated from Magnolia officinalis, to stimulate osteoblast function and inhibit the release of bone-resorbing mediators was investigated in osteoblastic MC3T3-E1 cells. Methods. Osteoblast function was measured by cell growth, alkaline phosphatase activity, collagen synthesis, and mineralization. Glutathione content was also measured in the cells. Bone-resorbing cytokines, receptor activator of nuclear factor-κB ligand (RANKL), TNF-α, and IL-6 were measured with an enzyme immunoassay system. Results. Magnolol caused a significant elevation of cell growth, alkaline phosphatase activity, collagen synthesis, mineralization, and glutathione content in the cells (P < 0.05). Skeletal turnover is orchestrated by a complex network of regulatory factors. Among cytokines, RANKL, TNF-α, and IL-6 were found to be key osteoclastogenetic molecules produced by osteoblasts. Magnolol significantly (P < 0.05) decreased the production of osteoclast differentiation inducing factors such as RANKL, TNF-α, and IL-6 in the presence of antimycin A, which inhibits mitochondrial electron transport and has been used as an ROS generator. Conclusion. Magnolol might be a candidate as an agent for the prevention of bone disorders such as osteoporosis. PMID:22474400

  8. 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. © 2015 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.

  9. Inhibition of prostate cancer osteoblastic progression with VEGF121/rGel, a single agent targeting osteoblasts, osteoclasts, and tumor neovasculature

    PubMed Central

    Mohamedali, Khalid A.; Li, Zhi Gang; Starbuck, Michael W.; Wan, Xinhai; Yang, Jun; Kim, Sehoon; Zhang, Wendy; Rosenblum, Michael G.; Navone, Nora M.

    2011-01-01

    Purpose A hallmark of prostate cancer (PCa) progression is the development of osteoblastic bone metastases, which respond poorly to available therapies. We previously reported that VEGF121/rGel targets osteoclast precursors and tumor neovasculature. Here we tested the hypothesis that targeting non-tumor cells expressing these receptors can inhibit tumor progression in a clinically relevant model of osteoblastic PCa. Experimental Design Cells from MDA PCa 118b, a PCa xenograft obtained from a bone metastasis in a patient with castrate-resistant PCa, were injected into the femurs of mice. Osteoblastic progression was monitored following systemic administration of VEGF121/rGel. Results VEGF121/rGel was cytotoxic in vitro to osteoblast precursor cells. This cytotoxicity was specific as VEGF121/rGel internalization into osteoblasts was VEGF121 receptor driven. Furthermore, VEGF121/rGel significantly inhibited PCa-induced bone formation in a mouse calvaria culture assay. In vivo, VEGF121/rGel significantly inhibited the osteoblastic progression of PCa cells in the femurs of nude mice. Microcomputed tomography analysis revealed that VEGF121/rGel restored the bone volume fraction of tumor-bearing femurs to values similar to those of the contralateral (non–tumor bearing) femurs. VEGF121/rGel significantly reduced the number of tumor-associated osteoclasts but did not change the numbers of peritumoral osteoblasts. Importantly, VEGF121/rGel-treated mice had significantly less tumor burden than control mice. Our results thus indicate that VEGF121/rGel inhibits osteoblastic tumor progression by targeting angiogenesis, osteoclastogenesis, and bone formation. Conclusions Targeting VEGFR-1 – or VEGFR-2–expressing cells is effective in controlling the osteoblastic progression of PCa in bone. These findings provide the basis for an effective multitargeted approach for metastatic PCa. PMID:21343372

  10. Inhibition of prostate cancer osteoblastic progression with VEGF121/rGel, a single agent targeting osteoblasts, osteoclasts, and tumor neovasculature.

    PubMed

    Mohamedali, Khalid A; Li, Zhi Gang; Starbuck, Michael W; Wan, Xinhai; Yang, Jun; Kim, Sehoon; Zhang, Wendy; Rosenblum, Michael G; Navone, Nora M

    2011-04-15

    A hallmark of prostate cancer (PCa) progression is the development of osteoblastic bone metastases, which respond poorly to available therapies. We previously reported that VEGF(121)/rGel targets osteoclast precursors and tumor neovasculature. Here we tested the hypothesis that targeting nontumor cells expressing these receptors can inhibit tumor progression in a clinically relevant model of osteoblastic PCa. Cells from MDA PCa 118b, a PCa xenograft obtained from a bone metastasis in a patient with castrate-resistant PCa, were injected into the femurs of mice. Osteoblastic progression was monitored following systemic administration of VEGF(121)/rGel. VEGF(121)/rGel was cytotoxic in vitro to osteoblast precursor cells. This cytotoxicity was specific as VEGF(121)/rGel internalization into osteoblasts was VEGF(121) receptor driven. Furthermore, VEGF(121)/rGel significantly inhibited PCa-induced bone formation in a mouse calvaria culture assay. In vivo, VEGF(121)/rGel significantly inhibited the osteoblastic progression of PCa cells in the femurs of nude mice. Microcomputed tomographic analysis revealed that VEGF(121)/rGel restored the bone volume fraction of tumor-bearing femurs to values similar to those of the contralateral (non-tumor-bearing) femurs. VEGF(121)/rGel significantly reduced the number of tumor-associated osteoclasts but did not change the numbers of peritumoral osteoblasts. Importantly, VEGF(121)/rGel-treated mice had significantly less tumor burden than control mice. Our results thus indicate that VEGF(121)/rGel inhibits osteoblastic tumor progression by targeting angiogenesis, osteoclastogenesis, and bone formation. Targeting VEGF receptor (VEGFR)-1- or VEGFR-2-expressing cells is effective in controlling the osteoblastic progression of PCa in bone. These findings provide the basis for an effective multitargeted approach for metastatic PCa. ©2011 AACR.

  11. 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.

  12. Zoledronic acid at subtoxic dose extends osteoblastic stage span of primary human osteoblasts.

    PubMed

    Zara, Susi; De Colli, Marianna; di Giacomo, Viviana; Zizzari, Vincenzo Luca; Di Nisio, Chiara; Di Tore, Umberto; Salini, Vincenzo; Gallorini, Marialucia; Tetè, Stefano; Cataldi, Amelia

    2015-04-01

    This study aimed to check the effect of zoledronic acid (ZA) at subtoxic dose on human osteoblasts (HOs) in terms of cell viability, apoptosis occurrence, and differentiation induction. ZA belongs to the family of bisphosphonates (BPs), largely used in the clinical practice for the treatment of bone diseases, often associated with jaw osteonecrosis onset. Their pharmacological action consists in the direct block of the osteoclast-mediated bone resorption along with indirect action on osteoblasts. HOs were treated choosing the highest limit concentration (10(-5) M) which does not induce toxic effects. Live/dead staining, flow cytometry, mitochondrial membrane potential assay, osteocalcin western blotting, gp38 RT-PCR, collagen type I, PGE2, and IL-6 ELISA assays were performed. Similar viability level between control and ZA-treated samples is found along with no significant increase of apoptotic and necrotic cells in ZA-treated sample. To establish if an early apoptotic pathway was triggered, Bax expression and mitochondrial membrane potential were evaluated finding a higher protein expression in control sample and a good integrity of mitochondrial membrane in both experimental points. Type I collagen secretion and alkaline phosphatase (ALP) activity appear increased in ZA-treated sample, osteocalcin expression level is reduced in ZA-treated cells, whereas no modifications of gp38 mRNA level are evidenced. No statistical differences are identified in PGE2 secretion level whereas IL-6 secretion is lower in ZA-treated HOs with respect to control ones. These results highlight that ZA, delaying the osteoblastic differentiation process versus the osteocytic lineage, strengthens its pharmacological activity enhancing bone density. The knowledge of ZA effects on osteoblasts at subtoxic dose allows to improve therapeutic protocols in order to strengthen drug pharmacological activity through a combined action on both osteoclastic and osteoblastic cells.

  13. UV-killed Staphylococcus aureus enhances adhesion and differentiation of osteoblasts on bone-associated biomaterials.

    PubMed

    Somayaji, Shankari N; Huet, Yvette M; Gruber, Helen E; Hudson, Michael C

    2010-11-01

    Titanium alloys (Ti) are the preferred material for orthopedic applications. However, very often, these metallic implants loosen over a long period and mandate revision surgery. For implant success, osteoblasts must adhere to the implant surface and deposit a mineralized extracellular matrix (ECM). Here, we utilized UV-killed Staphylococcus aureus as a novel osteoconductive coating for Ti surfaces. S. aureus expresses surface adhesins capable of binding to bone and biomaterials directly. Furthermore, interaction of S. aureus with osteoblasts activates growth factor-related pathways that potentiate osteogenesis. Although UV-killed S. aureus cells retain their bone-adhesive ability, they do not stimulate significant immune modulator expression. All of the abovementioned properties were utilized for a novel implant coating so as to promote osteoblast recruitment and subsequent cell functions on the bone-implant interface. In this study, osteoblast adhesion, proliferation, and mineralized ECM synthesis were measured on Ti surfaces coated with fibronectin with and without UV-killed bacteria. Osteoblast adhesion was enhanced on Ti alloy surfaces coated with bacteria compared to uncoated surfaces, while cell proliferation was sustained comparably on both surfaces. Osteoblast markers such as collagen, osteocalcin, alkaline phosphatase activity, and mineralized nodule formation were increased on Ti alloy coated with bacteria compared to uncoated surfaces.

  14. The antiepileptic medications carbamazepine and phenytoin inhibit native sodium currents in murine osteoblasts.

    PubMed

    Petty, Sandra J; Milligan, Carol J; Todaro, Marian; Richards, Kay L; Kularathna, Pamuditha K; Pagel, Charles N; French, Chris R; Hill-Yardin, Elisa L; O'Brien, Terence J; Wark, John D; Mackie, Eleanor J; Petrou, Steven

    2016-09-01

    Fracture risk is a serious comorbidity in epilepsy and may relate to the use of antiepileptic drugs (AEDs). Many AEDs inhibit ion channel function, and the expression of these channels in osteoblasts raises the question of whether altered bone signaling increases bone fragility. We aimed to confirm the expression of voltage-gated sodium (NaV ) channels in mouse osteoblasts, and to investigate the action of carbamazepine and phenytoin on NaV channels. Immunocytochemistry was performed on primary calvarial osteoblasts extracted from neonatal C57BL/6J mice and additional RNA sequencing (RNASeq) was included to confirm expression of NaV . Whole-cell patch-clamp recordings were made to identify the native currents expressed and to assess the actions of carbamazepine (50 μm) or phenytoin (50 μm). NaV expression was demonstrated with immunocytochemistry, RNA sequencing, and functionally, with demonstration of robust tetrodotoxin-sensitive and voltage-activated inward currents. Application of carbamazepine or phenytoin resulted in significant inhibition of current amplitude for carbamazepine (31.6 ± 5.9%, n = 9; p < 0.001), and for phenytoin (35.5 ± 6.9%, n = 7; p < 0.001). Mouse osteoblasts express NaV , and native NaV currents are blocked by carbamazepine and phenytoin, supporting our hypothesis that AEDs can directly influence osteoblast function and potentially affect bone strength. Wiley Periodicals, Inc. © 2016 International League Against Epilepsy.

  15. MicroRNA miR-23a cluster promotes osteocyte differentiation by regulating TGF-β signalling in osteoblasts

    PubMed Central

    Zeng, Huan-Chang; Bae, Yangjin; Dawson, Brian C.; Chen, Yuqing; Bertin, Terry; Munivez, Elda; Campeau, Philippe M.; Tao, Jianning; Chen, Rui; Lee, Brendan H.

    2017-01-01

    Osteocytes are the terminally differentiated cell type of the osteoblastic lineage and have important functions in skeletal homeostasis. Although the transcriptional regulation of osteoblast differentiation has been well characterized, the factors that regulate differentiation of osteocytes from mature osteoblasts are poorly understood. Here we show that miR-23a∼27a∼24-2 (miR-23a cluster) promotes osteocyte differentiation. Osteoblast-specific miR-23a cluster gain-of-function mice have low bone mass associated with decreased osteoblast but increased osteocyte numbers. By contrast, loss-of-function transgenic mice overexpressing microRNA decoys for either miR-23a or miR-27a, but not miR24-2, show decreased osteocyte numbers. Moreover, RNA-sequencing analysis shows altered transforming growth factor-β (TGF-β) signalling. Prdm16, a negative regulator of the TGF-β pathway, is directly repressed by miR-27a with concomitant alteration of sclerostin expression, and pharmacological inhibition of TGF-β rescues the phenotypes observed in the gain-of-function transgenic mice. Taken together, the miR-23a cluster regulates osteocyte differentiation by modulating the TGF-β signalling pathway through targeting of Prdm16. PMID:28397831

  16. Histone deacetylase inhibitors reduce differentiating osteoblast-mediated protection of acute myeloid leukemia cells from cytarabine

    PubMed Central

    Sterner, Rosalie M.; Kremer, Kimberly N.; Al-Kali, Aref; Patnaik, Mrinal M.; Gangat, Naseema; Litzow, Mark R.; Kaufmann, Scott H.; Westendorf, Jennifer J.; van Wijnen, Andre J.; Hedin, Karen E.

    2017-01-01

    The bone marrow microenvironment protects acute myeloid leukemia (AML) cells during chemotherapy and is a major factor in relapse. Here, we examined which type(s) of bone marrow cells are responsible for the relapse of AML following treatment with cytarabine (Ara-C), and we identified a means to inhibit this protection. To determine the protective cell type(s), AML cells were treated with Ara-C, and AML cell survival in the presence or absence of osteoblast lineage cells was assessed. Cultured AML cells and patient bone marrow isolates were each significantly protected from Ara-C-induced apoptosis by co-culture with differentiating osteoblasts. Moreover, pretreating differentiating osteoblasts with the histone deacetylase inhibitors (HDACi) vorinostat and panobinostat abrogated the ability of the differentiating osteoblasts to protect AML cells. Together, our results indicate that differentiating osteoblasts have the potential to promote residual AML in the bone marrow following standard chemotherapy and act via a mechanism requiring HDACi-sensitive gene expression. Using HDACi to target the leukemic microenvironment in combination with Ara-C could potentially improve treatment of AML. Moreover, other strategies for manipulating bone marrow osteoblasts may also help eradicate AML cells and reduce relapse. PMID:29212250

  17. 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

  18. 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.

  19. 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.

  20. Subcloning of three osteoblastic cell lines with distinct differentiation phenotypes from the mouse osteoblastic cell line KS-4.

    PubMed

    Yamashita, T; Ishii, H; Shimoda, K; Sampath, T K; Katagiri, T; Wada, M; Osawa, T; Suda, T

    1996-11-01

    Three distinct osteoblastic cell lines (KS418, KS460, and KS483) were subcloned from the mouse osteoblastic KS-4 cells, which possessed the abilities not only to differentiate into mature osteoblasts, but also to support osteoclast differentiation in coculture with spleen cells. The order of the magnitude of the basal alkaline phosphatase (ALP) activity was KS483 > KS418 > KS460. KS483 cells were also more differentiated than KS418 and KS460 in terms of ALP activity and osteocalcin production, when cultured in growth medium containing 10% fetal bovine serum. In long-term culture, KS418 and KS483 apparently differentiated into mature osteoblasts and formed calcified nodules without addition of beta-glycerophosphate. Electron microscopic analysis demonstrated that calcification occurring in the nodules was initiated in the matrix vesicles as observed in bone formation in vivo. Nodule formation and mineral deposition occurred simultaneously in the presence of beta-glycerophosphate, but the former always preceded the latter without addition of beta-glycerophosphate. In contrast, KS460 cells did not show time-dependent increases of ALP activity, type I collagen expression and osteocalcin production, which were induced by treatment with recombinant osteogenic protein-1 (OP-1). The three cell lines similarly supported osteoclast differentiation in coculture with spleen cells in response to 1,25-dihydroxyvitamin D3. These results indicate that the three cell lines subcloned from the original KS-4 cells represent phenotypically distinct osteoblasts during osteoblast differentiation, but are equipped similarly with the capacity to support osteoclast differentiation. The subcloned cells of the KS-4 series may provide useful systems in which to study osteoblast differentiation and function.

  1. Additively Manufactured 3D Porous Ti-6Al-4V Constructs Mimic Trabecular Bone Structure and Regulate Osteoblast Proliferation, Differentiation and Local Factor Production in a Porosity and Surface Roughness Dependent Manner

    PubMed Central

    Cheng, Alice; Humayun, Aiza; Cohen, David J.; Boyan, Barbara D.; Schwartz, Zvi

    2014-01-01

    Additive manufacturing by laser sintering is able to produce high resolution metal constructs for orthopaedic and dental implants. In this study, we used a human trabecular bone template to design and manufacture Ti-6Al-4V constructs with varying porosity via laser sintering. Characterization of constructs revealed interconnected porosities ranging from 15–70% with compressive moduli of 2063–2954 MPa. These constructs with macro porosity were further surface-treated to create a desirable multi-scale micro-/nano-roughness, which has been shown to enhance the osseointegration process. Osteoblasts (MG63 cells) exhibited high viability when grown on the constructs. Proliferation (DNA) and alkaline phosphatase specific activity (ALP), an early differentiation marker, decreased as porosity increased, while osteocalcin (OCN), a late differentiation marker, as well as osteoprotegerin (OPG), vascular endothelial growth factor (VEGF) and bone morphogenetic proteins 2 and 4 (BMP2, BMP4) increased with increasing porosity. 3D constructs with the highest porosity and surface modification supported the greatest osteoblast differentiation and local factor production. These results indicate that additively manufactured 3D porous constructs mimicking human trabecular bone and produced with additional surface treatment can be customized for increased osteoblast response. Increased factors for osteoblast maturation and differentiation on high porosity constructs suggest the enhanced performance of these surfaces for increasing osseointegration in vivo. PMID:25287305

  2. Forskolin Regulates L-Type Calcium Channel through Interaction between Actinin 4 and β3 Subunit in Osteoblasts.

    PubMed

    Zhang, Xuemei; Li, Fangping; 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.

  3. 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

  4. Skeletal (stromal) stem cells: an update on intracellular signaling pathways controlling osteoblast differentiation.

    PubMed

    Abdallah, Basem M; Jafari, Abbas; Zaher, Walid; Qiu, Weimin; Kassem, Moustapha

    2015-01-01

    Skeletal (marrow stromal) stem cells (BMSCs) are a group of multipotent cells that reside in the bone marrow stroma and can differentiate into osteoblasts, chondrocytes and adipocytes. Studying signaling pathways that regulate BMSC differentiation into osteoblastic cells is a strategy for identifying druggable targets for enhancing bone formation. This review will discuss the functions and the molecular mechanisms of action on osteoblast differentiation and bone formation; of a number of recently identified regulatory molecules: the non-canonical Notch signaling molecule Delta-like 1/preadipocyte factor 1 (Dlk1/Pref-1), the Wnt co-receptor Lrp5 and intracellular kinases. This article is part of a Special Issue entitled: Stem Cells and Bone. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. Reduced osteoblast activity in the mice lacking TR4 nuclear receptor leads to osteoporosis.

    PubMed

    Lin, Shin-Jen; Ho, Hsin-Chiu; Lee, Yi-Fen; Liu, Ning-Chun; Liu, Su; Li, Gonghui; Shyr, Chih-Rong; Chang, Chawnshang

    2012-06-07

    Early studies suggested that TR4 nuclear receptor might play important roles in the skeletal development, yet its detailed mechanism remains unclear. We generated TR4 knockout mice and compared skeletal development with their wild type littermates. Primary bone marrow cells were cultured and we assayed bone differentiation by alkaline phosphatase and alizarin red staining. Primary calvaria were cultured and osteoblastic marker genes were detected by quantitative PCR. Luciferase reporter assays, chromatin immunoprecipitation (ChIP) assays, and electrophoretic mobility shift assays (EMSA) were performed to demonstrate TR4 can directly regulate bone differentiation marker osteocalcin. We first found mice lacking TR4 might develop osteoporosis. We then found that osteoblast progenitor cells isolated from bone marrow of TR4 knockout mice displayed reduced osteoblast differentiation capacity and calcification. Osteoblast primary cultures from TR4 knockout mice calvaria also showed higher proliferation rates indicating lower osteoblast differentiation ability in mice after loss of TR4. Mechanism dissection found the expression of osteoblast markers genes, such as ALP, type I collagen alpha 1, osteocalcin, PTH, and PTHR was dramatically reduced in osteoblasts from TR4 knockout mice as compared to those from TR4 wild type mice. In vitro cell line studies with luciferase reporter assay, ChIP assay, and EMSA further demonstrated TR4 could bind directly to the promoter region of osteocalcin gene and induce its gene expression at the transcriptional level in a dose dependent manner. Together, these results demonstrate TR4 may function as a novel transcriptional factor to play pathophysiological roles in maintaining normal osteoblast activity during the bone development and remodeling, and disruption of TR4 function may result in multiple skeletal abnormalities.

  6. 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

  7. ATF4 mediation of NF1 functions in osteoblast reveals a nutritional basis for congenital skeletal dysplasiae.

    PubMed

    Elefteriou, Florent; Benson, M Douglas; Sowa, Hideaki; Starbuck, Michael; Liu, Xiuyun; Ron, David; Parada, Luis F; Karsenty, Gerard

    2006-12-01

    The transcription factor ATF4 enhances bone formation by favoring amino acid import and collagen synthesis in osteoblasts, a function requiring its phosphorylation by RSK2, the kinase inactivated in Coffin-Lowry Syndrome. Here, we show that in contrast, RSK2 activity, ATF4-dependent collagen synthesis, and bone formation are increased in mice lacking neurofibromin in osteoblasts (Nf1(ob)(-/-) mice). Independently of RSK2, ATF4 phosphorylation by PKA is enhanced in Nf1(ob)(-/-) mice, thereby increasing Rankl expression, osteoclast differentiation, and bone resorption. In agreement with ATF4 function in amino acid transport, a low-protein diet decreased bone protein synthesis and normalized bone formation and bone mass in Nf1(ob)(-/-) mice without affecting other organ weight, while a high-protein diet overcame Atf4(-/-) and Rsk2(-/-) mice developmental defects, perinatal lethality, and low bone mass. By showing that ATF4-dependent skeletal dysplasiae are treatable by dietary manipulations, this study reveals a molecular connection between nutrition and skeletal development.

  8. Microarray and Proteomic Analysis of Breast Cancer Cell and Osteoblast Co-cultures

    PubMed Central

    Morrison, Charlotte; Mancini, Stephanie; Cipollone, Jane; Kappelhoff, Reinhild; Roskelley, Calvin; Overall, Christopher

    2011-01-01

    Dynamic reciprocal interactions between a tumor and its microenvironment impact both the establishment and progression of metastases. These interactions are mediated, in part, through proteolytic sculpting of the microenvironment, particularly by the matrix metalloproteinases, with both tumors and stroma contributing to the proteolytic milieu. Because bone is one of the predominant sites of breast cancer metastases, we used a co-culture system in which a subpopulation of the highly invasive human breast cancer cell line MDA-MB-231, with increased propensity to metastasize to bone, was overlaid onto a monolayer of differentiated osteoblast MC3T3-E1 cells in a mineralized osteoid matrix. CLIP-CHIP® microarrays identified changes in the complete protease and inhibitor expression profile of the breast cancer and osteoblast cells that were induced upon co-culture. A large increase in osteoblast-derived MMP-13 mRNA and protein was observed. Affymetrix analysis and validation showed induction of MMP-13 was initiated by soluble factors produced by the breast tumor cells, including oncostatin M and the acute response apolipoprotein SAA3. Significant changes in the osteoblast secretomes upon addition of MMP-13 were identified by degradomics from which six novel MMP-13 substrates with the potential to functionally impact breast cancer metastasis to bone were identified and validated. These included inactivation of the chemokines CCL2 and CCL7, activation of platelet-derived growth factor-C, and cleavage of SAA3, osteoprotegerin, CutA, and antithrombin III. Hence, the influence of breast cancer metastases on the bone microenvironment that is executed via the induction of osteoblast MMP-13 with the potential to enhance metastases growth by generating a microenvironmental amplifying feedback loop is revealed. PMID:21784845

  9. Tridax procumbens flavonoids promote osteoblast differentiation and bone formation.

    PubMed

    Al Mamun, Md Abdullah; Hosen, Mohammad Jakir; Islam, Kamrul; Khatun, Amina; Alam, M Masihul; Al-Bari, Md Abdul Alim

    2015-11-18

    Tridax procumbens flavonoids (TPFs) are well known for their medicinal properties among local natives. Besides traditionally used for dropsy, anemia, arthritis, gout, asthma, ulcer, piles, and urinary problems, it is also used in treating gastric problems, body pain, and rheumatic pains of joints. TPFs have been reported to increase osteogenic functioning in mesenchymal stem cells. Our previous study showed that TPFs were significantly suppressed the RANKL-induced differentiation of osteoclasts and bone resorption. However, the effects of TPFs to promote osteoblasts differentiation and bone formation remain unclear. TPFs were isolated from Tridax procumbens and investigated for their effects on osteoblasts differentiation and bone formation by using primary mouse calvarial osteoblasts. TPFs promoted osteoblast differentiation in a dose-dependent manner demonstrated by up-regulation of alkaline phosphatase and osteocalcin. TPFs also upregulated osteoblast differentiation related genes, including osteocalcin, osterix, and Runx2 in primary osteoblasts. TPFs treated primary osteoblast cells showed significant upregulation of bone morphogenetic proteins (BMPs) including Bmp-2, Bmp-4, and Bmp-7. Addition of noggin, a BMP specific-antagonist, inhibited TPFs induced upregulation of the osteocalcin, osterix, and Runx2. Our findings point towards the induction of osteoblast differentiation by TPFs and suggested that TPFs could be a potential anabolic agent to treat patients with bone loss-associated diseases such as osteoporosis.

  10. Osteoblastic differentiation of human stem cells derived from bone marrow and periodontal ligament under the effect of enamel matrix derivative and transforming growth factor-beta.

    PubMed

    Houshmand, Behzad; Behnia, Hossein; Khoshzaban, Ahad; Morad, Golnaz; Behrouzi, Gholamreza; Dashti, Seyedeh Ghazaleh; Khojasteh, Arash

    2013-01-01

    To increase the understanding of the applicability of biomaterials and growth factors in enhancing stem cell-based bone regeneration modalities, this study evaluated the effects of enamel matrix derivative (EMD) and recombinant human transforming growth factor-beta (rhTGF-β) on osteoblastic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) as well as human periodontal ligament stem cells (hPDLSCs). hBMSCs and hPDLSCs were obtained, and identification of stem cell surface markers was performed according to the criteria of the International Society for Cellular Therapy. Each group of stem cells was separately treated with a serial dilution of EMD (10, 50, and 100 μg/mL) or rhTGF-β (10 ng/mL). Osteoblastic differentiation was examined through in vitro matrix mineralization by alizarin red staining, and mRNA expression of osteopontin and osteonectin was determined by quantitative reverse-transcriptase polymerase chain reaction. hPDLSCs were further assessed for osteocalcin mRNA expression. Stem cells cultured in osteogenic medium were employed as a standard positive control group. In none of the experimental groups were bone-related mRNAs detected subsequent to treatment with EMD for 5, 10, and 15 days. Alizarin red staining on day 21 was negative in EMD-treated BMSC and PDLSC cultures. In rhTGF-β-supplemented BMSC culture, expression of osteonectin mRNA was demonstrated on day 15, which was statistically comparable to the positive control group. Nevertheless, extracellular matrix mineralization was inhibited in both groups of stem cells. Within the limitations of this study, it could be concluded that EMD with a concentration of 10, 50, or 100 μg/mL has no appreciable effect on osteoblastic differentiation of BMSCs and PDLSCs. Application of rhTGF-β increased osteonectin mRNA expression in BMSCs. This finding corroborates the hypothesis that TGF-β might be involved in early osteoblastic maturation.

  11. Directing the osteoblastic and chondrocytic differentiations of mesenchymal stem cells: matrix vs. induction media

    PubMed Central

    He, Jing; Guo, Jianglong; Jiang, Bo; Yao, Ruijuan; Wu, Yao

    2017-01-01

    Abstract While both induction culture media and matrix have been reported to regulate the stem cell fate, little is known about which factor plays a more decisive role in directing the MSC differentiation lineage as well as the underlying mechanisms. To this aim, we seeded MSCs on HA-collagen and HA-synthetic hydrogel matrixes, which had demonstrated highly different potentials toward osteoblastic and chondrocytic differentiation lineages, respectively, and cultured them with osteogenic, chondrogenic and normal culture media, respectively. A systematic comparison has been carried out on the effects of induction media and matrix on MSC adhesion, cytoskeleton organization, proliferation, and in particular differentiation into the osteoblastic and chondrocytic lineages. The results demonstrated that the matrix selection had a much more profound effect on directing the differentiation lineage than the induction media did. The strong modulation effect on the transcription activities might be the critical factor contributing to the above observations in our study, where canonical Wnt-β-Catenin signal pathway was directly involved in the matrix-driven osteoblastic differentiation. Such findings not only provide a critical insight on natural cellular events leading to the osteoblastic and chondrocytic differentiations, but also have important implications in biomaterial design for tissue engineering applications. PMID:29026640

  12. 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

  13. Effects of extracellular magnesium extract on the proliferation and differentiation of human osteoblasts and osteoclasts in coculture.

    PubMed

    Wu, Lili; Feyerabend, Frank; Schilling, Arndt F; Willumeit-Römer, Regine; Luthringer, Bérengère J C

    2015-11-01

    Coculture of osteoblasts and osteoclasts is a subject of interest in the understanding of how magnesium (Mg)-based implants influence the bone metabolism and remodeling upon degradation. Human telomerase reverse transcriptase (hTERT) transduced mesenchymal stem cells (SCP-1) were first differentiated into osteoblasts with osteogenic supplements and then further cocultured with peripheral blood mononucleated cells (PBMC) without the addition of osteoclastogenesis promoting factors. Concomitantly, the cultures were exposed to variable Mg extract dilutions (0, 30×, 10×, 5×, 3×, 2× and 1×). Phenotype characterization documented that while 2× dilution of Mg extract was extremely toxic to osteoclast monoculture, monocytes in coculture with osteoblasts exhibited a greater tolerance to higher Mg extract concentration. The dense growth of osteoblasts in cultures with 1× dilution of Mg extract suggested that high concentration of Mg extract promoted osteoblast proliferation/differentiation behavior. The results of intracellular alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) activities as well as protein and gene expressions of receptor activator of nuclear factor kappa-B ligand (RANKL), macrophage colony-stimulating factor (M-CSF), and osteoclast-associated receptor (OSCAR) revealed significantly enhanced formation of osteoblasts whereas decreased osteoclastogenesis in the cultures with high concentrations of Mg extract (2× and 1× dilutions). In conclusion, while an increased osteoinductivity has been demonstrated, the impact of potentially decreased osteoclastogenesis around the Mg-based implants should be also taken into account. Cocultures containing both bone-forming osteoblasts and bone-resorbing osteoclasts should be preferentially performed for in vitro cytocompatibility assessment of Mg-based implants as they more closely mimic the in vivo environment. An attractive human osteoblasts and osteoclasts cocultivation regime was

  14. Porous hydroxyapatite and biphasic calcium phosphate ceramics promote ectopic osteoblast differentiation from mesenchymal stem cells

    NASA Astrophysics Data System (ADS)

    Zhang, Lingli; Hanagata, Nobutaka; Maeda, Megumi; Minowa, Takashi; Ikoma, Toshiyuki; Fan, Hongsong; Zhang, Xingdong

    2009-04-01

    Because calcium phosphate (Ca-P) ceramics have been used as bone substitutes, it is necessary to investigate what effects the ceramics have on osteoblast maturation. We prepared three types of Ca-P ceramics with different Ca-P ratios, i.e. hydroxyapatite (HA), beta-tricalcium phosphate (β-TCP), and biphasic calcium phosphate (BCP) ceramics with dense-smooth and porous structures. Comprehensive gene expression microarray analysis of mouse osteoblast-like cells cultured on these ceramics revealed that porous Ca-P ceramics considerably affected the gene expression profiles, having a higher potential for osteoblast maturation. In the in vivo study that followed, porous Ca-P ceramics were implanted into rat skeletal muscle. Sixteen weeks after the implantation, more alkaline-phosphatase-positive cells were observed in the pores of hydroxyapatite and BCP, and the expression of the osteocalcin gene (an osteoblast-specific marker) in tissue grown in pores was also higher in hydroxyapatite and BCP than in β-TCP. In the pores of any Ca-P ceramics, 16 weeks after the implantation, we detected the expressions of marker genes of the early differentiation stage of chondrocytes and the complete differentiation stage of adipocytes, which originate from mesenchymal stem cells, as well as osteoblasts. These marker gene expressions were not observed in the muscle tissue surrounding the implanted Ca-P ceramics. These observations indicate that porous hydroxyapatite and BCP had a greater potential for promoting the differentiation of mesenchymal stem cells into osteoblasts than β-TCP.

  15. Assessing the osteoblast transcriptome in a model of enhanced bone formation due to constitutive Gs-G protein signaling in osteoblasts

    PubMed Central

    Wattanachanya, Lalita; Wang, Liping; Millard, Susan M.; Lu, Wei-Dar; O’Carroll, Dylan; Hsiao, Edward C.; Conklin, Bruce R.; Nissenson, Robert A.

    2015-01-01

    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 Gs-coupled GPCR, under the control of the 2.3-kb Col I promoter. These mice showed a dramatic age-dependent increase in trabecular bone of femurs. Here, we further evaluated the effects of enhanced Gs signaling in OBs on intramembranous bone formation by examining calvariae of 1- and 9-week-old Col1(2.3)/Rs1 mice and characterized the in vivo gene expression specifically occurring in osteoblasts with activated Gs G protein–coupled receptor signaling, at the cellular level rather than in a whole bone. Rs1 calvariae displayed a dramatic increase in bone volume with partial loss of cortical structure. By immunohistochemistry, Osterix was detected in cells throughout the inter-trabecular space while Osteocalcin was expressed predominantly in cells along bone surfaces, suggesting the role of paracrine mediators secreted from OBs driven by 2.3-kb Col I promoter could influence early OB commitment, differentiation, and/or proliferation. Gene expression analysis of calvarial OBs revealed that genes affected by Rs1 signaling include those encoding proteins important for cell differentiation, cytokines and growth factors, angiogenesis, coagulation, and energy metabolism. The set of Gs-GPCRs and other GPCRs that may contribute to the observed skeletal phenotype and candidate paracrine mediators of the effect of Gs signaling in OBs were also determined. Our results identify novel detailed in vivo cellular changes of the anabolic response of the skeleton to Gs signaling in mature OBs. PMID:25704759

  16. Alendronate and raloxifene affect the osteoprotegerin/RANKL system in human osteoblast primary cultures from patients with osteoporosis and osteoarthritis.

    PubMed

    Giner, Mercè; Rios, Ma José; Montoya, Ma José; Vázquez, Ma Angeles; Miranda, Cristina; Pérez-Cano, Ramón

    2011-01-15

    The osteoprotegerin/RANKL system modulates bone remodelling. Alendronate and raloxifene are anti-resorptive drugs effective in osteoporotic disease. They reduce fracture risk, the activity of bone remodelling and increase bone mineral density. It is not known if they can exert a direct effect in osteoblasts via the osteoprotegerin/RANKL system. Our objective was to assess the effects of alendronate and raloxifene among osteoprotegerin production (ELISA), as well as osteoprotegerin and RANKL expression (RT-PCR), in primary cultures of human osteoblasts (hOB). We compared 17 osteoporotic patients with 16 patients affected by osteoarthritis in basal conditions and after incubation with alendronate (10(-6) M), raloxifene (10(-7) M) or 17-β estradiol (10(-7) M) for 24 h. The statistical analysis was determined by ANOVA. Osteoprotegerin protein secretion in hOB cultures was higher in patients with osteoporosis than osteoarthritis. Osteoprotegerin secretion levels remained unchanged after each treatment. The osteoporotic group was more sensitive to treatment. Both raloxifene (34%) and estradiol (37%) increased osteoprotegerin mRNA expression, and alendronate (118%) and raloxifene (61%) increased the mRNA expression of RANKL. The RANKL/osteoprotegerin mRNA ratio was higher in osteoporotic than osteoarthritic patients. In the osteoporotic group, the RANKL/osteoprotegerin mRNA ratio was significantly increased after treatment with alendronate (112%) and after treatment with raloxifene (60%). These results indicate a direct action of alendronate and raloxifene on hOB cultures from osteoporotic patients, and the cited drugs are able to modulate the osteoprotegerin/RANKL system. Copyright © 2010 Elsevier B.V. All rights reserved.

  17. Bone Talk: Activated Osteoblasts Promote Lung Cancer Growth.

    PubMed

    Bružas, Emilis; Egeblad, Mikala

    2018-03-01

    Cancer cells can directly stimulate the generation and recruitment of tumor-supportive bone marrow-derived cells (BMDCs), including neutrophils, via secreted factors. A new study demonstrates that lung tumors also remotely activate bone-residing osteoblasts, which in turn promote neutrophil production. This is a multistep mechanism of establishing a supportive tumor microenvironment. Copyright © 2018 Elsevier Ltd. All rights reserved.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kimira, Yoshifumi, E-mail: kimira@josai.ac.jp; Ogura, Kana; Taniuchi, Yuri

    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 indicatemore » 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.« less

  19. Hydraulic Pressure during Fluid Flow Regulates Purinergic Signaling and Cytoskeleton Organization of Osteoblasts.

    PubMed

    Gardinier, Joseph D; Gangadharan, Vimal; Wang, Liyun; Duncan, Randall L

    2014-06-01

    During physiological activities, osteoblasts experience a variety of mechanical forces that stimulate anabolic responses at the cellular level necessary for the formation of new bone. Previous studies have primarily investigated the osteoblastic response to individual forms of mechanical stimuli. However in this study, we evaluated the response of osteoblasts to two simultaneous, but independently controlled stimuli; fluid flow-induced shear stress (FSS) and static or cyclic hydrostatic pressure (SHP or CHP, respectively). MC3T3-E1 osteoblasts-like cells were subjected to 12dyn/cm 2 FSS along with SHP or CHP of varying magnitudes to determine if pressure enhances the anabolic response of osteoblasts during FSS. For both SHP and CHP, the magnitude of hydraulic pressure that induced the greatest release of ATP during FSS was 15 mmHg. Increasing the hydraulic pressure to 50 mmHg or 100 mmHg during FSS attenuated the ATP release compared to 15 mmHg during FSS. Decreasing the magnitude of pressure during FSS to atmospheric pressure reduced ATP release to that of basal ATP release from static cells and inhibited actin reorganization into stress fibers that normally occurred during FSS with 15 mmHg of pressure. In contrast, translocation of nuclear factor kappa B (NFκB) to the nucleus was independent of the magnitude of hydraulic pressure and was found to be mediated through the activation of phospholipase-C (PLC), but not src kinase. In conclusion, hydraulic pressure during FSS was found to regulate purinergic signaling and actin cytoskeleton reorganization in the osteoblasts in a biphasic manner, while FSS alone appeared to stimulate NFκB translocation. Understanding the effects of hydraulic pressure on the anabolic responses of osteoblasts during FSS may provide much needed insights into the physiologic effects of coupled mechanical stimuli on osteogenesis.

  20. Hydraulic Pressure during Fluid Flow Regulates Purinergic Signaling and Cytoskeleton Organization of Osteoblasts

    PubMed Central

    Gardinier, Joseph D.; Gangadharan, Vimal; Wang, Liyun; Duncan, Randall L.

    2014-01-01

    During physiological activities, osteoblasts experience a variety of mechanical forces that stimulate anabolic responses at the cellular level necessary for the formation of new bone. Previous studies have primarily investigated the osteoblastic response to individual forms of mechanical stimuli. However in this study, we evaluated the response of osteoblasts to two simultaneous, but independently controlled stimuli; fluid flow-induced shear stress (FSS) and static or cyclic hydrostatic pressure (SHP or CHP, respectively). MC3T3-E1 osteoblasts-like cells were subjected to 12dyn/cm2 FSS along with SHP or CHP of varying magnitudes to determine if pressure enhances the anabolic response of osteoblasts during FSS. For both SHP and CHP, the magnitude of hydraulic pressure that induced the greatest release of ATP during FSS was 15 mmHg. Increasing the hydraulic pressure to 50 mmHg or 100 mmHg during FSS attenuated the ATP release compared to 15 mmHg during FSS. Decreasing the magnitude of pressure during FSS to atmospheric pressure reduced ATP release to that of basal ATP release from static cells and inhibited actin reorganization into stress fibers that normally occurred during FSS with 15 mmHg of pressure. In contrast, translocation of nuclear factor kappa B (NFκB) to the nucleus was independent of the magnitude of hydraulic pressure and was found to be mediated through the activation of phospholipase-C (PLC), but not src kinase. In conclusion, hydraulic pressure during FSS was found to regulate purinergic signaling and actin cytoskeleton reorganization in the osteoblasts in a biphasic manner, while FSS alone appeared to stimulate NFκB translocation. Understanding the effects of hydraulic pressure on the anabolic responses of osteoblasts during FSS may provide much needed insights into the physiologic effects of coupled mechanical stimuli on osteogenesis. PMID:24910719

  1. Regulation of Extracellular Matrix Remodeling Proteins by Osteoblasts in Titanium Nanoparticle-Induced Aseptic Loosening Model.

    PubMed

    Xie, Jing; Hou, Yanhua; Fu, Na; Cai, Xiaoxiao; Li, Guo; Peng, Qiang; Lin, Yunfeng

    2015-10-01

    Titanium (Ti)-wear particles, formed at the bone-implant interface, are responsible for aseptic loosening, which is a main cause of total joint replacement failure. There have been many studies on Ti particle-induced function changes in mono-cultured osteoblasts and synovial cells. However, little is known on extracellular matrix remodeling displayed by osteoblasts when in coexistence with Synovial cells. To further mimic the bone-implant interface environment, we firstly established a nanoscaled-Ti particle-induced aseptic loosening system by co-culturing osteoblasts and Synovial cells. We then explored the impact of the Synovial cells on Ti particle-engulfed osteoblasts in the mimicked flamed niche. The matrix metalloproteinases and lysyl oxidases expression levels, two protein families which are critical in osseointegration, were examined under induction by tumor necrosis factor-alpha. It was found that the co-culture between the osteoblasts and Synovial cells markedly increased the migration and proliferation of the osteoblasts, even in the Ti-particle engulfed osteoblasts. Importantly, the Ti-particle engulfed osteoblasts, induced by TNF-alpha after the co-culture, enhanced the release of the matrix metalloproteinases and reduced the expressions of lysyl oxidases. The regulation of extracellular matrix remodeling at the protein level was further assessed by investigations on gene expression of the matrix metalloproteinases and lysyl oxidases, which also suggested that the regulation started at the genetic level. Our research work has therefore revealed the critical role of multi cell-type interactions in the extracellular matrix remodeling within the peri-prosthetic tissues, which provides new insights on aseptic loosening and brings new clues about incomplete osseointegration between the implantation materials and their surrounding bones.

  2. Value of Osteoblast-Derived Exosomes in Bone Diseases.

    PubMed

    Ge, Min; Wu, Yingzhi; Ke, Ronghu; Cai, Tianyi; Yang, Junyi; Mu, Xiongzheng

    2017-06-01

    The authors' purpose is to reveal the value of osteoblast-derived exosomes in bone diseases. Microvesicles from supernatants of mouse Mc3t3 were isolated by ultracentrifugation and then the authors presented the protein profile by proteomics analysis. The authors detected a total number of 1536 proteins by mass spectrometry and found 172 proteins overlap with bone database. The Ingenuity Pathway Analysis shows network of "Skeletal and Muscular System Development and Function, Developmental Disorder, Hereditary Disorder" and pathway about osteogenesis. EFNB1 and transforming growth factor beta receptor 3 in the network, LRP6, bone morphogenetic protein receptor type-1, and SMURF1 in the pathway seemed to be valuable in the exosome research of related bone disease. The authors' study unveiled the content of osteoblast-derived exosome and discussed valuable protein in it which might provide novel prospective in bone diseases research.

  3. Bradykinin regulates osteoblast differentiation by Akt/ERK/NFκB signaling axis.

    PubMed

    Srivastava, Swati; Sharma, Kirti; Kumar, Narender; Roy, Partha

    2014-12-01

    Bradykinin (BK), a well known mediator of pain and inflammation, is also known to be involved in the process of bone resorption. The present study therefore evaluated the role of BK in osteoblast lineage commitment. Our data showed that BK inhibits the migration of bone marrow mesenchymal stem cells, but does not affect their viability. Moreover, BK also inhibits osteoblastic differentiation by significantly downregulating the levels of mRNAs for osteopontin, runX2, col24, osterix, osteocalcin genes and bone mineralization (P < 0.05). Further, BK was found to elicit the BK receptors (BDKR1 and BDKR2) mediated activation of ERK1/2 and Akt pathways, which finally led to the activation of NFκB. BK also promoted the osteoclast differentiation of bone marrow derived preosteoclast cells by upregulating the expression of c-fos, NFATC1, TRAP, clcn7, cathK, and OSCAR genes and increasing TRAP activity through NFκB pathway. In conclusion, our data suggest that BK decreases the differentiation of osteoblasts with concomitant increase in osteoclast formation and thus provides new insight into the mechanism of action of BK in modulating bone resorption. © 2014 Wiley Periodicals, Inc.

  4. Glucocorticoid suppresses the canonical Wnt signal in cultured human osteoblasts

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ohnaka, Keizo; Tanabe, Mizuho; Kawate, Hisaya

    2005-04-01

    To explore the mechanism of glucocorticoid-induced osteoporosis, we investigated the effect of glucocorticoid on canonical Wnt signaling that emerged as a novel key pathway for promoting bone formation. Wnt3a increased the T-cell factor (Tcf)/lymphoid enhancer factor (Lef)-dependent transcriptional activity in primary cultured human osteoblasts. Dexamethasone suppressed this transcriptional activity in a dose-dependent manner, while 1,25-dihydroxyvitamin D3 increased this transcriptional activity. LiCl, an inhibitor of glycogen synthase kinase-3{beta}, also enhanced the Tcf/Lef-dependent transcriptional activity, which was, however, not inhibited by dexamethasone. The addition of anti-dickkopf-1 antibody partially restored the transcriptional activity suppressed by dexamethasone. Dexamethasone decreased the cytosolic amount of {beta}-cateninmore » accumulated by Wnt3a and also inhibited the nuclear translocation of {beta}-catenin induced by Wnt3a. These data suggest that glucocorticoid suppresses the canonical Wnt signal in cultured human osteoblasts, partially through the enhancement of the dickkopf-1 production.« less

  5. 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

  6. Mechanisms of Mechano-Transduction within Osteoblasts

    DTIC Science & Technology

    1999-09-01

    inositol trisphosphate levels (Reich and Frangos , 1993) with increasing shear stress. In a study concerning the effect of fluid shear...stress on cultured rat calvaria 34 osteoblasts, Hillsley and Frangos (1997) reported no change in collagen or osteopontin expression...Hillsley MV and Frangos JA (1997). Alkaline phosphatase in osteoblasts is down-regulated by pulsatile fluid flow. Calcif Tissue Int 60

  7. High Capacity Na+/H+ Exchange Activity in Mineralizing Osteoblasts

    PubMed Central

    Liu, Li; Schlesinger, Paul H.; Slack, Nicole M.; Friedman, Peter A.; Blair, Harry C.

    2015-01-01

    Osteoblasts synthesize bone in polarized groups of cells sealed by tight junctions. Large amounts of acid are produced as bone mineral is precipitated. We addressed the mechanism by which cells manage this acid load by measuring intracellular pH (pHi) in non-transformed osteoblasts in response to weak acid or bicarbonate loading. Basal pHi in mineralizing osteoblasts was ∼7.3 and decreased by ∼ 1.4 units upon replacing extracellular Na+ with N-methyl-d-glucamine. Loading with 40 mM acetic or propionic acids, in normal extracellular Na+, caused only mild cytosolic acidification. In contrast, in Na+-free solutions, weak acids reduced pHi dramatically. After Na+ reintroduction, pHi recovered rapidly, in keeping with Na+/H+exchanger (NHE) activity. Sodium-dependent pHi recovery from weak acid loading was inhibited by amiloride with the Ki consistent with NHEs. NHE1 and NHE6 were expressed strongly, and expression was upregulated highly, by mineralization, in human osteoblasts. Antibody labeling of mouse bone showed NHE1 on basolateral surfaces of all osteoblasts. NHE6 occurred on basolateral surfaces of osteoblasts mainly in areas of mineralization. Conversely, elevated HCO3- alkalinized osteoblasts, and pH recovered in medium containing CI-, with or without Na+, in keeping with Na+-independent CI-/HCO3- exchange. The exchanger AE2 also occurred on the basolateral surface of osteoblasts, consistent with CI-/HCO3- exchange for elimination of metabolic carbonate. Overexpression of NHE6 or knockdown of NHE1 in MG63 human osteosarcoma cells confirmed roles of NHE1 and NHE6 in maintaining pHi. We conclude that in mineralizing osteoblasts, slightly basic basal pHi is maintained, and external acid load is dissipated, by high-capacity Na+/H+ exchange via NHE1 and NHE6. PMID:21413028

  8. 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.

  9. Chondroitin and glucosamine sulfate in combination decrease the pro-resorptive properties of human osteoarthritis subchondral bone osteoblasts: a basic science study

    PubMed Central

    Tat, Steeve Kwan; Pelletier, Jean-Pierre; Vergés, Josep; Lajeunesse, Daniel; Montell, Eulàlia; Fahmi, Hassan; Lavigne, Martin; Martel-Pelletier, Johanne

    2007-01-01

    Early in the pathological process of osteoarthritis (OA), subchondral bone remodelling, which is related to altered osteoblast metabolism, takes place. In the present study, we explored in human OA subchondral bone whether chondroitin sulfate (CS), glucosamine sulfate (GS), or both together affect the major bone biomarkers, osteoprotegerin (OPG), receptor activator of nuclear factor-kappa B ligand (RANKL), and the pro-resorptive activity of OA osteoblasts. The effect of CS (200 μg/mL), GS (50 and 200 μg/mL), or both together on human OA subchondral bone osteoblasts, in the presence or absence of 1,25(OH)2D3 (vitamin D3) (50 nM), was determined on the bone biomarkers alkaline phosphatase and osteocalcin, on the expression (mRNA) and production (enzyme-linked immunosorbent assay) of bone remodelling factors OPG and RANKL, and on the pro-resorptive activity of these cells. For the latter experiments, human OA osteoblasts were incubated with differentiated peripheral blood mononuclear cells on a sub-micron synthetic calcium phosphate thin film. Data showed that CS and GS affected neither basal nor vitamin D3-induced alkaline phosphatase or osteocalcin release. Interestingly, OPG expression and production under basal conditions or vitamin D3 treatment were upregulated by CS and by both CS and GS incubated together. Under basal conditions, RANKL expression was significantly reduced by CS and by both drugs incubated together. Under vitamin D3, these drugs also showed a decrease in RANKL level, which, however, did not reach statistical significance. Importantly, under basal conditions, CS and both compounds combined significantly upregulated the expression ratio of OPG/RANKL. Vitamin D3 decreased this ratio, and GS further decreased it. Both drugs reduced the resorption activity, and statistical significance was reached for GS and when CS and GS were incubated together. Our data indicate that CS and GS do not overly affect cell integrity or bone biomarkers. Yet CS and

  10. 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.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kornu, R.; 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%more » (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.« less

  12. miR-96 promotes osteogenic differentiation by suppressing HBEGF-EGFR signaling in osteoblastic cells.

    PubMed

    Yang, Mingfu; Pan, Yong; Zhou, Yue

    2014-12-20

    MicroRNAs (miRNAs) are a class of small non-coding RNAs with important roles in various biological and pathological processes, including osteoblast differentiation. Here, we identified miR-96 as a positive regulator of osteogenic differentiation in a mouse osteoblastic cell line (MC3T3-E1) and in mouse bone marrow-derived mesenchymal stem cells. Moreover, we found that miR-96 down-regulates post-transcriptional expression of heparin-binding EGF-like growth factor (HB-EGF) by specifically binding to the 3'untranslated region of HB-EGF mRNA. Furthermore, in MC3T3-E1 cells, miR-96-induced HB-EGF down-regulation suppressed the phosphorylation of epidermal growth factor receptor (EGFR) and of extracellular signal-regulated kinase 1 (ERK1) and AKT, which both lie downstream of EGFR activation. Taken together, miR-96 promotes osteogenic differentiation by inhibiting HB-EGF and by blocking the HB-EGF-EGFR signaling pathway in osteoblastic cells. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  13. Effects of titanium surface anodization with CaP incorporation on human osteoblastic response

    PubMed Central

    OLIVEIRA, Natássia Cristina Martins; MOURA, Camilla Christian Gomes; ZANETTA-BARBOSA, Darceny; MENDONÇA, Daniela Baccelli Silveira; MENDONÇA, Gustavo; DECHICHI, Paula

    2015-01-01

    In this study we investigated whether anodization with calcium phosphate (CaP) incorporation (Vulcano®) enhances growth factors secretion, osteoblast-specific gene expression, and cell viability, when compared to acid etched surfaces (Porous®) and machined surfaces (Screw®) after 3 and 7 days. Results showed significant cell viability for Porous and Vulcano at day 7, when compared with Screw (p=0.005). At the same time point, significant differences regarding runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP) and bone sialoprotein (BSP) expression were found for all surfaces (p<0.05), but with greater fold induction for Porous and Vulcano. The secretion of transforming growth factor β1 (TGF-β1) and bone morphogenetic protein 2 (BMP-2) was not significantly affected by surface treatment in any experimental time (p>0.05). Although no significant correlation was found for growth factors secretion and Runx2 expression, a significant positive correlation between this gene and ALP/BSP expression showed that their strong association is independent on the type of surface. The incorporation of CaP affected the biological parameters evaluated similar to surfaces just acid etched. The results presented here support the observations that roughness also may play an important role in determining cell response. PMID:23498218

  14. 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. © 2015 Wiley Periodicals, Inc.

  15. Altered osteoblast structure and function in parabolic flight

    NASA Astrophysics Data System (ADS)

    Zhong-Quan, Dai; Ying-Hui, Li; Fen, Yang; Bai, Ding; Ying-Jun, Tan

    Introduction Bone loss has a significant impact on astronauts during spaceflight being one of the main obstacles preventing interplanetary missions However the exact mechanism is not well understood In the present study we investigated the effects of acute gravitational changes generated by parabolic flight on the structure and function of osteoblasts ROS17 2 8 carried by airbus A300 Methods The alteration of microfilament cytoskeleton was observed by the Texas red conjugated Phalloidin and Alexa Fluor 488 conjugated DNase I immunofluorescence stain ALP activity and expression COL1A1 expression osteocalcin secrete which presenting the osteoblast function were detected by modified calcium and cobalt method RT-PCR and radioimmunity methods respectively Results The changed gravity induced the reorganization of microfilament cytoskeleton of osteoblast After 3 hours parabolic flight F-actin of osteoblast cytoskeleton became more thickness and directivity whereas G-actin reduced and relatively concentrated at the edge of nucleus observed by confocal fluorescence microscopy This phenomenon is identical with structure alternation observed in hypergravity but the osteoblast function decrease The excretion of osteocalcin the activity and mRNA expression of ALP decrease but the COL1A1 expression has no changes These results were similar to the changes in simulated or real microgravity Conclusion Above results suggest that short time gravity alternative change induce osteoblast structure and function

  16. Priming integrin α5 promotes human mesenchymal stromal cell osteoblast differentiation and osteogenesis

    PubMed Central

    Hamidouche, Zahia; Fromigué, Olivia; Ringe, Jochen; Häupl, Thomas; Vaudin, Pascal; Pagès, Jean-Christophe; Srouji, Samer; Livne, Erella; Marie, Pierre J.

    2009-01-01

    Adult human mesenchymal stromal cells (hMSCs) have the potential to differentiate into chondrogenic, adipogenic, or osteogenic lineages, providing a potential source for tissue regeneration. An important issue for efficient bone regeneration is to identify factors that can be targeted to promote the osteogenic potential of hMSCs. Using transcriptome analysis, we found that integrin α5 (ITGA5) expression is up-regulated during dexamethasone-induced osteoblast differentiation of hMSCs. Gain-of-function studies showed that ITGA5 promotes the expression of osteoblast phenotypic markers and in vitro osteogenesis of hMSCs. Down-regulation of endogenous ITGA5 using specific shRNAs blunted osteoblast marker gene expression and osteogenic differentiation. Molecular analyses showed that the enhanced osteoblast differentiation induced by ITGA5 was mediated by activation of focal adhesion kinase/ERK1/2-MAPKs and PI3K signaling pathways. Remarkably, activation of endogenous ITGA5 using agonists such as a specific antibody that primes the integrin or a peptide that specifically activates ITGA5 was sufficient to enhance ERK1/2-MAPKs and PI3K signaling and to promote osteoblast differentiation and osteogenic capacity of hMSCs. Importantly, we demonstrated that hMSCs engineered to overexpress ITGA5 exhibited a marked increase in their osteogenic potential in vivo. Taken together, these findings not only reveal that ITGA5 is required for osteoblast differentiation of adult hMSCs but also provide a targeted strategy using ITGA5 agonists to promote the osteogenic capacity of hMSCs. This may be used for tissue regeneration in bone disorders where the recruitment or capacity of hMSCs is compromised. PMID:19843692

  17. Priming integrin alpha5 promotes human mesenchymal stromal cell osteoblast differentiation and osteogenesis.

    PubMed

    Hamidouche, Zahia; Fromigué, Olivia; Ringe, Jochen; Häupl, Thomas; Vaudin, Pascal; Pagès, Jean-Christophe; Srouji, Samer; Livne, Erella; Marie, Pierre J

    2009-11-03

    Adult human mesenchymal stromal cells (hMSCs) have the potential to differentiate into chondrogenic, adipogenic, or osteogenic lineages, providing a potential source for tissue regeneration. An important issue for efficient bone regeneration is to identify factors that can be targeted to promote the osteogenic potential of hMSCs. Using transcriptome analysis, we found that integrin alpha5 (ITGA5) expression is up-regulated during dexamethasone-induced osteoblast differentiation of hMSCs. Gain-of-function studies showed that ITGA5 promotes the expression of osteoblast phenotypic markers and in vitro osteogenesis of hMSCs. Down-regulation of endogenous ITGA5 using specific shRNAs blunted osteoblast marker gene expression and osteogenic differentiation. Molecular analyses showed that the enhanced osteoblast differentiation induced by ITGA5 was mediated by activation of focal adhesion kinase/ERK1/2-MAPKs and PI3K signaling pathways. Remarkably, activation of endogenous ITGA5 using agonists such as a specific antibody that primes the integrin or a peptide that specifically activates ITGA5 was sufficient to enhance ERK1/2-MAPKs and PI3K signaling and to promote osteoblast differentiation and osteogenic capacity of hMSCs. Importantly, we demonstrated that hMSCs engineered to overexpress ITGA5 exhibited a marked increase in their osteogenic potential in vivo. Taken together, these findings not only reveal that ITGA5 is required for osteoblast differentiation of adult hMSCs but also provide a targeted strategy using ITGA5 agonists to promote the osteogenic capacity of hMSCs. This may be used for tissue regeneration in bone disorders where the recruitment or capacity of hMSCs is compromised.

  18. 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

  19. Osteoblast response to magnesium ion-incorporated nanoporous titanium oxide surfaces.

    PubMed

    Park, Jin-Woo; Kim, Youn-Jeong; Jang, Je-Hee; Song, Hwangjun

    2010-11-01

    This study investigated the surface characteristics and in vitro osteoconductivity of a titanium (Ti) surface incorporated with the magnesium ions (Mg) produced by hydrothermal treatment for future application as an endosseous implant surface. Mg-incorporated Ti oxide surfaces were produced by hydrothermal treatment using Mg-containing solution on two different microstructured surfaces--abraded minimally rough (Ma) or grit-blasted moderately rough (RBM) samples. The surface characteristics were evaluated using scanning electron microscopy, thin-film X-ray diffractometry, X-ray photoelectron spectroscopy, optical profilometry, and inductively coupled plasma atomic emission spectroscopy (ICP-AES). MC3T3-E1 pre-osteoblast cell attachment, proliferation, alkaline phosphatase (ALP) activity, and quantitative analysis of osteoblastic gene expression on Ma, RBM, Mg-incorporated Ma (Mg), and Mg-incorporated grit-blasted (RBM/Mg) Ti surfaces were evaluated. Hydrothermal treatment produced an Mg-incorporated Ti oxide layer with nanoporous surface structures. Mg-incorporated surfaces showed surface morphologies and surface roughness values almost identical to those of untreated smooth or micro-rough surfaces at the micron scale. ICP-AES analysis showed Mg ions released from treated surfaces into the solution. Mg incorporation significantly increased cellular attachment (P=0 at 0.5 h, P=0.01 at 1 h) on smooth surfaces, but no differences were found on micro-rough surfaces. Mg incorporation further increased ALP activity in cells grown on both smooth and micro-rough surfaces at 7 and 14 days of culture (P=0). Real-time polymerase chain reaction analysis showed higher mRNA expressions of the osteoblast transcription factor gene (Dlx5), various integrins, and the osteoblast phenotype genes (ALP, bone sialoprotein and osteocalcin) in cells grown on micro-rough (RBM) and Mg-incorporated (Mg and RBM/Mg) surfaces than those on Ma surfaces. Mg incorporation further increased the m

  20. ATF4 mediation of NF1 functions in osteoblast reveals a nutritional basis for congenital skeletal dysplasiae

    PubMed Central

    Elefteriou, Florent; Benson, M. Douglas; Sowa, Hideaki; Starbuck, Michael; Liu, Xiuyun; Ron, David; Parada, Luis F.; Karsenty, Gerard

    2009-01-01

    Summary The transcription factor ATF4 enhances bone formation by favoring amino acid import and collagen synthesis in osteoblasts, a function requiring its phosphorylation by RSK2, the kinase inactivated in Coffin-Lowry Syndrome. Here, we show that in contrast, RSK2 activity, ATF4-dependent collagen synthesis, and bone formation are increased in mice lacking neurofibromin in osteoblasts (Nf1ob−/− mice). Independently of RSK2, ATF4 phosphorylation by PKA is enhanced in Nf1ob−/− mice, thereby increasing Rankl expression, osteoclast differentiation, and bone resorption. In agreement with ATF4 function in amino acid transport, a low-protein diet decreased bone protein synthesis and normalized bone formation and bone mass in Nf1ob−/− mice without affecting other organ weight, while a high-protein diet overcame Atf4−/− and Rsk2−/− mice developmental defects, perinatal lethality, and low bone mass. By showing that ATF4-dependent skeletal dysplasiae are treatable by dietary manipulations, this study reveals a molecular connection between nutrition and skeletal development. PMID:17141628

  1. Osteoblast fibronectin mRNA, protein synthesis, and matrix are unchanged after exposure to microgravity

    NASA Technical Reports Server (NTRS)

    Hughes-Fulford, M.; Gilbertson, V.

    1999-01-01

    The well-defined osteoblast line, MC3T3-E1 was used to examine fibronectin (FN) mRNA levels, protein synthesis, and extracellular FN matrix accumulation after growth activation in spaceflight. These osteoblasts produce FN extracellular matrix (ECM) known to regulate adhesion, differentiation, and function in adherent cells. Changes in bone ECM and osteoblast cell shape occur in spaceflight. To determine whether altered FN matrix is a factor in causing these changes in spaceflight, quiescent osteoblasts were launched into microgravity and were then sera activated with and without a 1-gravity field. Synthesis of FN mRNA, protein, and matrix were measured after activation in microgravity. FN mRNA synthesis is significantly reduced in microgravity (0-G) when compared to ground (GR) osteoblasts flown in a centrifuge simulating earth's gravity (1-G) field 2.5 h after activation. However, 27.5 h after activation there were no significant differences in mRNA synthesis. A small but significant reduction of FN protein was found in the 0-G samples 2.5 h after activation. Total FN protein 27.5 h after activation showed no significant difference between any of the gravity conditions, however, there was a fourfold increase in absolute amount of protein synthesized during the incubation. Using immunofluorescence, we found no significant differences in the amount or in the orientation of the FN matrix after 27.5 h in microgravity. These results demonstrate that FN is made by sera-activated osteoblasts even during exposure to microgravity. These data also suggest that after a total period of 43 h of spaceflight FN transcription, translation, or altered matrix assembly is not responsible for the altered cell shape or altered matrix formation of osteoblasts.

  2. Histone H4 Methyltransferase Suv420h2 Maintains Fidelity of Osteoblast Differentiation.

    PubMed

    Khani, Farzaneh; Thaler, Roman; Paradise, Christopher R; Deyle, David R; Kruijthof-de Julio, Marianne; Galindo, Mario; Gordon, Jonathan A; Stein, Gary S; Dudakovic, Amel; van Wijnen, Andre J

    2017-05-01

    Osteogenic lineage commitment and progression is controlled by multiple signaling pathways (e.g., WNT, BMP, FGF) that converge on bone-related transcription factors. Access of osteogenic transcription factors to chromatin is controlled by epigenetic regulators that generate post-translational modifications of histones ("histone code"), as well as read, edit and/or erase these modifications. Our understanding of the biological role of epigenetic regulators in osteoblast differentiation remains limited. Therefore, we performed next-generation RNA sequencing (RNA-seq) and established which chromatin-related proteins are robustly expressed in mouse bone tissues (e.g., fracture callus, calvarial bone). These studies also revealed that cells with increased osteogenic potential have higher levels of the H4K20 methyl transferase Suv420h2 compared to other methyl transferases (e.g., Suv39h1, Suv39h2, Suv420h1, Ezh1, Ezh2). We find that all six epigenetic regulators are transiently expressed at different stages of osteoblast differentiation in culture, with maximal mRNAs levels of Suv39h1 and Suv39h2 (at day 3) preceding maximal expression of Suv420h1 and Suv420h2 (at day 7) and developmental stages that reflect, respectively, early and later collagen matrix deposition. Loss of function analysis of Suv420h2 by siRNA depletion shows loss of H4K20 methylation and decreased expression of bone biomarkers (e.g., alkaline phosphatase/Alpl) and osteogenic transcription factors (e.g., Sp7/Osterix). Furthermore, Suv420h2 is required for matrix mineralization during osteoblast differentiation. We conclude that Suv420h2 controls the H4K20 methylome of osteoblasts and is critical for normal progression of osteoblastogenesis. J. Cell. Biochem. 118: 1262-1272, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  3. Histone H4 methyltransferase Suv420h2 maintains fidelity of osteoblast differentiation

    PubMed Central

    Farzaneh, Khani; Thaler, Roman; Paradise, Christopher R.; Deyle, David R.; Julio, Marianne Kruijthof-de; Galindo, Mario; Gordon, Jonathan A.; Stein, Gary S.; Dudakovic, Amel; van Wijnen, Andre J.

    2017-01-01

    Osteogenic lineage commitment and progression is controlled by multiple signaling pathways (e.g., WNT, BMP, FGF) that converge on bone-related transcription factors. Access of osteogenic transcription factors to chromatin is controlled by epigenetic regulators that generate post-translational modifications of histones (‘histone code’), as well as read, edit and/or erase these modifications. Our understanding of the biological role of epigenetic regulators in osteoblast differentiation remains limited. Therefore, we performed next-generation RNA sequencing (RNA-seq) and established which chromatin-related proteins are robustly expressed in mouse bone tissues (e.g., fracture callus, calvarial bone). These studies also revealed that cells with increased osteogenic potential have higher levels of the H4K20 methyl transferase Suv420h2 compared to other methyl transferases (e.g., Suv39h1, Suv39h2, Suv420h1, Ezh1, Ezh2). We find that all six epigenetic regulators are transiently expressed at different stages of osteoblast differentiation in culture, with maximal mRNAs levels of Suv39h1 and Suv39h2 (at day 3) preceding maximal expression of Suv420h1 and Suv420h2 (at day 7) and developmental stages that reflect, respectively, early and later collagen matrix deposition. Loss of function analysis of Suv420h2 by siRNA depletion shows loss of H4K20 methylation and decreased expression of bone biomarkers (e.g., alkaline phosphatase/Alpl) and osteogenic transcription factors (e.g., Sp7/Osterix). Furthermore, Suv420h2 is required for matrix mineralization during osteoblast differentiation. We conclude that Suv420h2 controls the H4K20 methylome of osteoblasts and is critical for normal progression of osteoblastogenesis. PMID:27862226

  4. Proteinase-activated receptor (PAR)-2 activation impacts bone resorptive properties of human osteoarthritic subchondral bone osteoblasts.

    PubMed

    Amiable, Nathalie; Tat, Steeve Kwan; Lajeunesse, Daniel; Duval, Nicolas; Pelletier, Jean-Pierre; Martel-Pelletier, Johanne; Boileau, Christelle

    2009-06-01

    In osteoarthritis (OA), the subchondral bone undergoes a remodelling process involving several factors synthesized by osteoblasts. In this study, we investigated the expression, production, modulation, and role of PAR-2 in human OA subchondral bone osteoblasts. PAR-2 expression and production were determined by real-time PCR and flow cytometry, respectively. PAR-2 modulation was investigated in OA subchondral bone osteoblasts treated with IL-1 beta (100 pg/ml), TNF-alpha (5 ng/ml), TGF-beta1 (10 ng/ml), PGE(2) (500 nM), IL-6 (10 ng/ml) and IL-17 (10 ng/ml). Membranous RANKL protein was assessed by flow cytometry, and OPG, MMP-1, MMP-9, MMP-13, IL-6 and intracellular signalling pathways by specific ELISAs. Bone resorptive activity was measured by using a co-culture model of human PBMC and OA subchondral bone osteoblasts. PAR-2 expression and production (p<0.05) were markedly increased when human OA subchondral bone osteoblasts were compared to normal. On OA osteoblasts, PAR-2 production was significantly increased by IL-1 beta, TNF-alpha and PGE(2). Activation of PAR-2 with a specific agonist, SLIGKV-NH(2), induced a significant up-regulation of MMP-1, MMP-9, IL-6, and membranous RANKL, but had no effect on MMP-13 or OPG production. Interestingly, bone resorptive activity was also significantly enhanced following PAR-2 activation. The PAR-2 effect was mediated by activation of the MAP kinases Erk1/2 and JNK. This study is the first to demonstrate that PAR-2 activation plays a role in OA subchondral bone resorption via an up-regulation of major bone remodelling factors. These results shed new light on the potential of PAR-2 as a therapeutic target in OA.

  5. Stepwise differentiation of pluripotent stem cells into osteoblasts using four small molecules under serum-free and feeder-free conditions.

    PubMed

    Kanke, Kosuke; Masaki, Hideki; Saito, Taku; Komiyama, Yuske; Hojo, Hironori; Nakauchi, Hiromitsu; Lichtler, Alexander C; Takato, Tsuyoshi; Chung, Ung-Il; Ohba, Shinsuke

    2014-06-03

    Pluripotent stem cells are a promising tool for mechanistic studies of tissue development, drug screening, and cell-based therapies. Here, we report an effective and mass-producing strategy for the stepwise differentiation of mouse embryonic stem cells (mESCs) and mouse and human induced pluripotent stem cells (miPSCs and hiPSCs, respectively) into osteoblasts using four small molecules (CHIR99021 [CHIR], cyclopamine [Cyc], smoothened agonist [SAG], and a helioxanthin-derivative 4-(4-methoxyphenyl)pyrido[4',3':4,5]thieno[2,3-b]pyridine-2-carboxamide [TH]) under serum-free and feeder-free conditions. The strategy, which consists of mesoderm induction, osteoblast induction, and osteoblast maturation phases, significantly induced expressions of osteoblast-related genes and proteins in mESCs, miPSCs, and hiPSCs. In addition, when mESCs defective in runt-related transcription factor 2 (Runx2), a master regulator of osteogenesis, were cultured by the strategy, they molecularly recapitulated osteoblast phenotypes of Runx2 null mice. The present strategy will be a platform for biological and pathological studies of osteoblast development, screening of bone-augmentation drugs, and skeletal regeneration.

  6. FGF2 Stimulation of the Pyrophosphate-Generating Enzyme, PC-1, in Pre-Osteoblast Cells Is Mediated by RUNX2

    PubMed Central

    Hatch, Nan E; Li, Yan; Franceschi, Renny T

    2009-01-01

    Pyrophosphate is an established inhibitor of hydroxyapatite deposition and crystal growth, yet when hydrolyzed into phosphate, it becomes a substrate for hydroxyapatite deposition. Pyrophosphate-generating enzyme (PC-1), Ank, and tissue nonspecific alkaline phosphatase (Tnap) are three factors that regulate extracellular pyrophosphate levels through its generation, transport, and hydrolysis. We previously showed that fibroblast growth factor 2 (FGF2) induces PC-1 and Ank while inhibiting Tnap expression and mineralization in MC3T3E1(C4) calvarial pre-osteoblast cells. In this study, we showed similar FGF2 regulation of these genes in primary pre-osteoblast cultures. In contrast to Ank and Tnap that are regulated by FGF2 in multiple cell types, we found regulation of PC-1 to be selective to pre-osteoblastic cells and to require the osteoblast-related transcription factor, Runx2. Specifically, FGF2 was unable to induce PC-1 expression in Runx2-negative nonbone cells or in calvarial cells from Runx2-deficient mice. Transfection of these cells with a Runx2 expression vector restored FGF2 responsiveness. FGF2 was also shown to stimulate recruitment of Runx2 to the endogenous PC-1 promoter in MC3T3E1(C4) cells, as measured by chromatin immunoprecipitation. Taken together, our results establish that FGF2 is a specific inducer of PC-1 in pre-osteoblast cells and that FGF2 induces PC-1 expression through a mechanism involving Runx2. PMID:19049325

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

    PubMed

    Thiolloy, Sophie; Edwards, James R; Fingleton, Barbara; Rifkin, Daniel B; Matrisian, Lynn M; Lynch, Conor C

    2012-01-01

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

  8. An Osteoblast-Derived Proteinase Controls Tumor Cell Survival via TGF-beta Activation in the Bone Microenvironment

    PubMed Central

    Thiolloy, Sophie; Edwards, James R.; Fingleton, Barbara; Rifkin, Daniel B.; Matrisian, Lynn M.; Lynch, Conor C.

    2012-01-01

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

  9. Lactate induces osteoblast differentiation by stabilization of HIF1α.

    PubMed

    Wu, Yu; Wang, Miaomiao; Feng, Haihua; Peng, Ying; Sun, Jieyun; Qu, Xiuxia; Li, Chunping

    2017-09-05

    Aerobic glycolysis is involved in osteoblast differentiation induced by Wnt signaling or PTH treatment. However, it is still unclear whether lactate, the end product of aerobic glycolysis, plays any role in osteoblast differentiation. Herein we report that in cultures of osteoblast-lineage cells, lactate promoted alkaline phosphatase-positive cell formation, increased the activity of alkaline phosphatase, and induced the expression of osteocalcin. This osteoblast differentiation-inducing effect of lactate can be inhibited by blocking its entry into cells with MCT1 siRNA or inhibitors, and by interfering with its metabolism by using specific siRNAs for LDHB and PDH. Moreover, lactate stabilized HIF1α expression and inhibited HIF1α activity, with BAY87-2243 lowering the osteoblast differentiation-inducing effect of lactate. Thus, these findings reveal an unrecognized role for aerobic glycolysis in osteoblast differentiation via its end product, lactate. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Pneumolysin-induced autophagy contributes to inhibition of osteoblast differentiation through downregulation of Sp1 in human osteosarcoma cells.

    PubMed

    Kim, Jinwook; Lee, Hee-Weon; Rhee, Dong Kwon; Paton, James C; Pyo, Suhkneung

    2017-11-01

    The 53kDa protein pneumolysin (PLY) is the main virulence factor of Streptococcus pneumoniae, a leading cause of invasive pneumococcal diseases. PLY forms pores in cholesterol-containing membranes, thereby interfering with the function of cells. Bone destruction is a serious matter in chronic inflammatory diseases such as septic arthritis and osteomyelitis. S. pneumoniae is increasingly being recognized as a common cause of septic arthritis, but its pathogenesis is poorly defined. We examined the effect of PLY on osteoblast differentiation and its mechanisms of action. The effect of PLY on osteoblast differentiation was evaluated by qRT-PCR, ALP activity assay, flow cytometric analysis, and Western blotting. We also examined the role of PLY-induced autophagy in osteoblast differentiation using RNA interference analysis. PLY inhibited osteoblast differentiation by decreasing the expression of osteoblast marker genes such as Runx2 and OCN, along with ALP activity. ROS production was increased by PLY during osteoblast differentiation. PLY induced autophagy through ROS-mediated regulation of AMPK and mTOR, which downregulated the expression of Sp1 and subsequent inhibition of differentiation. Treatment with autophagy inhibitors or Atg5 siRNA alleviated the PLY-induced inhibition of differentiation. The results suggest that PLY inhibits osteoblast differentiation by downregulation of Sp1 accompanied by induction of autophagy through ROS-mediated regulation of the AMPK/mTOR pathway. This study proposes a molecular mechanism for inhibition of osteoblast differentiation in response to PLY. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Changes in osteocyte density correspond with changes in osteoblast and osteoclast activity in an osteoporotic sheep model.

    PubMed

    Zarrinkalam, M R; Mulaibrahimovic, A; Atkins, G J; Moore, R J

    2012-04-01

    Histomorphometric assessment of trabecular bone in osteoporotic sheep showed that bone volume, osteoid surface area, bone formation rate, and osteocyte density were reduced. In contrast, eroded surface area and empty lacunae density were increased. Changes in osteocyte density correlated with changes in osteoblast and osteoclast activity. Osteocytes contribute to the regulation of the activity of osteoclasts and osteoblasts that together control bone mass. Osteocytes therefore likely play a role in the loss of bone mass associated with osteoporosis. The purpose of this study was to investigate the relationships between osteocyte lacunar density and other bone histomorphometric parameters in the iliac crest (IC) and lumbar spine (LS) of osteoporotic sheep. Osteoporosis was induced in ten mature ewes by an established protocol involving a combination of ovariectomy, dexamethasone injection, and low calcium diet for 6 months. Five ewes were used as controls. Post-mortem IC and LS biopsies were collected and processed for further histomorphometric assessment. Bone volume, osteoid surface, and bone formation rate in the IC and LS of osteoporotic sheep were reduced compared to those of the controls. In contrast, eroded surface area was increased in osteoporotic sheep. In the osteoporotic group, osteocyte density was reduced in the LS region and to a greater extent in the IC region. The empty osteocyte lacunae were increased 1.7-fold in LS and 2.1-fold in IC in the osteoporotic group. The osteocyte density correlated positively with markers of osteoblast activity and negatively with those of osteoclast activity. Depletion of osteocytes and an increase in the empty lacunae could be important factors contributing to bone loss in this model since they may adversely affect intercellular communication between osteoblasts and osteoclasts. The regional differences in histology suggest that there may be different pathological mechanisms operating at different anatomical sites.

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

    PubMed Central

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

    2010-01-01

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

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Xu, Yong; Fang, Shi-ji; Zhu, Li-juan

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

  14. Regulation of DMT1 on autophagy and apoptosis in osteoblast

    PubMed Central

    Liu, Fei; Zhang, Wei-Lin; Meng, Hong-Zheng; Cai, Zheng-Yu; Yang, Mao-Wei

    2017-01-01

    Iron overload has recently been associated with the changes in the bone microstructure that occur in osteoporosis. However, the effect of iron overload on osteoblasts is unclear. The purpose of this study was to explore the function of divalent metal transporter 1 (DMT1) in the pathological processes of osteoporosis. Osteoblast hFOB1.19 cells were cultured in medium supplemented with different concentrations (0, 50, 100, 200, 300, 400, 500 μmol/L) of ferric ammonium citrate (FAC) as a donor of ferric ions. We used western blotting and immunofluorescence to determine the levels of DMT1 after treatment with FAC. Apoptosis was evaluated by detecting the levels of cleaved caspase 3, BCL2, and BAX with western blotting. Autophagy was evaluated by detecting the levels of LC3 with western blotting and immunofluorescence. Beclin-1 expression was also assessed with western blotting. The autophagy inhibitor 3-methyladenine was used to determine whether autophagy affects the apoptosis induced by FAC. Our results show that FAC increased the levels of DMT1, upregulated the expression of BCL2, and downregulated the apoptosis-related proteins cleaved caspase 3 and BAX. Both LC3I/LC3II levels and beclin-1 were also increased, indicating that FAC increases the accumulation of autophagosomes in hFOB1.19 cells. FAC-induced autophagy was increased by the apoptosis inhibitor 3-MA but was reduced in DMT1 shRNA hFOB1.19 cells. These results suggest that the increased expression of DMT1 induces iron overload and iron overload induces osteoblast autophagy and apoptosis, thus affecting the pathological processes of osteoporosis. Clarifying the mechanisms underlying the effects of DMT1 will allow the identification of novel targets for the prevention and treatment of osteoporosis. PMID:28367088

  15. Mechanisms in endocrinology: micro-RNAs: targets for enhancing osteoblast differentiation and bone formation.

    PubMed

    Taipaleenmäki, Hanna; Bjerre Hokland, Lea; Chen, Li; Kauppinen, Sakari; Kassem, Moustapha

    2012-03-01

    Osteoblast differentiation and bone formation (osteogenesis) are regulated by transcriptional and post-transcriptional mechanisms. Recently, a novel class of regulatory factors termed micro-RNAs (miRNAs) has been identified as playing an important role in the regulation of many aspects of osteoblast biology including proliferation, differentiation, metabolism and apoptosis. Also, preliminary data from animal disease models suggest that targeting miRNAs in bone can be a novel approach to increase bone mass. This review highlights the current knowledge of miRNA biology and their role in bone formation and discusses their potential use in future therapeutic applications for metabolic bone diseases.

  16. Magnesium and zinc borate enhance osteoblastic differentiation of stem cells from human exfoliated deciduous teeth in vitro.

    PubMed

    Liu, Yao-Jen; Su, Wen-Ta; Chen, Po-Hung

    2018-01-01

    Various biocompatible and biodegradable scaffolds blended with biochemical signal molecules with adequate osteoinductive and osteoconductive properties have attracted significant interest in hard tissue engineering regeneration. We evaluated the distinct effects of magnesium borate, zinc borate, and boric acid blended into chitosan scaffold for osteogenic differentiation of stem cells from exfoliated deciduous teeth. Stem cells from exfoliated deciduous teeth cells are a potential source of functional osteoblasts for applications in bone tissue engineering, but the efficiency of osteoblastic differentiation is low, thereby significantly limiting their clinical applications. Divalent metal borates have potential function in bone remodeling because they can simulate bone formation and decrease bone resorption. These magnesium, zinc, and B ions can gradually be released into the culture medium from the scaffold and induce advanced osteoblastic differentiation from stem cells from exfoliated deciduous teeth. Stem cells from exfoliated deciduous teeth with magnesium borate or zinc borate as inducer demonstrated more osteoblastic differentiation after 21 days of culture. Differentiated cells exhibited activity of alkaline phosphatase, bone-related gene expression of collagen type I, runt-related transcription factor 2, osteopontin, osteocalcin, vascular endothelial growth factor, and angiopoietin-1, as noted via real-time polymerase chain reaction analysis, as well as significant deposits of calcium minerals. Divalent mental magnesium and zinc and nonmetal boron can be an effective inducer of osteogenesis for stem cells from exfoliated deciduous teeth. This experiment might provide useful inducers for osteoblastic differentiation of stem cells from exfoliated deciduous teeth for tissue engineering and bone repair.

  17. Osteoblast Production by Reserved Progenitor Cells in Zebrafish Bone Regeneration and Maintenance.

    PubMed

    Ando, Kazunori; Shibata, Eri; Hans, Stefan; Brand, Michael; Kawakami, Atsushi

    2017-12-04

    Mammals cannot re-form heavily damaged bones as in large fracture gaps, whereas zebrafish efficiently regenerate bones even after amputation of appendages. However, the source of osteoblasts that mediate appendage regeneration is controversial. Several studies in zebrafish have shown that osteoblasts are generated by dedifferentiation of existing osteoblasts at injured sites, but other observations suggest that de novo production of osteoblasts also occurs. In this study, we found from cell-lineage tracing and ablation experiments that a group of cells reserved in niches serves as osteoblast progenitor cells (OPCs) and has a significant role in fin ray regeneration. Besides regeneration, OPCs also supply osteoblasts for normal bone maintenance. We further showed that OPCs are derived from embryonic somites, as is the case with embryonic osteoblasts, and are replenished from mesenchymal precursors in adult zebrafish. Our findings reveal that reserved progenitors are a significant and complementary source of osteoblasts for zebrafish bone regeneration. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Osteoblast-specific factor 2: cloning of a putative bone adhesion protein with homology with the insect protein fasciclin I.

    PubMed Central

    Takeshita, S; Kikuno, R; Tezuka, K; Amann, E

    1993-01-01

    A cDNA library prepared from the mouse osteoblastic cell line MC3T3-E1 was screened for the presence of specifically expressed genes by employing a combined subtraction hybridization/differential screening approach. A cDNA was identified and sequenced which encodes a protein designated osteoblast-specific factor 2 (OSF-2) comprising 811 amino acids. OSF-2 has a typical signal sequence, followed by a cysteine-rich domain, a fourfold repeated domain and a C-terminal domain. The protein lacks a typical transmembrane region. The fourfold repeated domain of OSF-2 shows homology with the insect protein fasciclin I. RNA analyses revealed that OSF-2 is expressed in bone and to a lesser extent in lung, but not in other tissues. Mouse OSF-2 cDNA was subsequently used as a probe to clone the human counterpart. Mouse and human OSF-2 show a high amino acid sequence conservation except for the signal sequence and two regions in the C-terminal domain in which 'in-frame' insertions or deletions are observed, implying alternative splicing events. On the basis of the amino acid sequence homology with fasciclin I, we suggest that OSF-2 functions as a homophilic adhesion molecule in bone formation. Images Figure 3 Figure 4 Figure 5 Figure 6 PMID:8363580

  19. Osthole Stimulates Osteoblast Differentiation and Bone Formation by Activation of β-Catenin–BMP Signaling

    PubMed Central

    Tang, De-Zhi; Hou, Wei; Zhou, Quan; Zhang, Minjie; Holz, Jonathan; Sheu, Tzong-Jen; Li, Tian-Fang; Cheng, Shao-Dan; Shi, Qi; Harris, Stephen E; Chen, Di; Wang, Yong-Jun

    2010-01-01

    Osteoporosis is defined as reduced bone mineral density with a high risk of fragile fracture. Current available treatment regimens include antiresorptive drugs such as estrogen receptor analogues and bisphosphates and anabolic agents such as parathyroid hormone (PTH). However, neither option is completely satisfactory because of adverse effects. It is thus highly desirable to identify novel anabolic agents to improve future osteoporosis treatment. Osthole, a coumarin-like derivative extracted from Chinese herbs, has been shown to stimulate osteoblast proliferation and differentiation, but its effect on bone formation in vivo and underlying mechanism remain unknown. In this study, we found that local injection of Osthole significantly increased new bone formation on the surface of mouse calvaria. Ovariectomy caused evident bone loss in rats, whereas Osthole largely prevented such loss, as shown by improved bone microarchitecture, histomorphometric parameters, and biomechanical properties. In vitro studies demonstrated that Osthole activated Wnt/β-catenin signaling, increased Bmp2 expression, and stimulated osteoblast differentiation. Targeted deletion of the β-catenin and Bmp2 genes abolished the stimulatory effect of Osthole on osteoblast differentiation. Since deletion of the Bmp2 gene did not affect Osthole-induced β-catenin expression and the deletion of the β-catenin gene inhibited Osthole-regulated Bmp2 expression in osteoblasts, we propose that Osthole acts through β-catenin–BMP signaling to promote osteoblast differentiation. Our findings demonstrate that Osthole could be a potential anabolic agent to stimulate bone formation and prevent estrogen deficiency–induced bone loss. © 2010 American Society for Bone and Mineral Research. PMID:20200936

  20. Insulin-like growth factor-1 receptor in mature osteoblasts is required for periosteal bone formation induced by reloading

    NASA Astrophysics Data System (ADS)

    Kubota, Takuo; Elalieh, Hashem Z.; Saless, Neema; Fong, Chak; Wang, Yongmei; Babey, Muriel; Cheng, Zhiqiang; Bikle, Daniel D.

    2013-11-01

    Skeletal loading and unloading has a pronounced impact on bone remodeling, a process also regulated by insulin-like growth factor-1 (IGF-1) signaling. Skeletal unloading leads to resistance to the anabolic effect of IGF-1, while reloading after unloading restores responsiveness to IGF-1. However, a direct study of the importance of IGF-1 signaling in the skeletal response to mechanical loading remains to be tested. In this study, we assessed the skeletal response of osteoblast-specific Igf-1 receptor deficient (Igf-1r-/-) mice to unloading and reloading. The mice were hindlimb unloaded for 14 days and then reloaded for 16 days. Igf-1r-/- mice displayed smaller cortical bone and diminished periosteal and endosteal bone formation at baseline. Periosteal and endosteal bone formation decreased with unloading in Igf-1r+/+ mice. However, the recovery of periosteal bone formation with reloading was completely inhibited in Igf-1r-/- mice, although reloading-induced endosteal bone formation was not hampered. These changes in bone formation resulted in the abolishment of the expected increase in total cross-sectional area with reloading in Igf-1r-/- mice compared to the control mice. These results suggest that the Igf-1r in mature osteoblasts has a critical role in periosteal bone formation in the skeletal response to mechanical loading.

  1. Distribution of type VI collagen in association with osteoblast lineages in the groove of Ranvier during rat postnatal development.

    PubMed

    Kohara, Yukihiro; Soeta, Satoshi; Izu, Yayoi; Arai, Kiyotaka; Amasaki, Hajime

    2016-11-01

    In the groove of Ranvier (GOR), osteoblast lineages form bone bark, which develops into endosteal cortical bone. This ossification process is thought to be regulated by the microenvironment in the GOR. Type VI collagen (Col VI), an extracellular matrix (ECM) protein found in the periosteum/perichondrium, mediates osteoblast differentiation via the cell-surface receptor neural/glial antigen 2 (NG2) chondroitin sulfate proteoglycan. In order to clarify the function of Col VI during osteoblast differentiation in the GOR, in the present study, we examined the distribution of Col VI and osteoblast lineages expressing NG2 in the rat tibia proximal end during postnatal growing periods by immunohistochemistry. Our data revealed that Col VI accumulated in the ECM of the GOR middle layer and that Col VI accumulation was reduced and disappeared in the inner and middle lower regions. Runt-related transcription factor 2-immunoreactive pre-osteoblasts expressed NG2 in Col VI-immunopositive areas. However, Osterix-immunoreactive mature osteoblasts were only found in the Col VI-immunonegative area. These findings indicate that Col VI provided a characteristic microenvironment in the GOR and that NG2-Col VI interactions may regulate the differentiation of osteoblast lineages prior to terminal maturation. Copyright © 2016 Elsevier GmbH. All rights reserved.

  2. Osteoclast-derived microRNA-containing exosomes selectively inhibit osteoblast activity

    PubMed Central

    Sun, Weijia; Zhao, Chenyang; Li, Yuheng; Wang, Liang; Nie, Guangjun; Peng, Jiang; Wang, Aiyuan; Zhang, Pengfei; Tian, Weiming; Li, Qi; Song, Jinping; Wang, Cheng; Xu, Xiaolong; Tian, Yanhua; Zhao, Dingsheng; Xu, Zi; Zhong, Guohui; Han, Bingxing; Ling, Shukuan; Chang, Yan-Zhong; Li, Yingxian

    2016-01-01

    MicroRNAs have an important role in bone homeostasis. However, the detailed mechanism of microRNA-mediated intercellular communication between bone cells remains elusive. Here, we report that osteoclasts secrete microRNA-enriched exosomes, by which miR-214 is transferred into osteoblasts to inhibit their function. In a coculture system, inhibition of exosome formation and secretion prevented miR-214 transportation. Exosomes specifically recognized osteoblasts through the interaction between ephrinA2 and EphA2. In osteoclast-specific miR-214 transgenic mice, exosomes were secreted into the serum, and miR-214 and ephrinA2 levels were elevated. Therefore, these exosomes have an inhibitory role in osteoblast activity. miR-214 and ephrinA2 levels in serum exosomes from osteoporotic patients and mice were upregulated substantially. These exosomes may significantly inhibit osteoblast activity. Inhibition of exosome secretion via Rab27a small interfering RNA prevented ovariectomized-induced osteoblast dysfunction in vivo. Taken together, these findings suggest that exosome-mediated transfer of microRNA plays an important role in the regulation of osteoblast activity. Circulating miR-214 in exosomes not only represents a biomarker for bone loss but could selectively regulate osteoblast function. PMID:27462462

  3. BG60S dissolution interferes with osteoblast calcium signals.

    PubMed

    Valério, P; Pereira, M M; Goes, A M; Leite, M F

    2007-02-01

    We investigated the influence of extracellular calcium concentration, caused by the dissolution of a bioactive glass with 60% of silicon (BG60S), on intracellular calcium (Ca(i) (2 +)) signals and expression of inositol 1, 4, 5-triphosphate receptors (InsP(3)R) in primary culture of osteoblasts. We found that BG60S caused an increase in Ca(i) (2 +) signals in this cell type. Additionally, osteoblasts pre-incubated in the presence of BG60S showed an increase in Ca(i) (2 +) when cells were stimulated with vasopressin. On the other hand, a decrease in Ca(i) (2 +) signals were observed in osteoblasts pre-treated with BG60S and stimulated with KCl. We furher found that in osteoblasts, the type I InsP(3)R is preferentially distributed in the nucleus while the type II InsP(3)R in the cytoplasm. Preincubation of osteoblasts with BG60S altered the receptor expression level, increasing the type I InsP(3)R in the nucleus and decreasing type II InsP(3)R in the cytosol. Together, our results showed that in osteoblasts, BG60S increased Ca(i) (2 +)signals and altered Ca(i) (2 +) machinery.

  4. Premature Osteoblast Clustering by Enamel Matrix Proteins Induces Osteoblast Differentiation through Up-Regulation of Connexin 43 and N-Cadherin

    PubMed Central

    Miron, Richard J.; Hedbom, Erik; Ruggiero, Sabrina; Bosshardt, Dieter D.; Zhang, Yufeng; Mauth, Corinna; Gemperli, Anja C.; Iizuka, Tateyuki; Buser, Daniel; Sculean, Anton

    2011-01-01

    In recent years, enamel matrix derivative (EMD) has garnered much interest in the dental field for its apparent bioactivity that stimulates regeneration of periodontal tissues including periodontal ligament, cementum and alveolar bone. Despite its widespread use, the underlying cellular mechanisms remain unclear and an understanding of its biological interactions could identify new strategies for tissue engineering. Previous in vitro research has demonstrated that EMD promotes premature osteoblast clustering at early time points. The aim of the present study was to evaluate the influence of cell clustering on vital osteoblast cell-cell communication and adhesion molecules, connexin 43 (cx43) and N-cadherin (N-cad) as assessed by immunofluorescence imaging, real-time PCR and Western blot analysis. In addition, differentiation markers of osteoblasts were quantified using alkaline phosphatase, osteocalcin and von Kossa staining. EMD significantly increased the expression of connexin 43 and N-cadherin at early time points ranging from 2 to 5 days. Protein expression was localized to cell membranes when compared to control groups. Alkaline phosphatase activity was also significantly increased on EMD-coated samples at 3, 5 and 7 days post seeding. Interestingly, higher activity was localized to cell cluster regions. There was a 3 fold increase in osteocalcin and bone sialoprotein mRNA levels for osteoblasts cultured on EMD-coated culture dishes. Moreover, EMD significantly increased extracellular mineral deposition in cell clusters as assessed through von Kossa staining at 5, 7, 10 and 14 days post seeding. We conclude that EMD up-regulates the expression of vital osteoblast cell-cell communication and adhesion molecules, which enhances the differentiation and mineralization activity of osteoblasts. These findings provide further support for the clinical evidence that EMD increases the speed and quality of new bone formation in vivo. PMID:21858092

  5. Vitamins D3 and K2 may partially counterbalance the detrimental effects of pentosidine in ex vivo human osteoblasts.

    PubMed

    Sanguineti, R; Monacelli, F; Parodi, A; Furfaro, A L; Borghi, R; Pacini, D; Pronzato, M A; Odetti, P; Molfetta, L; Traverso, N

    2016-01-01

    Osteoporosis is a metabolic multifaceted disorder, characterized by insufficient bone strength. It has been recently shown that advanced glycation end products (AGEs) play a role in senile osteoporosis, through bone cell impairment and altered biomechanical properties. Pentosidine (PENT), a wellcharacterized AGE, is also considered a biomarker of bone fracture. Adequate responses to various hormones, such as 1,25-dihydroxyvitamin D 3 , are prerequisites for optimal osteoblasts functioning. Vitamin K 2 is known to enhance in vitro and in vitro vitamin D-induced bone formation. The aim of the study was to assess the effects of Vitamins D 3 and K 2 and PENT on in vitro osteoblast activity, to convey a possible translational clinical message. Ex vivo human osteoblasts cultured, for 3 weeks, with vitamin D 3 and vitamin K 2 were exposed to PENT, a well-known advanced glycoxidation end product for the last 72 hours. Experiments with PENT alone were also carried out. Gene expression of specific markers of bone osteoblast maturation [alkaline phosphatase, ALP; collagen I, COL Iα1; and osteocalcin (bone-Gla-protein) BGP] was measured, together with the receptor activator of nuclear factor kappa-B ligand/osteoproteregin (RANKL/OPG) ratio to assess bone remodeling. Expression of RAGE, a well-characterized receptor of AGEs, was also assessed. PENT+vitamins slightly inhibited ALP secretion while not affecting gene expression, indicating hampered osteoblast functional activity. PENT+vitamins up-regulated collagen gene expression, while protein secretion was unchanged. Intracellular collagen levels were partially decreased, and a significant reduction in BGP gene expression and intracellular protein concentration were both reported after PENT exposure. The RANKL/OPG ratio was increased, favouring bone reabsorption. RAGE gene expression significantly decreased. These results were confirmed by a lower mineralization rate. We provided in vitro evidence that glycoxidation might

  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.

  7. The impact of diamond nanocrystallinity on osteoblast functions.

    PubMed

    Yang, Lei; Sheldon, Brian W; Webster, Thomas J

    2009-07-01

    Nanocrystalline diamond has been proposed as an anti-abrasive film on orthopedic implants. In this study, osteoblast (bone forming cells) functions including adhesion (up to 4h), proliferation (up to 5 days) and differentiation (up to 21 days) on different diamond film topographies were systematically investigated. In order to exclude interferences from changes in surface chemistry and wettability (energy), diamond films with nanometer and micron scale topographies were fabricated through microwave plasma enhanced chemical-vapor-deposition and hydrogen plasma treatment. Scanning electron microscopy (SEM), atomic force microscopy (AFM), Raman spectroscopy and water contact angle measurements verified the similar surface chemistry and wettability but varied topographies for all of the diamond films prepared on silicon in this study. Cytocompatibility assays demonstrated enhanced osteoblast functions (including adhesion, proliferation, intracellular protein synthesis, alkaline phosphatase activity and extracellular calcium deposition) on nanocrystalline diamond compared to submicron diamond grain size films for all time periods tested up to 21 days. An SEM study of osteoblast attachment helped to explain the topographical impact diamond had on osteoblast functions by showing altered filopodia extensions on the different diamond topographies. In summary, these results provided insights into understanding the role diamond nanotopography had on osteoblast interactions and more importantly, the application of diamond films to improve orthopedic implant lifetimes.

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

    NASA Astrophysics Data System (ADS)

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

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

  9. Collagen production of osteoblasts revealed by ultra-high voltage electron microscopy.

    PubMed

    Hosaki-Takamiya, Rumiko; Hashimoto, Mana; Imai, Yuichi; Nishida, Tomoki; Yamada, Naoko; Mori, Hirotaro; Tanaka, Tomoyo; Kawanabe, Noriaki; Yamashiro, Takashi; Kamioka, Hiroshi

    2016-09-01

    In the bone, collagen fibrils form a lamellar structure called the "twisted plywood-like model." Because of this unique structure, bone can withstand various mechanical stresses. However, the formation of this structure has not been elucidated because of the difficulty of observing the collagen fibril production of the osteoblasts via currently available methods. This is because the formation occurs in the very limited space between the osteoblast layer and bone matrix. In this study, we used ultra-high-voltage electron microscopy (UHVEM) to observe collagen fibril production three-dimensionally. UHVEM has 3-MV acceleration voltage and enables us to use thicker sections. We observed collagen fibrils that were beneath the cell membrane of osteoblasts elongated to the outside of the cell. We also observed that osteoblasts produced collagen fibrils with polarity. By using AVIZO software, we observed collagen fibrils produced by osteoblasts along the contour of the osteoblasts toward the bone matrix area. Immediately after being released from the cell, the fibrils run randomly and sparsely. But as they recede from the osteoblast, the fibrils began to run parallel to the definite direction and became thick, and we observed a periodical stripe at that area. Furthermore, we also observed membrane structures wrapped around filamentous structures inside the osteoblasts. The filamentous structures had densities similar to the collagen fibrils and a columnar form and diameter. Our results suggested that collagen fibrils run parallel and thickly, which may be related to the lateral movement of the osteoblasts. UHVEM is a powerful tool for observing collagen fibril production.

  10. Rotary culture enhances pre-osteoblast aggregation and mineralization.

    PubMed

    Facer, S R; Zaharias, R S; Andracki, M E; Lafoon, J; Hunter, S K; Schneider, G B

    2005-06-01

    Three-dimensional environments have been shown to enhance cell aggregation and osteoblast differentiation. Thus, we hypothesized that three-dimensional (3D) growth environments would enhance the mineralization rate of human embryonic palatal mesenchymal (HEPM) pre-osteoblasts. The objective of this study was to investigate the potential use of rotary cell culture systems (RCCS) as a means to enhance the osteogenic potential of pre-osteoblast cells. HEPM cells were cultured in a RCCS to create 3D enviroments. Tissue culture plastic (2D) cultures served as our control. 3D environments promoted three-dimensional aggregate formations. Increased calcium and phosphorus deposition was significantly enhanced three- to 18-fold (P < 0.001) in 3D cultures as compared with 2D environments. 3D cultures mineralized in 1 wk as compared with the 2D cultures, which took 4 wks, a decrease in time of nearly 75%. In conclusion, our studies demonstrated that 3D environments enhanced osteoblast cell aggregation and mineralization.

  11. 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.

  12. 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.

  13. Mechanical loading increases detection of estrogen receptor-alpha in osteocytes and osteoblasts despite chronic energy restriction.

    PubMed

    Swift, Sibyl N; Swift, Joshua M; Bloomfield, Susan A

    2014-12-01

    Estrogen receptor-α (ER-α) is an important mediator of the bone response to mechanical loading. We sought to determine whether restricting dietary energy intake by 40% limits the bone formation rate (BFR) response to mechanical loading (LOAD) by downregulating ER-α-expressing osteocytes, or osteoblasts, or both. Female rats (n = 48, 7 mo old) were randomized to ADLIB-SHAM and ADLIB-LOAD groups fed AIN-93M purified diet ad libitum or to ER40-SHAM and ER40-LOAD groups fed modified AIN-93M with 40% less energy (100% of all other nutrients). After 12 wk, LOAD rats were subjected to a muscle contraction protocol three times every third day. ER40 produced lower proximal tibia bone volume (-22%), trabecular thickness (-14%), and higher trabecular separation (+127%) in SHAM but not LOAD rats. ER40 rats exhibited reductions in mineral apposition rate, but not percent mineralizing surface or BFR. LOAD induced similar relative increases in these kinetic measures of osteoblast activity/recruitment in both diet groups., but absolute values for ER40 LOAD rats were lower vs. ADLIB-LOAD. There were fourfold and eightfold increases in proportion of estrogen receptor-α protein-positive osteoblast and osteocytes, respectively, in LOAD vs. SHAM rats, with no effect of ER40. These data suggest that a brief period of mechanical loading significantly affects estrogen receptor-α in cancellous bone osteoblasts and osteocytes. Chronic energy restriction does result in lower absolute values in indices of osteoblast activity after mechanical loading, but not by a smaller increment relative to unloaded bones; this change is not explained by an associated downregulation of ER-α in osteoblasts or osteocytes.

  14. 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. © 2016 Wiley Periodicals, Inc.

  15. EDA-Fibronectin Originating from Osteoblasts Inhibits the Immune Response against Cancer

    PubMed Central

    Rossnagl, Stephanie; Altrock, Eva; Sens, Carla; Kraft, Sabrina; Rau, Katrin; Giese, Thomas; Samstag, Yvonne; Nakchbandi, Inaam A.

    2016-01-01

    Osteoblasts lining the inner surface of bone support hematopoietic stem cell differentiation by virtue of proximity to the bone marrow. The osteoblasts also modify their own differentiation by producing various isoforms of fibronectin (FN). Despite evidence for immune regulation by osteoblasts, there is limited knowledge of how osteoblasts modulate cells of the immune system. Here, we show that extra domain A (EDA)-FN produced by osteoblasts increases arginase production in myeloid-derived cells, and we identify α5β1 as the mediating receptor. In different mouse models of cancer, osteoblasts or EDA-FN was found to up-regulate arginase-1 expression in myeloid-derived cells, resulting in increased cancer growth. This harmful effect can be reduced by interfering with the integrin α5β1 receptor or inhibiting arginase. Conversely, in tissue injury, the expression of arginase-1 is normally beneficial as it dampens the immune response to allow wound healing. We show that EDA-FN protects against excessive fibrotic tissue formation in a liver fibrosis model. Our results establish an immune regulatory function for EDA-FN originating from the osteoblasts and identify new avenues for enhancing the immune reaction against cancer. PMID:27653627

  16. Synergistic effects of high dietary calcium and exogenous parathyroid hormone in promoting osteoblastic bone formation in mice.

    PubMed

    Feng, Yuxu; Zhou, Min; Zhang, Qunhu; Liu, Huan; Xu, Yong; Shu, Lei; Zhang, Jue; Miao, Dengshun; Ren, Yongxin

    2015-03-28

    In the present study, we investigated whether high dietary Ca and exogenous parathyroid hormone 1-34 fragments (PTH 1-34) have synergistic effects on bone formation in adult mice, and explored the related mechanisms. Adult male mice were fed a normal diet, a high-Ca diet, a PTH-treated diet, or a high-Ca diet combined with subcutaneously injected PTH 1-34 (80 μg/kg per d) for 4 weeks. Bone mineral density, trabecular bone volume, osteoblast number, alkaline phosphatase (ALP)- and type I collagen-positive areas, and the expression levels of osteoblastic bone formation-related genes and proteins were increased significantly in mice fed the high-Ca diet, the PTH-treated diet, and, even more dramatically, the high-Ca diet combined with PTH. Osteoclast number and surface and the ratio of receptor activator for nuclear factor-κB ligand (RANKL):osteoprotegerin (OPG) were decreased in the high-Ca diet treatment group, increased in the PTH treatment group, but not in the combined treatment group. Furthermore, third-passage osteoblasts were treated with high Ca (5 mM), PTH 1-34 (10⁻⁸ M) or high Ca combined with PTH 1-34. Osteoblast viability and ALP activity were increased in either the high Ca-treated or PTH-treated cultures and, even more dramatically, in the cultures treated with high Ca plus PTH, with consistent up-regulation of the expression levels of osteoblast proliferation and differentiation-related genes and proteins. These results indicate that dietary Ca and PTH play synergistic roles in promoting osteoblastic bone formation by stimulating osteoblast proliferation and differentiation.

  17. Prostate cancer cells induce osteoblastic differentiation via semaphorin 3A.

    PubMed

    Liu, Fuzhou; Shen, Weiwei; Qiu, Hao; Hu, Xu; Zhang, Chao; Chu, Tongwei

    2015-03-01

    Prostate cancer metastasis to bone is the second most commonly diagnosed malignant disease among men worldwide. Such metastatic disease is characterized by the presence of osteoblastic bone lesions, and is associated with high rates of mortality. However, the various mechanisms involved in prostate cancer-induced osteoblastic differentiation have not been fully explored. Semaphorin 3A (Sema 3A) is a newly identified regulator of bone metabolism which stimulates differentiation of pre-osteoblastic cells under physiological conditions. We investigated in this study whether prostate cancer cells can mediate osteoblastic activity through Sema 3A. We cultured osteoprogenitor MC3T3-E1 cells in prostate cancer-conditioned medium, and analyzed levels of Sema 3A protein in diverse prostate cancer cell lines to identify cell lines in which Sema 3A production showed a positive correlation with osteo-stimulation. C4-2 cells were stably transfected with Sema 3A short hairpin RNA to further determine whether Sema 3A contributes to the ability of C4-2 cells to induce osteoblastic differentiation. Down-regulation of Sema 3A expression decreased indicators of C4-2 CM-induced osteoblastic differentiation, including alkaline phosphatase production and mineralization. Additionally, silencing or neutralizing Sema 3A in C4-2 cells resulted in diminished β-catenin expression in osteogenitor MC3T3-E1 cells. Our results suggest that prostate cancer-induced osteoblastic differentiation is at least partially mediated by Sema 3A, and may be regulated by the β-catenin signalling pathway. Sema 3A may represent a novel target for treatment of prostate cancer-induced osteoblastic lesions. © 2014 Wiley Periodicals, Inc.

  18. Adhesion of osteoblasts to a nanorough titanium implant surface

    PubMed Central

    Gongadze, Ekaterina; Kabaso, Doron; Bauer, Sebastian; Slivnik, Tomaž; Schmuki, Patrik; van Rienen, Ursula; Iglič, Aleš

    2011-01-01

    This work considers the adhesion of cells to a nanorough titanium implant surface with sharp edges. The basic assumption was that the attraction between the negatively charged titanium surface and a negatively charged osteoblast is mediated by charged proteins with a distinctive quadrupolar internal charge distribution. Similarly, cation-mediated attraction between fibronectin molecules and the titanium surface is expected to be more efficient for a high surface charge density, resulting in facilitated integrin mediated osteoblast adhesion. We suggest that osteoblasts are most strongly bound along the sharp convex edges or spikes of nanorough titanium surfaces where the magnitude of the negative surface charge density is the highest. It is therefore plausible that nanorough regions of titanium surfaces with sharp edges and spikes promote the adhesion of osteoblasts. PMID:21931478

  19. The sp7 gene is required for maturation of osteoblast-lineage cells in medaka (Oryzias latipes) vertebral column development.

    PubMed

    Azetsu, Yuki; Inohaya, Keiji; Takano, Yoshiro; Kinoshita, Masato; Tasaki, Mai; Kudo, Akira

    2017-11-15

    Sp7 is a zinc finger transcription factor that is essential for osteoblast differentiation in mammals. To verify the characteristic features of osteoblast-lineage cells in teleosts, we established medaka sp7 mutants using a transcription activator-like effector nuclease (TALEN) genome editing system. These mutants showed severe defects in the formation of skeletal structures. In particular, the neural and the hemal arches were not formed, although the chordal centra were formed. Analysis of the transgenic medaka revealed that sp7 mutant had normal distribution of type X collagen a1 a (col10a1a)-positive osteoblast-like cells around the centrum and at the proximal region of the vertebral arch. The sp7 mutant phenotype could be rescued by exogenous sp7 expression in col10a1a-positive cells, as well as in sp7-positive osteoblast cells. Furthermore, runx2-positive osteoblast progenitors were observed on the vertebral arches, but not on the centrum, during vertebral column development. In addition, these osteoblast progenitors differentiated into the col10a1a-positive cells. In sp7 mutant, the runx2-positive cells were normally distributed at the region of unformed vertebral arch but failed to differentiate into col10a1a-positive cells. These results indicate that osteoblast-lineage cells undergo two distinct differentiation processes during development of the vertebral arch and the centrum. Nevertheless, our results verified that sp7 gene expression in osteoblast-lineage cells is required for differentiation into mature osteoblasts to form the vertebral column and other skeletal structures. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. 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.

  1. Anabolic activity of ursolic acid in bone: Stimulating osteoblast differentiation in vitro and inducing new bone formation in vivo.

    PubMed

    Lee, Su-Ui; Park, Sang-Joon; Kwak, Han Bok; Oh, Jaemin; Min, Yong Ki; Kim, Seong Hwan

    2008-01-01

    In the field of osteoporosis, there has been growing interest in anabolic agents that enhance bone mass and improve bone architecture. In this study, we demonstrated that the ubiquitous plant triterpenoid, ursolic acid, enhances differentiation and mineralization of osteoblasts in vitro. We found that ursolic acid induced the expression of osteoblast-specific genes with the activation of mitogen-activated protein kinases, nuclear factor-kappaB, and activator protein-1. Additionally, noggin, an antagonist of bone morphogenetic proteins (BMPs), inhibited ursolic acid-induced osteoblast differentiation. Noggin also inhibited the activation of Smad and the induction of BMP-2 mRNA expression by ursolic acid in the late stage of osteoblast differentiation. Importantly, ursolic acid was shown to have bone-forming activity in vivo in a mouse calvarial bone formation model. A high proportion of positive immunostaining of BMP-2 was found in the nuclear region of woven bone formed by ursolic acid. These results suggested that ursolic acid has the anabolic potential to stimulate osteoblast differentiation and enhance new bone formation.

  2. DYSAPOPTOSIS OF OSTEOBLASTS AND OSTEOCYTES INCREASES CANCELLOUS BONE FORMATION BUT EXAGGERATES BONE POROSITY WITH AGE

    PubMed Central

    Jilka, Robert L.; O’Brien, Charles A.; Roberson, Paula K.; Bonewald, Lynda F.; Weinstein, Robert S.; Manolagas, Stavros C.

    2013-01-01

    Skeletal aging is accompanied by decreased cancellous bone mass and increased formation of pores within cortical bone. The latter accounts for a large portion of the increase in non-vertebral fractures after age 65 in humans. We selectively deleted Bak and Bax, two genes essential for apoptosis, in two types of terminally differentiated bone cells: the short-lived osteoblasts that elaborate the bone matrix, and the long-lived osteocytes that are immured within the mineralized matrix and choreograph the regeneration of bone. Attenuation of apoptosis in osteoblasts increased their working lifespan and thereby cancellous bone mass in the femur. In long-lived osteocytes, however, it caused dysfunction with advancing age and greatly magnified intracortical femoral porosity associated with increased production of receptor activator of nuclear factor-κB ligand and vascular endothelial growth factor. Increasing bone mass by artificial prolongation of the inherent lifespan of short-lived osteoblasts, while exaggerating the adverse effects of aging on long-lived osteocytes, highlights the seminal role of cell age in bone homeostasis. In addition, our findings suggest that distress signals produced by old and/or dysfunctional osteocytes are the culprits of the increased intracortical porosity in old age. PMID:23761243

  3. Regulation of expression of collagenase-3 in normal, differentiating rat osteoblasts

    NASA Technical Reports Server (NTRS)

    Winchester, S. K.; Bloch, S. R.; Fiacco, G. J.; Partridge, N. C.

    1999-01-01

    We investigated the regulation of collagenase-3 expression in normal, differentiating rat osteoblasts. Fetal rat calvarial cell cultures showed an increase in alkaline phosphatase activity reaching maximal levels between 7-14 days post-confluence, then declining with the onset of mineralization. Collagenase-3 mRNA was just detectable after proliferation ceased at day 7, increased up to day 21, and declined at later ages. Postconfluent cells maintained in non-mineralizing medium expressed collagenase-3 but did not show the developmental increase exhibited by cells switched to mineralization medium. Cells maintained in non-mineralizing medium continued to proliferate; cells in mineralization medium ceased proliferation. In addition, collagenase-3 mRNA was not detected in subcultured cells allowed to remineralize. These results suggest that enhanced accumulation of collagenase-3 mRNA is triggered by cessation of proliferation or acquisition of a mineralized extracellular matrix and that other factors may also be required. After initiation of basal expression, parathyroid hormone (PTH) caused a dose-dependent increase in collagenase-3 mRNA. Both the cyclic adenosine monophosphate (cAMP) analogue, 8-bromo-cAMP (8-Br-cAMP), and the protein kinase C (PKC) activator, phorbol myristate acetate, increased collagenase-3 expression, while the calcium ionophore, ionomycin, did not, suggesting that PTH was acting through the protein kinase A (PKA) and PKC pathways. Inhibition of protein synthesis with cycloheximide caused an increase in basal collagenase-3 expression but blocked the effect of PTH, suggesting that an inhibitory factor prevents basal expression while an inductive factor is involved with PTH action. In summary, collagenase-3 is expressed in mineralized osteoblasts and cessation of proliferation and initiation of mineralization are triggers for collagenase-3 expression. PTH also stimulates expression of the enzyme through both PKA and PKC pathways in the

  4. Mediator MED23 cooperates with RUNX2 to drive osteoblast differentiation and bone development.

    PubMed

    Liu, Zhen; Yao, Xiao; Yan, Guang; Xu, YiChi; Yan, Jun; Zou, Weiguo; Wang, Gang

    2016-04-01

    How lineage specifiers are regulated during development is an outstanding question, and the molecular regulation of osteogenic factor RUNX2 remains to be fully understood. Here we report that the Mediator subunit MED23 cooperates with RUNX2 to regulate osteoblast differentiation and bone development. Med23 deletion in mesenchymal stem cells or osteoblast precursors results in multiple bone defects similar to those observed in Runx2(+/-) mice. In vitro, Med23-deficient progenitor cells are refractory to osteoblast differentiation, and Med23 deficiency reduces Runx2-target gene activity without changing Runx2 expression. Mechanistically, MED23 binds to RUNX2 and modulates its transcriptional activity. Moreover, Med23 deficiency in osteoprogenitor cells exacerbates the skeletal abnormalities observed in Runx2(+/-) mice. Collectively, our results establish a genetic and physical interaction between RUNX2 and MED23, suggesting that MED23 constitutes a molecular node in the regulatory network of anabolic bone formation and related diseases.

  5. Arsenic induces cell apoptosis in cultured osteoblasts through endoplasmic reticulum stress

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tang, C.-H., E-mail: chtang@mail.cmu.edu.t; Graduate Institute of Basic Medical Science, China Medical University, Taichung Taiwan; Chiu, Y.-C.

    Osteoporosis is characterized by low bone mass resulting from an imbalance between bone resorption by osteoclasts and bone formation by osteoblasts. Therefore, decreased bone formation by osteoblasts may lead to the development of osteoporosis, and rate of apoptosis is responsible for the regulation of bone formation. Arsenic (As) exists ubiquitously in our environment and increases the risk of neurotoxicity, liver injury, peripheral vascular disease and cancer. However, the effect of As on apoptosis of osteoblasts is mostly unknown. Here, we found that As induced cell apoptosis in osteoblastic cell lines (including hFOB, MC3T3-E1 and MG-63) and mouse bone marrow stromalmore » cells (M2-10B4). As also induced upregulation of Bax and Bak, downregulation of Bcl-2 and dysfunction of mitochondria in osteoblasts. As also triggered endoplasmic reticulum (ER) stress, as indicated by changes in cytosolic-calcium levels. We found that As increased the expression and activities of glucose-regulated protein 78 (GRP78) and calpain. Transfection of cells with GRP78 or calpain siRNA reduced As-mediated cell apoptosis in osteoblasts. Therefore, our results suggest that As increased cell apoptosis in cultured osteoblasts and increased the risk of osteoporosis.« less

  6. LIF inhibits osteoblast differentiation at least in part by regulation of HAS2 and its product hyaluronan.

    PubMed

    Falconi, Dominic; Aubin, Jane E

    2007-08-01

    LIF arrests osteogenesis in fetal rat calvaria cells in a differentiation stage-specific manner. Differential display identified HAS2 as a LIF-induced gene and its product, HA, modulated osteoblast differentiation similarly to LIF. Our data suggest that LIF arrests osteoblast differentiation by altering HA content of the extracellular matrix. Leukemia inhibitory factor (LIF) elicits both anabolic and catabolic effects on bone. We previously showed in the fetal rat calvaria (RC) cell system that LIF inhibits osteoblast differentiation at the late osteoprogenitor/early osteoblast stage. To uncover potential molecular mediators of this inhibitory activity, we used a positive-negative genome-wide differential display screen to identify LIF-induced changes in the developing osteoblast transcriptome. Although LIF signaling is active throughout the RC cell proliferation-differentiation sequence, only a relatively small number of genes, in several different functional clusters, are modulated by LIF specifically during the LIF-sensitive inhibitory time window. Based on their known and predicted functions, most of the LIF-regulated genes identified are plausible candidates to be involved in the LIF-induced arrest of osteoprogenitor differentiation. To test this hypothesis, we further analyzed the function of one of the genes identified, hyaluronan synthase 2 (HAS2), in the LIF-induced inhibition. Synthesis of hyaluronan (HA), the product of HAS enzymatic activity, was stimulated by LIF and mimicked the HAS2 expression profile, with highest expression in early/proliferative and late/maturing cultures and lowest levels in intermediate/late osteoprogenitor-early osteoblast cultures. Exogenously added high molecular weight HA, the product of HAS2, dose-dependently inhibited osteoblast differentiation, with pulse-treatment effective in the same differentiation stage-specific inhibitory window as seen with LIF. In addition, however, pulse treatment with HA in early cultures

  7. Alpha-adrenergic blocker mediated osteoblastic stem cell differentiation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Choi, Yoon Jung; Lee, Jue Yeon; Research Center, Nano Intelligent Biomedical Engineering Corporation

    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 clinicallymore » 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

  8. Primary human osteoblasts grow into porous tantalum and maintain an osteoblastic phenotype.

    PubMed

    Welldon, Katie J; Atkins, Gerald J; Howie, Donald W; Findlay, David M

    2008-03-01

    Porous tantalum (Ta) has found application in orthopedics, although the interaction of human osteoblasts (HOB) with this material has not been reported. The aim of this study was to investigate the interaction of primary HOB with porous tantalum, using 5-mm thick discs of porous tantalum. Comparison was made with discs of solid tantalum and tissue culture plastic. Confocal microscopy was used to investigate the attachment and growth of cells on porous Ta, and showed that HOB attached successfully to the metal "trabeculae," underwent extensive cell division, and penetrated into the Ta pores. The maturation of HOB on porous Ta was determined in terms of cell expression of the osteoblast phenotypic markers, STRO-1, and alkaline phosphatase. Despite some donor-dependent variation in STRO-1/AlkPhos expression, growth of cells grown on porous Ta either promoted, or did not impede, the maturation of HOB. In addition, the expression of key osteoblastic genes was investigated after 14 days of culture. The relative levels of mRNA encoding osteocalcin, osteopontin and receptor activator of NFkappaB ligand (RANKL) was not different between porous or solid Ta or plastic, although these genes were expressed differently by cells of different donors. However, bone sialoprotein and type I collagen mRNA species showed a decreased expression on porous Ta compared with expression on plastic. No substrate-dependent differences were seen in the extent of in vitro mineralization by HOB. These results indicate that porous Ta is a good substrate for the attachment, growth, and differentiated function of HOB. (c) 2007 Wiley Periodicals, Inc.

  9. Induction of osteoblast differentiation by selective activation of kinase-mediated actions of the estrogen receptor.

    PubMed

    Kousteni, Stavroula; Almeida, Maria; Han, Li; Bellido, Teresita; Jilka, Robert L; Manolagas, Stavros C

    2007-02-01

    Estrogens control gene transcription by cis or trans interactions of the estrogen receptor (ER) with target DNA or via the activation of cytoplasmic kinases. We report that selective activation of kinase-mediated actions of the ER with 4-estren-3alpha,17beta-diol (estren) or an estradiol-dendrimer conjugate, each a synthetic compound that stimulates kinase-mediated ER actions 1,000 to 10,000 times more potently than direct DNA interactions, induced osteoblastic differentiation in established cell lines of uncommitted osteoblast precursors and primary cultures of osteoblast progenitors by stimulating Wnt and BMP-2 signaling in a kinase-dependent manner. In sharp contrast, 17beta-estradiol (E(2)) suppressed BMP-2-induced osteoblast progenitor commitment and differentiation. Consistent with the in vitro findings, estren, but not E(2), stimulated Wnt/beta-catenin-mediated transcription in T-cell factor-lacZ transgenic mice. Moreover, E(2) stimulated BMP signaling in mice in which ERalpha lacks DNA binding activity and classical estrogen response element-mediated transcription (ERalpha(NERKI/-)) but not in wild-type controls. This evidence reveals for the first time the existence of a large signalosome in which inputs from the ER, kinases, bone morphogenetic proteins, and Wnt signaling converge to induce differentiation of osteoblast precursors. ER can either induce it or repress it, depending on whether the activating ligand (and presumably the resulting conformation of the receptor protein) precludes or accommodates ERE-mediated transcription.

  10. Low-intensity pulsed ultrasound stimulation promotes osteoblast differentiation through hedgehog signaling.

    PubMed

    Matsumoto, Kenichi; Shimo, Tsuyoshi; Kurio, Naito; Okui, Tatsuo; Ibaragi, Soichiro; Kunisada, Yuki; Obata, Kyoichi; Masui, Masanori; Pai, Pang; Horikiri, Yuu; Yamanaka, Nobuyuki; Takigawa, Masaharu; Sasaki, Akira

    2018-06-01

    Low-intensity pulsed ultrasound (LIPUS) has been used as an adjunct to fracture healing therapies, but the mechanisms underlying its action are not known. We reported that sonic hedgehog (SHH) signaling was activated in osteoblasts at the dynamic remodeling site of a bone fracture. Mechanical stimulation is a crucial factor in bone remodeling, and it is related to the primary cilia as a sensor of hedgehog signaling. Here we observed that LIPUS promoted callus formation in accord with Gli2-positive cells after 14 days at the mouse femur fractured site compared with a control group. An immunofluorescence analysis showed that the numbers of primary cilia and cilia/osterix double-positive osteoblasts were increased at the fracture site by LIPUS. LIPUS stimulated not only the number and the length of primary cilia, but also the levels of ciliated protein, Ift88 mRNA, and SHH, Gli1, and Gli2 in MC3T3-E1 cells. Further experiments revealed that LIPUS stimulated osteogenic differentiation in the presence of smoothened agonist (SAG) treatment. These results indicate that LIPUS stimulates osteogenic differentiation and the maturation of osteoblasts by a primary cilium-mediated activation of hedgehog signaling. © 2017 Wiley Periodicals, Inc.

  11. Reduced proliferation and osteocalcin expression in osteoblasts of male idiopathic osteoporosis.

    PubMed

    Ruiz-Gaspà, Sílvia; Blanch-Rubió, Josep; Ciria-Recasens, Manuel; Monfort, Jordi; Tío, Laura; Garcia-Giralt, Natàlia; Nogués, Xavier; Monllau, Joan C; Carbonell-Abelló, Jordi; Pérez-Edo, Lluis

    2010-03-01

    Osteoporosis is characterized by low bone mineral density (BMD), resulting in increasing susceptibility to bone fractures. In men, it has been related to some diseases and toxic habits, but in some instances the cause of the primary--or idiopathic--osteoporosis is not apparent. In a previous study, our group compared histomorphometric measurements in cortical and cancellous bones from male idiopathic osteoporosis (MIO) patients to those of control subjects and found reduced bone formation without major differences in bone resorption. To confirm these results, this study analyzed the etiology of this pathology, examining the osteoblast behavior in vitro. We compared two parameters of osteoblast activity in MIO patients and controls: osteoblastic proliferation and gene expression of COL1A1 and osteocalcin, in basal conditions and with vitamin D(3) added. All these experiments were performed from a first-passage osteoblastic culture, obtained from osteoblasts that had migrated from the transiliac explants to the plate. The results suggested that the MIO osteoblast has a slower proliferation rate and decreased expression of genes related to matrix formation, probably due to a lesser or slower response to some stimulus. We concluded that, contrary to female osteoporosis, in which loss of BMD is predominantly due to increased resorption, low BMD in MIO seems to be due to an osteoblastic defect.

  12. Lactoferrin – A Novel Bone Growth Factor

    PubMed Central

    Naot, Dorit; Grey, Andrew; Reid, Ian R; Cornish, Jillian

    2005-01-01

    Lactoferrin is an iron-binding glycoprotein that belongs to the transferrin family. It is present in breast milk, in epithelial secretions, and in the secondary granules of neutrophils. In healthy subjects lactoferrin circulates at concentrations of 2–7 x 10−6 g/ml. Lactoferrin is a pleiotropic factor with potent antimicrobial and immunomodulatory activities. Recently, we have shown that lactoferrin can also promote bone growth. At physiological concentrations, lactoferrin potently stimulates the proliferation and differentiation of primary osteoblasts and also acts as a survival factor inhibiting apoptosis induced by serum withdrawal. Lactoferrin also affects osteoclast formation and, in murine bone marrow culture, lactoferrin potently inhibits osteoclastogenesis. In vivo, local injection of lactoferrin above the hemicalvaria of adult mice results in substantial increases in the dynamic histomorphometric indices of bone formation and bone area. The mitogenic effect of lactoferrin in osteoblast-like cells is mediated mainly through LRP1, a member of the family of low-density lipoprotein receptor-related proteins that are primarily known as endocytic receptors. Using confocal laser scanning microscopy, we demonstrated that fluorescently labeled lactoferrin is endocytosed and can be visualized in the cytoplasm of primary osteoblastic cells. Lactoferrin also induces activation of p42/44 MAPK signaling in primary osteoblasts, but the two pathways seem to operate independently as activation of MAPK signaling, but not endocytosis, is necessary for the mitogenic effect of lactoferrin. We conclude that lactoferrin may have a physiological role in bone growth and healing, and a potential therapeutic role as an anabolic factor in osteoporosis. PMID:16012127

  13. Pyk2 and Megakaryocytes Regulate Osteoblast Differentiation and Migration via Distinct and Overlapping Mechanisms

    PubMed Central

    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

    2016-01-01

    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 (Pyk2Y402F) and kinase-mutant (Pyk2K457A) in Pyk2-KO osteoblasts. Both Pyk2Y402F and Pyk2K457A reduced ALP activity, whereas only kinase-inactive Pyk2K457A 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 WT osteoblasts, Pyk2 expression was not required 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. PMID:26552846

  14. Transduction of Oct6 or Oct9 gene concomitant with Myc family gene induced osteoblast-like phenotypic conversion in normal human fibroblasts

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mizoshiri, N.; Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto; Kishida, T.

    Introduction: Osteoblasts play essential roles in bone formation and regeneration, while they have low proliferation potential. Recently we established a procedure to directly convert human fibroblasts into osteoblasts (dOBs). Transduction of Runx2 (R), Osterix (X), Oct3/4 (O) and L-myc (L) genes followed by culturing under osteogenic conditions induced normal human fibroblasts to express osteoblast-specific genes and produce calcified bone matrix both in vitro and in vivo Intriguingly, a combination of only two factors, Oct3/4 and L-myc, significantly induced osteoblast-like phenotype in fibroblasts, but the mechanisms underlying the direct conversion remains to be unveiled. Materials and Methods: We examined which Oct family genesmore » and Myc family genes are capable of inducing osteoblast-like phenotypic conversion. Results: As result Oct3/4, Oct6 and Oct9, among other Oct family members, had the capability, while N-myc was the most effective Myc family gene. The Oct9 plus N-myc was the best combination to induce direct conversion of human fibroblasts into osteoblast-like cells. Discussion: The present findings may greatly contribute to the elucidation of the roles of the Oct and Myc proteins in osteoblast direct reprogramming. The results may also lead to establishment of novel regenerative therapy for various bone resorption diseases. - Highlights: • Introducing L-myc in a combination with either Oct3/4, Oct6 or Oct9 enables the conversion of fibroblasts to osteoblasts. • A combination of L-myc with Oct3/4 or Oct9 can induce the cells to a phenotype closer to normal osteoblasts. • N-myc was considered the most appropriate Myc family gene for induction of osteoblast-like phenotype in fibroblasts. • The combination of Oct9 plus N-myc has the strongest capability of inducing osteoblast-like phenotype.« less

  15. 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.

  16. Genome-wide DNase hypersensitivity, and occupancy of RUNX2 and CTCF reveal a highly dynamic gene regulome during MC3T3 pre-osteoblast differentiation.

    PubMed

    Tai, Phillip W L; Wu, Hai; van Wijnen, André J; Stein, Gary S; Stein, Janet L; Lian, Jane B

    2017-01-01

    The ability to discover regulatory sequences that control bone-related genes during development has been greatly improved by massively parallel sequencing methodologies. To expand our understanding of cis-regulatory regions critical to the control of gene expression during osteoblastogenesis, we probed the presence of open chromatin states across the osteoblast genome using global DNase hypersensitivity (DHS) mapping. Our profiling of MC3T3 mouse pre-osteoblasts during differentiation has identified more than 224,000 unique DHS sites. Approximately 65% of these sites are dynamic during temporal stages of osteoblastogenesis, and a majority of them are located within non-promoter (intergenic and intronic) regions. Nearly half of all DHS sites (both constitutive and dynamic) overlap binding events of the bone-essential RUNX2 and/or the chromatin-related CTCF transcription factors. This finding reinforces the role of these regulatory proteins as essential components of the bone gene regulome. We observe a reduction in chromatin accessibility throughout the genome between pre-osteoblast and early osteoblasts. Our analysis also defined a class of differentially expressed genes that harbor DHS peaks centered within 1 kb downstream of transcriptional end sites (TES). These DHSs at the 3'-flanks of genes exhibit dynamic changes during differentiation that may impact regulation of the osteoblast genome. Taken together, the distribution of DHS regions within non-promoter locations harboring osteoblast and chromatin related transcription factor binding motifs, reflect novel cis-regulatory requirements to support temporal gene expression in differentiating osteoblasts.

  17. Molecular mechanisms underlying the actions of dietary factors on the skeleton

    USDA-ARS?s Scientific Manuscript database

    This book chapter summarizes the current state of knowledge on molecular mechanisms whereby nutritional status and dietary factors found in fruits, vegetables, and grains affect bone turnover and skeletal quality. The Wnt-beta catenin and bone morphogenic protein (BMP) pathways in osteoblast bone ce...

  18. GATA4 negatively regulates bone sialoprotein expression in osteoblasts.

    PubMed

    Song, Insun; Jeong, Byung-Chul; Choi, Yong Jun; Chung, Yoon-Sok; Kim, Nacksung

    2016-06-01

    GATA4 has been reported to act as a negative regulator in osteoblast differentiation by inhibiting the Dlx5 transactivation of Runx2 via the attenuation of the binding ability of Dlx5 to the Runx2 promoter region. Here, we determine the role of GATA4 in the regulation of bone sialoprotein (Bsp) in osteoblasts. We observed that the overexpression of Runx2 or Sox9 induced the Bsp expression in osteoblastic cells. Silencing GATA4 further enhanced the Runx2- and Sox9-mediated Bsp promoter activity, whereas GATA4 overexpression down-regulated Bsp promoter activity mediated by Runx2 and Sox9. GATA4 also interacted with Runx2 and Sox9, by attenuating the binding ability of Runx2 and Sox9 to the Bsp promoter region. Our data suggest that GATA4 acts as a negative regulator of Bsp expression in osteoblasts. [BMB Reports 2016; 49(6): 343-348].

  19. Maf promotes osteoblast differentiation in mice by mediating the age-related switch in mesenchymal cell differentiation

    PubMed Central

    Nishikawa, Keizo; Nakashima, Tomoki; Takeda, Shu; Isogai, Masashi; Hamada, Michito; Kimura, Ayako; Kodama, Tatsuhiko; Yamaguchi, Akira; Owen, Michael J.; Takahashi, Satoru; Takayanagi, Hiroshi

    2010-01-01

    Aging leads to the disruption of the homeostatic balance of multiple biological systems. In bone marrow multipotent mesenchymal cells undergo differentiation into various anchorage-dependent cell types, including osteoblasts and adipocytes. With age as well as with treatment of antidiabetic drugs such as thiazolidinediones, mesenchymal cells favor differentiation into adipocytes, resulting in an increased number of adipocytes and a decreased number of osteoblasts, causing osteoporosis. The mechanism behind this differentiation switch is unknown. Here we show an age-related decrease in the expression of Maf in mouse mesenchymal cells, which regulated mesenchymal cell bifurcation into osteoblasts and adipocytes by cooperating with the osteogenic transcription factor Runx2 and inhibiting the expression of the adipogenic transcription factor Pparg. The crucial role of Maf in both osteogenesis and adipogenesis was underscored by in vivo observations of delayed bone formation in perinatal Maf–/– mice and an accelerated formation of fatty marrow associated with bone loss in aged Maf+/– mice. This study identifies a transcriptional mechanism for an age-related switch in cell fate determination and may provide a molecular basis for novel therapeutic strategies against age-related bone diseases. PMID:20877012

  20. 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.

  1. Obesity does not aggravate osteoporosis or osteoblastic insulin resistance in orchiectomized rats.

    PubMed

    Potikanond, Saranyapin; Rattanachote, Pinyada; Pintana, Hiranya; Suntornsaratoon, Panan; Charoenphandhu, Narattaphol; Chattipakorn, Nipon; Chattipakorn, Siriporn

    2016-02-01

    The present study aimed to test the hypothesis that testosterone deprivation impairs osteoblastic insulin signaling, decreases osteoblast survival, reduces bone density, and that obesity aggravates those deleterious effects in testosterone-deprived rats. Twenty four male Wistar rats underwent either a bilateral orchiectomy (O, n=12) or a sham operation (S, n=12). Then the rats in each group were further divided into two subgroups fed with either a normal diet (ND) or a high-fat diet (HF) for 12 weeks. At the end of the protocol, blood samples were collected to determine metabolic parameters and osteocalcin ratios. The tibiae were collected to determine bone mass using microcomputed tomography and for osteoblast isolation. The results showed that rats fed with HF (sham-operated HF-fed rats (HFS) and ORX HF-fed rats (HFO)) developed peripheral insulin resistance and had decreased trabecular bone density. In ND-fed rats, only the ORX ND-fed rats (NDO) group had decreased trabecular bone density. In addition, osteoblastic insulin resistance, as indicated by a decrease in tyrosine phosphorylation of the insulin receptor and Akt, were observed in all groups except the sham-operated ND-fed rats (NDS) rats. Those groups, again with the exception of the NDS rats, also had decreased osteoblastic survival. No differences in the levels of osteoblastic insulin resistance and osteoblastic survival were found among the NDO, HFS, and HFO groups. These findings suggest that either testosterone deprivation or obesity alone can impair osteoblastic insulin signaling and decrease osteoblastic survival leading to the development of osteoporosis. However, obesity does not aggravate those deleterious effects in the bone of testosterone-deprived rats. © 2016 Society for Endocrinology.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Nishimura, Noriko; Nishimura, Hisao; Ito, Tomohiro

    2009-05-01

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

  3. Epigenetic Library Screen Identifies Abexinostat as Novel Regulator of Adipocytic and Osteoblastic Differentiation of Human Skeletal (Mesenchymal) Stem Cells

    PubMed Central

    Ali, Dalia; Hamam, Rimi; Alfayez, Musaed; Kassem, Moustapha; Aldahmash, Abdullah

    2016-01-01

    The epigenetic mechanisms promoting lineage-specific commitment of human skeletal (mesenchymal or stromal) stem cells (hMSCs) into adipocytes or osteoblasts are still not fully understood. Herein, we performed an epigenetic library functional screen and identified several novel compounds, including abexinostat, which promoted adipocytic and osteoblastic differentiation of hMSCs. Using gene expression microarrays, chromatin immunoprecipitation for H3K9Ac combined with high-throughput DNA sequencing (ChIP-seq), and bioinformatics, we identified several key genes involved in regulating stem cell proliferation and differentiation that were targeted by abexinostat. Concordantly, ChIP-quantitative polymerase chain reaction revealed marked increase in H3K9Ac epigenetic mark on the promoter region of AdipoQ, FABP4, PPARγ, KLF15, CEBPA, SP7, and ALPL in abexinostat-treated hMSCs. Pharmacological inhibition of focal adhesion kinase (PF-573228) or insulin-like growth factor-1R/insulin receptor (NVP-AEW51) signaling exhibited significant inhibition of abexinostat-mediated adipocytic differentiation, whereas inhibition of WNT (XAV939) or transforming growth factor-β (SB505124) signaling abrogated abexinostat-mediated osteogenic differentiation of hMSCs. Our findings provide insight into the understanding of the relationship between the epigenetic effect of histone deacetylase inhibitors, transcription factors, and differentiation pathways governing adipocyte and osteoblast differentiation. Manipulating such pathways allows a novel use for epigenetic compounds in hMSC-based therapies and tissue engineering. Significance This unbiased epigenetic library functional screen identified several novel compounds, including abexinostat, that promoted adipocytic and osteoblastic differentiation of human skeletal (mesenchymal or stromal) stem cells (hMSCs). These data provide new insight into the understanding of the relationship between the epigenetic effect of histone deacetylase

  4. Three dimensional culture of the murine osteoblastic cell line OCT-1 on collagen coated microcarriers

    NASA Astrophysics Data System (ADS)

    Lau, P.; Hellweg, C. E.; Kirchner, S.; Baumstark-Khan, C.

    2005-08-01

    During long-term space missions, astronauts suffer from the loss of minerals especially from weightbearing bones due to prolonged sojourn under microgravity. Bone loss during space flight is about 1-2% per month. Bone is continually being remodelled under the influence of three types of highly specialized cells. Osteoblasts, the bone forming cells, osteoclasts, the bone resorbing cells and finally osteocytes preserve the homeostasis of bone formation and resorption. In vitro 3- dimensional cell culture of osteoblastic cell lines on microcarrier beads might be a better model to evaluate changes in bone cell morphology, function and differentiation under influence of spaceflight related factors than the conventional 2-D monolayer culture technique. Furthermore, it allows production of a greater amount of cells compared to the monolayer culture. Aim of this study is to examine the effects of culturing the immortalized murine osteoblastic cell line OCT-1 in a 3- dimensional environment on cell morphology and proliferation rate.

  5. The effects of tumour necrosis factor-α on bone cells involved in periodontal alveolar bone loss; osteoclasts, osteoblasts and osteocytes.

    PubMed

    Algate, K; Haynes, D R; Bartold, P M; Crotti, T N; Cantley, M D

    2016-10-01

    Periodontitis is the most common bone loss pathology in adults and if left untreated is responsible for premature tooth loss. Cytokines, such as tumour necrosis factor-α (TNFα), involved in the chronic inflammatory response within the periodontal gingiva, significantly influence the normal bone remodelling processes. In this review, the effects of TNFα on bone metabolism in periodontitis are evaluated in relation to its direct and indirect actions on bone cells including osteoclasts, osteoblasts and osteocytes. Evidence published to date suggests a potent catabolic role for TNFα through the stimulation of osteoclastic bone resorption as well as the suppression of osteoblastic bone formation and osteocytic survival. However, the extent and timing of TNFα exposure in vitro and in vivo greatly influences its effect on skeletal cells, with contradictory anabolic activity observed with TNFα in a number of studies. None the less, it is evident that managing the chronic inflammatory response in addition to the deregulated bone metabolism is required to improve periodontal and inflammatory bone loss treatments‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  6. Osteoblasts derived from osteophytes produce interleukin-6, interleukin-8, and matrix metalloproteinase-13 in osteoarthritis.

    PubMed

    Sakao, Kei; Takahashi, Kenji A; Arai, Yuji; Saito, Masazumi; Honjo, Kuniaki; Hiraoka, Nobuyuki; Asada, Hidetsugu; Shin-Ya, Masaharu; Imanishi, Jiro; Mazda, Osam; Kubo, Toshikazu

    2009-01-01

    To clarify the significance of the osteophytes that appear during the progression of osteoarthritis (OA), we investigated the expression of inflammatory cytokines and proteases in osteoblasts from osteophytes. We also examined the influence of mechanical stress loading on osteoblasts on the expression of inflammatory cytokines and proteases. Osteoblasts were isolated from osteophytes in 19 patients diagnosed with knee OA and from subchondral bone in 4 patients diagnosed with femoral neck fracture. Messenger RNA expression and protein production of inflammatory cytokines and proteases were analyzed using real-time RT-PCR and ELISA, respectively. To examine the effects of mechanical loading, continuous hydrostatic pressure was applied to the osteoblasts. We determined the mRNA expression and protein production of IL-6, IL-8, and MMP-13, which are involved in the progression of OA, were increased in the osteophytes. Additionally, when OA pathological conditions were simulated by applying a nonphysiological mechanical stress load, the gene expression of IL-6 and IL-8 increased. Our results suggested that nonphysiological mechanical stress may induce the expression of biological factors in the osteophytes and is involved in OA progression. By controlling the expression of these genes in the osteophytes, the progression of cartilage degeneration in OA may be reduced, suggesting a new treatment strategy for OA.

  7. The interfacial pH of acidic degradable polymeric biomaterials and its effects on osteoblast behavior.

    PubMed

    Ruan, Changshun; Hu, Nan; Ma, Yufei; Li, Yuxiao; Liu, Juan; Zhang, Xinzhou; Pan, Haobo

    2017-07-28

    A weak alkaline environment is established to facilitate the growth of osteoblasts. Unfortunately, this is inconsistent with the application of biodegradable polymer in bone regeneration, as the degradation products are usually acidic. In this study, the variation of the interfacial pH of poly (D, L-lactide) and piperazine-based polyurethane ureas (P-PUUs), as the representations of acidic degradable materials, and the behavior of osteoblasts on these substrates with tunable interfacial pH were investigated in vitro. These results revealed that the release of degraded products caused a rapid decrease in the interfacial pH, and this could be relieved by the introduction of alkaline segments. On the contrary, when culturing with osteoblasts, the variation of the interfacial pH revealed an upward tendency, indicating that cell could construct the microenvironment by secreting cellular metabolites to satisfy its own survival. In addition, the behavior of osteoblasts on substrates exhibited that P-PUUs with the most PP units were better for cell growth and osteogenic differentiation of cells. This is due to the hydrophilic surface and the moderate N% in P-PUUs, key factors in the promotion of the early stages of cellular responses, and the interfacial pH contributing to the enhanced effect on osteogenic differentiation.

  8. 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.

  9. SLM Produced Porous Titanium Implant Improvements for Enhanced Vascularization and Osteoblast Seeding

    PubMed Central

    Matena, Julia; Petersen, Svea; Gieseke, Matthias; Kampmann, Andreas; Teske, Michael; Beyerbach, Martin; Murua Escobar, Hugo; Haferkamp, Heinz; Gellrich, Nils-Claudius; Nolte, Ingo

    2015-01-01

    To improve well-known titanium implants, pores can be used for increasing bone formation and close bone-implant interface. Selective Laser Melting (SLM) enables the production of any geometry and was used for implant production with 250-µm pore size. The used pore size supports vessel ingrowth, as bone formation is strongly dependent on fast vascularization. Additionally, proangiogenic factors promote implant vascularization. To functionalize the titanium with proangiogenic factors, polycaprolactone (PCL) coating can be used. The following proangiogenic factors were examined: vascular endothelial growth factor (VEGF), high mobility group box 1 (HMGB1) and chemokine (C-X-C motif) ligand 12 (CXCL12). As different surfaces lead to different cell reactions, titanium and PCL coating were compared. The growing into the porous titanium structure of primary osteoblasts was examined by cross sections. Primary osteoblasts seeded on the different surfaces were compared using Live Cell Imaging (LCI). Cross sections showed cells had proliferated, but not migrated after seven days. Although the cell count was lower on titanium PCL implants in LCI, the cell count and cell spreading area development showed promising results for titanium PCL implants. HMGB1 showed the highest migration capacity for stimulating the endothelial cell line. Future perspective would be the incorporation of HMGB1 into PCL polymer for the realization of a slow factor release. PMID:25849656

  10. SLM produced porous titanium implant improvements for enhanced vascularization and osteoblast seeding.

    PubMed

    Matena, Julia; Petersen, Svea; Gieseke, Matthias; Kampmann, Andreas; Teske, Michael; Beyerbach, Martin; Murua Escobar, Hugo; Haferkamp, Heinz; Gellrich, Nils-Claudius; Nolte, Ingo

    2015-04-02

    To improve well-known titanium implants, pores can be used for increasing bone formation and close bone-implant interface. Selective Laser Melting (SLM) enables the production of any geometry and was used for implant production with 250-µm pore size. The used pore size supports vessel ingrowth, as bone formation is strongly dependent on fast vascularization. Additionally, proangiogenic factors promote implant vascularization. To functionalize the titanium with proangiogenic factors, polycaprolactone (PCL) coating can be used. The following proangiogenic factors were examined: vascular endothelial growth factor (VEGF), high mobility group box 1 (HMGB1) and chemokine (C-X-C motif) ligand 12 (CXCL12). As different surfaces lead to different cell reactions, titanium and PCL coating were compared. The growing into the porous titanium structure of primary osteoblasts was examined by cross sections. Primary osteoblasts seeded on the different surfaces were compared using Live Cell Imaging (LCI). Cross sections showed cells had proliferated, but not migrated after seven days. Although the cell count was lower on titanium PCL implants in LCI, the cell count and cell spreading area development showed promising results for titanium PCL implants. HMGB1 showed the highest migration capacity for stimulating the endothelial cell line. Future perspective would be the incorporation of HMGB1 into PCL polymer for the realization of a slow factor release.

  11. 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.

  12. Modifying the osteoblastic niche with zoledronic acid in vivo—Potential implications for breast cancer bone metastasis

    PubMed Central

    Haider, Marie-Therese; Holen, Ingunn; Dear, T. Neil; Hunter, Keith; Brown, Hannah K.

    2014-01-01

    affecting the total number of tumour cells. The number of circulating tumour cells was reduced in ZOL treated animals. Conclusion A single dose of zoledronic acid caused significant changes in the bone area suggested to contain the metastatic niche. Tumour cells arriving in this modified bone microenvironment appeared to preferentially locate to osteoblast-rich areas, supporting that osteoblasts may be key components of the bone metastasis niche and therefore a potential therapeutic target in breast cancer. PMID:24971713

  13. Osteoblast Differentiation and Bone Matrix Formation In Vivo and In Vitro.

    PubMed

    Blair, Harry C; Larrouture, Quitterie C; Li, Yanan; Lin, Hang; Beer-Stoltz, Donna; Liu, Li; Tuan, Rocky S; Robinson, Lisa J; Schlesinger, Paul H; Nelson, Deborah J

    2017-06-01

    We review the characteristics of osteoblast differentiation and bone matrix synthesis. Bone in air breathing vertebrates is a specialized tissue that developmentally replaces simpler solid tissues, usually cartilage. Bone is a living organ bounded by a layer of osteoblasts that, because of transport and compartmentalization requirements, produce bone matrix exclusively as an organized tight epithelium. With matrix growth, osteoblasts are reorganized and incorporated into the matrix as living cells, osteocytes, which communicate with each other and surface epithelium by cell processes within canaliculi in the matrix. The osteoblasts secrete the organic matrix, which are dense collagen layers that alternate parallel and orthogonal to the axis of stress loading. Into this matrix is deposited extremely dense hydroxyapatite-based mineral driven by both active and passive transport and pH control. As the matrix matures, hydroxyapatite microcrystals are organized into a sophisticated composite in the collagen layer by nucleation in the protein lattice. Recent studies on differentiating osteoblast precursors revealed a sophisticated proton export network driving mineralization, a gene expression program organized with the compartmentalization of the osteoblast epithelium that produces the mature bone matrix composite, despite varying serum calcium and phosphate. Key issues not well defined include how new osteoblasts are incorporated in the epithelial layer, replacing those incorporated in the accumulating matrix. Development of bone in vitro is the subject of numerous projects using various matrices and mesenchymal stem cell-derived preparations in bioreactors. These preparations reflect the structure of bone to variable extents, and include cells at many different stages of differentiation. Major challenges are production of bone matrix approaching the in vivo density and support for trabecular bone formation. In vitro differentiation is limited by the organization and

  14. Osteoblast Differentiation and Bone Matrix Formation In Vivo and In Vitro

    PubMed Central

    Larrouture, Quitterie C.; Li, Yanan; Lin, Hang; Beer-Stoltz, Donna; Liu, Li; Tuan, Rocky S.; Robinson, Lisa J.; Schlesinger, Paul H.; Nelson, Deborah J.

    2017-01-01

    We review the characteristics of osteoblast differentiation and bone matrix synthesis. Bone in air breathing vertebrates is a specialized tissue that developmentally replaces simpler solid tissues, usually cartilage. Bone is a living organ bounded by a layer of osteoblasts that, because of transport and compartmentalization requirements, produce bone matrix exclusively as an organized tight epithelium. With matrix growth, osteoblasts are reorganized and incorporated into the matrix as living cells, osteocytes, which communicate with each other and surface epithelium by cell processes within canaliculi in the matrix. The osteoblasts secrete the organic matrix, which are dense collagen layers that alternate parallel and orthogonal to the axis of stress loading. Into this matrix is deposited extremely dense hydroxyapatite-based mineral driven by both active and passive transport and pH control. As the matrix matures, hydroxyapatite microcrystals are organized into a sophisticated composite in the collagen layer by nucleation in the protein lattice. Recent studies on differentiating osteoblast precursors revealed a sophisticated proton export network driving mineralization, a gene expression program organized with the compartmentalization of the osteoblast epithelium that produces the mature bone matrix composite, despite varying serum calcium and phosphate. Key issues not well defined include how new osteoblasts are incorporated in the epithelial layer, replacing those incorporated in the accumulating matrix. Development of bone in vitro is the subject of numerous projects using various matrices and mesenchymal stem cell-derived preparations in bioreactors. These preparations reflect the structure of bone to variable extents, and include cells at many different stages of differentiation. Major challenges are production of bone matrix approaching the in vivo density and support for trabecular bone formation. In vitro differentiation is limited by the organization and

  15. Sequential delivery of BMP-2 and IGF-1 using a chitosan gel with gelatin microspheres enhances early osteoblastic differentiation

    PubMed Central

    Kim, Sungwoo; Kang, Yunqing; Krueger, Chad A.; Sen, Milan; Holcomb, John B.; Chen, Di; Wenke, Joseph C.; Yang, Yunzhi

    2012-01-01

    The purpose of this study was to develop and characterize a chitosan gel/gelatin microspheres (MSs) dual delivery system for sequential release of bone morphogenetic protein-2 (BMP-2) and insulin-like growth factor-1 (IGF-1) to enhance osteoblast differentiation in vitro. We made and characterized the delivery system based on its degree of cross-linking, degradation, and release kinetics. We also evaluated the cytotoxicity of the delivery system and the effect of growth factors on cell response using pre-osteoblast W-20-17 mouse bone marrow stromal cells. IGF-1 was first loaded into MSs, and then the IGF-1 containing MSs were encapsulated into the chitosan gel which contained BMP-2. Cross-linking of gelatin with glyoxal via Schiff bases significantly increased thermal stability and decreased the solubility of the MSs, leading to a significant decrease in the initial release of IGF-1. Encapsulation of the MSs into the chitosan gel generated polyelectrolyte complexes by intermolecular interactions, which further affected the release kinetics of IGF-1. This combinational delivery system provided an initial release of BMP-2 followed by a slow and sustained release of IGF-1. Significantly greater alkaline phosphatase activity was found in W-20-17 cells treated with the sequential delivery system than other treatments (p<0.05) after a week of culture. PMID:22293583

  16. 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.

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

    PubMed Central

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

    2015-01-01

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

  18. Osteoblast histogenesis in periodontal ligament and tibial metaphysis during simulated weightlessness

    NASA Technical Reports Server (NTRS)

    Fielder, Paul J.; Morey, Emily R.; Roberts, W. Eugene

    1986-01-01

    Utilizing the nuclear morphometric assay for osteoblast histogenesis, the effect of simulated weightlessness (SW) on the relative numbers of the periodontal ligament (PDL) osteoblast progenitors and on the total number of osteogenic cells was determined in rats. Weightlessness was simulated by subjecting rats to continuous 30-deg head-down posture using a modified back-harness device of Morey (1979). The response of a partially unloaded, weight-bearing bone, tibial primary spongiosa (PS), was compared to a normally loaded, nonweight-bearing PDL bone. Data indicated a similar differentiation sequence in PS and PDL, which suggests that these bones might be sensitive to the same systemic factors. Preosteoblast numbers were seen to decrease in both nonweight-bearing and weight-bearing bones during SW (compared with rats not exposed to SW), indicating the importance of systemic mediators, such as cephalad fluid shift, physiological stress, and/or growth retardation.

  19. Increased osteoblastic activity and expression of receptor activator of NF-kappaB ligand in nonuremic nephrotic syndrome.

    PubMed

    Freundlich, Michael; Alonzo, Evelyn; Bellorin-Font, Ezequiel; Weisinger, Jose R

    2005-07-01

    Patients with nephrotic syndrome (NS), even with normal GFR, often display altered mineral homeostasis and abnormal bone histology. However, the latter, mostly osteomalacia and increased bone resorption, cannot be readily explained by the prevalent concentrations of parathyroid hormone and vitamin D metabolites. The transmembrane receptor activator of NF-kappaB ligand (RANKL) of osteoblasts is essential for osteoclast formation and differentiation. Osteoblasts activity and the expression of RANKL were tested in cultures of normal human osteoblasts with sera obtained from patients with NS and normal GFR (129 +/- 26 ml/min per 1.73 m2) during relapse and remission of their NS. Osteoblasts that were cultured in vitro with sera during relapse displayed elevated concentrations of alkaline phosphatase (AP) and increased expression of RANKL. By contrast, during remission, AP concentrations were significantly lower (P < 0.05) and RANKL expression notably attenuated or absent. AP correlated with the proteinuria (r = 0.5, P < 0.05) and was not significantly affected by the therapeutic administration of corticosteroids. Whereas parathyroid hormone levels were normal (35 +/- 21 pg/ml), the serum markers of bone formation (osteocalcin and bone-specific alkaline phosphatase) were lower during relapse compared with remission. Thus, sera from patients with NS and normal GFR stimulate the activity of osteoblasts and upregulate their expression of RANKL. These alterations, more prominent during clinically active NS, are transient and reversible upon remission. These disturbances of bone biology may play an important pathogenic role in the abnormal bone histology observed in patients with NS even before a decline in GFR occurs.

  20. Specific Biomimetic Hydroxyapatite Nanotopographies Enhance Osteoblastic Differentiation and Bone Graft Osteointegration

    PubMed Central

    Loiselle, Alayna E.; Wei, Lai; Faryad, Muhammad; Paul, Emmanuel M.; Lewis, Gregory S.; Gao, Jun; Lakhtakia, Akhlesh

    2013-01-01

    Impaired healing of cortical bone grafts represents a significant clinical problem. Cadaveric bone grafts undergo extensive chemical processing to decrease the risk of disease transmission; however, these processing techniques alter the bone surface and decrease the osteogenic potential of cells at the healing site. Extensive work has been done to optimize the surface of bone grafts, and hydroxyapatite (HAP) and nanotopography both increase osteoblastic differentiation. HAP is the main mineral component of bone and can enhance osteoblastic differentiation and bone implant healing in vivo, while nanotopography can enhance osteoblastic differentiation, adhesion, and proliferation. This is the first study to test the combined effects of HAP and nanotopographies on bone graft healing. With the goal of identifying the optimized surface features to improve bone graft healing, we tested the hypothesis that HAP-based nanotopographic resurfacing of bone grafts improves integration of cortical bone grafts by enhancing osteoblastic differentiation. Here we show that osteoblastic cells cultured on processed bones coated with specific-scale (50–60 nm) HAP nanotopographies display increased osteoblastic differentiation compared to cells on uncoated bone, bones coated with poly-l-lactic acid nanotopographies, or other HAP nanotopographies. Further, bone grafts coated with 50–60-nm HAP exhibited increased formation of new bone and improved healing, with mechanical properties equivalent to live autografts. These data indicate the potential for specific HAP nanotopographies to not only increase osteoblastic differentiation but also improve bone graft incorporation, which could significantly increase patient quality of life after traumatic bone injuries or resection of an osteosarcoma. PMID:23510012

  1. Mature osteoblasts dedifferentiate in response to traumatic bone injury in the zebrafish fin and skull.

    PubMed

    Geurtzen, Karina; Knopf, Franziska; Wehner, Daniel; Huitema, Leonie F A; Schulte-Merker, Stefan; Weidinger, Gilbert

    2014-06-01

    Zebrafish have an unlimited capacity to regenerate bone after fin amputation. In this process, mature osteoblasts dedifferentiate to osteogenic precursor cells and thus represent an important source of newly forming bone. By contrast, differentiated osteoblasts do not appear to contribute to repair of bone injuries in mammals; rather, osteoblasts form anew from mesenchymal stem cells. This raises the question whether osteoblast dedifferentiation is specific to appendage regeneration, a special feature of the lepidotrichia bone of the fish fin, or a process found more generally in fish bone. Here, we show that dedifferentiation of mature osteoblasts is not restricted to fin regeneration after amputation, but also occurs during repair of zebrafish fin fractures and skull injuries. In both models, mature osteoblasts surrounding the injury downregulate the expression of differentiation markers, upregulate markers of the pre-osteoblast state and become proliferative. Making use of photoconvertible Kaede protein as well as Cre-driven genetic fate mapping, we show that osteoblasts migrate to the site of injury to replace damaged tissue. Our findings suggest a fundamental role for osteoblast dedifferentiation in reparative bone formation in fish and indicate that adult fish osteoblasts display elevated cellular plasticity compared with mammalian bone-forming cells. © 2014. Published by The Company of Biologists Ltd.

  2. [Study of signal transduction pathway in the expression of inflammatory factors stimulated by lipopolysaccharides from Porphyromonas endodontalis in osteoblasts].

    PubMed

    Yang, Di; Qiu, Li-hong; Li, Ren; Li, Zi-mu; Li, Chen

    2010-04-01

    To quantify the interleukin (IL)-1beta mRNA and IL-6 mRNA expression induced by lipopolysaccharides ([PS) extracted from Porphyromonoas endodontalis (P. endodontalis) in osteoblasts, and to relate P. endodontalis LPS to the bone resorptive pathogenesis in the lesions of chronic apical periodontitis. MG63 cells was pretreated with PD98059 or SB203580 for 1 h and then treated with P. endodontolis LPS for 6 h. The expression of IL-1beta mRNA and IL-6 mRNA were detected by reverse transcription polymerase chain reaction (RT-PCR) technique. The production of IL-1beta mRNA induced by P. endodontalis LPS decreased in osteoblasts pretreated with PD98059. Both of the production of IL-1beta mRNA and JL-6 mRNA induced by P. endodontalis LPS decreased in osteoblasts pretreated with SB203580. The synthesis of IL-1beta mRNA stimulated by Pendodontalis LPS in MG63 probably occur via extracellular signal-regulated kinase (ERK) 1/2 and p38 mitogen activated protein kinase (MAPK) signal transduction system. The synthesis of IL-6 mRNA stimulated by P.endodontalis LPS in MG63 probahly occur via p38MAPK signal transduction system.

  3. Single cell gene expression profiling of cortical osteoblast lineage cells.

    PubMed

    Flynn, James M; Spusta, Steven C; Rosen, Clifford J; Melov, Simon

    2013-03-01

    In tissues with complex architectures such as bone, it is often difficult to purify and characterize specific cell types via molecular profiling. Single cell gene expression profiling is an emerging technology useful for characterizing transcriptional profiles of individual cells isolated from heterogeneous populations. In this study we describe a novel procedure for the isolation and characterization of gene expression profiles of single osteoblast lineage cells derived from cortical bone. Mixed populations of different cell types were isolated from adult long bones of C57BL/6J mice by enzymatic digestion, and subsequently subjected to FACS to purify and characterize osteoblast lineage cells via a selection strategy using antibodies against CD31, CD45, and alkaline phosphatase (AP), specific for mature osteoblasts. The purified individual osteoblast lineage cells were then profiled at the single cell level via nanofluidic PCR. This method permits robust gene expression profiling on single osteoblast lineage cells derived from mature bone, potentially from anatomically distinct sites. In conjunction with this technique, we have also shown that it is possible to carry out single cell profiling on cells purified from fixed and frozen bone samples without compromising the gene expression signal. The latter finding means the technique can be extended to biopsies of bone from diseased individuals. Our approach for single cell expression profiling provides a new dimension to the transcriptional profile of the primary osteoblast lineage population in vivo, and has the capacity to greatly expand our understanding of how these cells may function in vivo under normal and diseased states. Copyright © 2012 Elsevier Inc. All rights reserved.

  4. Osteoblasts Protect AML Cells from SDF-1-Induced Apoptosis

    PubMed Central

    Kremer, Kimberly N.; Dudakovic, Amel; McGee-Lawrence, Meghan E.; Philips, Rachael L.; Hess, Allan D.; Smith, B. Douglas; van Wijnen, Andre J.; Karp, Judith E.; Kaufmann, Scott H.; Westendorf, Jennifer J.; Hedin, Karen E.

    2014-01-01

    The bone marrow provides a protective environment for acute myeloid leukemia (AML) cells that often allows leukemic stem cells to survive standard chemotherapeutic regimens. Targeting these leukemic stem cells within the bone marrow is critical for preventing relapse. We recently demonstrated that SDF-1, a chemokine abundant in the bone marrow, induces apoptosis in AML cell lines and in patient samples expressing high levels of its receptor, CXCR4. Here we show that a subset of osteoblast lineage cells within the bone marrow can protect AML cells from undergoing apoptosis in response to the SDF-1 naturally present in that location. In co-culture systems, osteoblasts at various stages of differentiation protected AML cell lines and patient isolates from SDF-1-induced apoptosis. The differentiation of the osteoblast cell lines, MC3T3 and W-20-17, mediated this protection via a cell contact-independent mechanism. In contrast, bone marrow-derived mesenchymal cells, the precursors of osteoblasts, induced apoptosis in AML cells via a CXCR4-dependent mechanism and failed to protect AML cells from exogenously added SDF-1. These results indicate that osteoblasts in the process of differentiation potently inhibit the SDF-1-driven apoptotic pathway of CXCR4-expressing AML cells residing in the bone marrow. Drugs targeting this protective mechanism could potentially provide a new approach to treating AML by enhancing the SDF-1-induced apoptosis of AML cells residing within the bone marrow microenvironment. PMID:24851270

  5. Osteoblasts Growth Behaviour on Bio-Based Calcium Carbonate Aragonite Nanocrystal

    PubMed Central

    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

  6. Expression Profiles of TGF-β and TLR Pathways in Porphyromonas gingivalis and Prevotella intermedia Challenged Osteoblasts.

    PubMed

    Aydin, Kubra; Ekinci, Fatma Yesim; Korachi, May

    2015-04-01

    The presence of certain oral pathogens at implant sites can hinder the osseointegration process. However, it is unclear how and by what microorganisms it happens. This study investigated whether the presence of oral pathogens of Porphyromonas gingivalis and Prevotella intermedia individually, play a role in the failure of bone formation by determining the expression profiles of Transforming Growth Factor Beta (TGF-β/Bone Morphogenic Protein (BMP) and Toll-Like Receptor (TLR) pathways in challenged osteoblasts. Cell viability of P. gingivalis and P. intermedia challenged osteoblasts were determined by WST assay. Changes in osteoblast morphology and inhibition of mineralization were observed by Scanning Electron Microscopy (SEM) and Von Kossa staining, respectively. Expression of TGF-β and TLR pathway genes on challenged cells were identified by RT profiler array. Both P. gingivalis and P. intermedia challenges resulted in reduced viability and mineralization of osteoblasts. Viability was reduced to 56.8% (P. gingivalis) and 52.75% (P. intermedia) at 1000 multiplicity. Amongst 48 genes examined, expressions of BMPER, SMAD1, IL8 and NFRKB were found to be highly upregulated by both bacterial challenges (Fold Change > 4). P. gingivalis and P. intermedia could play a role in implant failure by changing the expression profiles of genes related to bone formation and resorption.

  7. ERβ induces the differentiation of cultured osteoblasts by both Wnt/β-catenin signaling pathway and estrogen signaling pathways

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yin, Xinhua; Wang, Xiaoyuan; Hu, Xiongke

    Although 17β-estradial (E2) is known to stimulate bone formation, the underlying mechanisms are not fully understood. Recent studies have implicated the Wnt/β-catenin pathway as a major signaling cascade in bone biology. The interactions between Wnt/β-catenin signaling pathway and estrogen signaling pathways have been reported in many tissues. In this study, E2 significantly increased the expression of β-catenin by inducing phosphorylations of GSK3β at serine 9. ERβ siRNAs were transfected into MC3T3-E1 cells and revealed that ERβ involved E2-induced osteoblasts proliferation and differentiation via Wnt/β-catenin signaling. The osteoblast differentiation genes (BGP, ALP and OPN) and proliferation related gene (cyclin D1) expressionmore » were significantly induced by E2-mediated ERβ. Furthermore immunofluorescence and immunoprecipitation analysis demonstrated that E2 induced the accumulation of β-catenin protein in the nucleus which leads to interaction with T-cell-specific transcription factor/lymphoid enhancer binding factor (TCF/LEF) transcription factors. Taken together, these findings suggest that E2 promotes osteoblastic proliferation and differentiation by inducing proliferation-related and differentiation-related gene expression via ERβ/GSK-3β-dependent Wnt/β-catenin signaling pathway. Our findings provide novel insights into the mechanisms of action of E2 in osteoblastogenesis. - Highlights: • 17β-estradial (E2) promotes GSK3-β phosphorylation. • E2 activates the Wnt/β-catenin signaling pathway. • The Wnt/β-catenin signaling pathway interacts with estrogen signaling pathways. • E2-mediated ER induced osteoblast differentiation and proliferation related genes expression.« less

  8. Osteoclast-derived exosomal miR-214-3p inhibits osteoblastic bone formation

    PubMed Central

    Li, Defang; Liu, Jin; Guo, Baosheng; Liang, Chao; Dang, Lei; Lu, Cheng; He, Xiaojuan; Cheung, Hilda Yeuk-Siu; Xu, Liang; Lu, Changwei; He, Bing; Liu, Biao; Shaikh, Atik Badshah; Li, Fangfei; Wang, Luyao; Yang, Zhijun; Au, Doris Wai-Ting; Peng, Songlin; Zhang, Zongkang; Zhang, Bao-Ting; Pan, Xiaohua; Qian, Airong; Shang, Peng; Xiao, Lianbo; Jiang, Baohong; Wong, Chris Kong-Chu; Xu, Jiake; Bian, Zhaoxiang; Liang, Zicai; Guo, De-an; Zhu, Hailong; Tan, Weihong; Lu, Aiping; Zhang, Ge

    2016-01-01

    Emerging evidence indicates that osteoclasts direct osteoblastic bone formation. MicroRNAs (miRNAs) have a crucial role in regulating osteoclast and osteoblast function. However, whether miRNAs mediate osteoclast-directed osteoblastic bone formation is mostly unknown. Here, we show that increased osteoclastic miR-214-3p associates with both elevated serum exosomal miR-214-3p and reduced bone formation in elderly women with fractures and in ovariectomized (OVX) mice. Osteoclast-specific miR-214-3p knock-in mice have elevated serum exosomal miR-214-3p and reduced bone formation that is rescued by osteoclast-targeted antagomir-214-3p treatment. We further demonstrate that osteoclast-derived exosomal miR-214-3p is transferred to osteoblasts to inhibit osteoblast activity in vitro and reduce bone formation in vivo. Moreover, osteoclast-targeted miR-214-3p inhibition promotes bone formation in ageing OVX mice. Collectively, our results suggest that osteoclast-derived exosomal miR-214-3p transfers to osteoblasts to inhibit bone formation. Inhibition of miR-214-3p in osteoclasts may be a strategy for treating skeletal disorders involving a reduction in bone formation. PMID:26947250

  9. Action potentials in primary osteoblasts and in the MG-63 osteoblast-like cell line.

    PubMed

    Pangalos, Maria; Bintig, Willem; Schlingmann, Barbara; Feyerabend, Frank; Witte, Frank; Begandt, Daniela; Heisterkamp, Alexander; Ngezahayo, Anaclet

    2011-06-01

    Whole-cell patch-clamp analysis revealed a resting membrane potential of -60 mV in primary osteoblasts and in the MG-63 osteoblast-like cells. Depolarization-induced action potentials were characterized by duration of 60 ms, a minimal peak-to-peak distance of 180 ms, a threshold value of -20 mV and a repolarization between the spikes to -45 mV. Expressed channels were characterized by application of voltage pulses between -150 mV and 90 mV in 10 mV steps, from a holding potential of -40 mV. Voltages below -60 mV induced an inward current. Depolarizing voltages above -30 mV evoked two currents: (a) a fast activated and inactivated inward current at voltages between -30 and 30 mV, and (b) a delayed-activated outward current that was induced by voltages above -30 mV. Electrophysiological and pharmacological parameters indicated that hyperpolarization activated strongly rectifying K(+) (K(ir)) channels, whereas depolarization activated tetrodotoxin sensitive voltage gated Na(+) (Na(v)) channels as well as delayed, slowly activated, non-inactivating, and tetraethylammonium sensitive voltage gated K(+) (K(v)) channels. In addition, RT-PCR showed expression of Na(v)1.3, Na(v)1.4, Na(v)1.5, Na(v)1.6, Na(v)1.7, and K(ir)2.1, K(ir)2.3, and K(ir)2.4 as well as K(v)2.1. We conclude that osteoblasts express channels that allow firing of action potentials.

  10. Differential expression of neuroleukin in osseous tissues and its involvement in mineralization during osteoblast differentiation

    NASA Technical Reports Server (NTRS)

    Zhi, J.; Sommerfeldt, D. W.; Rubin, C. T.; Hadjiargyrou, M.

    2001-01-01

    Osteoblast differentiation is a multistep process that involves critical spatial and temporal regulation of cellular processes marked by the presence of a large number of differentially expressed molecules. To identify key functional molecules, we used differential messenger RNA (mRNA) display and compared RNA populations isolated from the defined transition phases (proliferation, matrix formation, and mineralization) of the MC3T3-E1 osteoblast-like cell line. Using this approach, a complementary DNA (cDNA) fragment was isolated and identified as neuroleukin (NLK), a multifunctional cytokine also known as autocrine motility factor (AMF), phosphoglucose isomerase (PGI; phosphohexose isomerase [PHI]), and maturation factor (MF). Northern analysis showed NLK temporal expression during MC3T3-E1 cell differentiation with a 3.5-fold increase during matrix formation and mineralization. Immunocytochemical studies revealed the presence of NLK in MC3T3-E1 cells as well as in the surrounding matrix, consistent with a secreted molecule. In contrast, the NLK receptor protein was detected primarily on the cell membrane. In subsequent studies, a high level of NLK expression was identified in osteoblasts and superficial articular chondrocytes in bone of 1-, 4-, and 8-month-old normal mice, as well as in fibroblasts, proliferating chondrocytes, and osteoblasts within a fracture callus. However, NLK was not evident in hypertrophic chondrocytes or osteocytes. In addition, treatment of MC3T3 cells with 6-phosphogluconic acid (6PGA; a NLK inhibitor) resulted in diminishing alkaline phosphatase (ALP) activity and mineralization in MC3T3-E1 cells, especially during the matrix formation stage of differentiating cells. Taken together, these data show specific expression of NLK in discrete populations of bone and cartilage cells and suggest a possible role for this secreted protein in bone development and regeneration.

  11. Estrogen and estrogen receptor alpha promotes malignancy and osteoblastic tumorigenesis in prostate cancer.

    PubMed

    Mishra, Sweta; Tai, Qin; Gu, Xiang; Schmitz, James; Poullard, Ashley; Fajardo, Roberto J; Mahalingam, Devalingam; Chen, Xiaodong; Zhu, Xueqiong; Sun, Lu-Zhe

    2015-12-29

    The role of estrogen signaling in regulating prostate tumorigenesis is relatively underexplored. Although, an increasing body of evidence has linked estrogen receptor beta (ERß) to prostate cancer, the function of estrogen receptor alpha (ERα) in prostate cancer is not very well studied. We have discovered a novel role of ERα in the pathogenesis of prostate tumors. Here, we show that prostate cancer cells express ERα and estrogen induces oncogenic properties in prostate cancer cells through ERα. Importantly, ERα knockdown in the human prostate cancer PacMetUT1 cells as well as pharmacological inhibition of ERα with ICI 182,780 inhibited osteoblastic lesion formation and lung metastasis in vivo. Co-culture of pre-osteoblasts with cancer cells showed a significant induction of osteogenic markers in the pre-osteoblasts, which was attenuated by knockdown of ERα in cancer cells suggesting that estrogen/ERα signaling promotes crosstalk between cancer and osteoblastic progenitors to stimulate osteoblastic tumorigenesis. These results suggest that ERα expression in prostate cancer cells is essential for osteoblastic lesion formation and lung metastasis. Thus, inhibition of ERα signaling in prostate cancer cells may be a novel therapeutic strategy to inhibit the osteoblastic lesion development as well as lung metastasis in patients with advanced prostate cancer.

  12. Effect of acetaminophen on osteoblastic differentiation and migration of MC3T3-E1 cells.

    PubMed

    Nakatsu, Yoshihiro; Nakagawa, Fumio; Higashi, Sen; Ohsumi, Tomoko; Shiiba, Shunji; Watanabe, Seiji; Takeuchi, Hiroshi

    2018-02-01

    N-acetyl-p-aminophenol (APAP, acetaminophen, paracetamol) is a widely used analgesic/antipyretic with weak inhibitory effects on cyclooxygenase (COX) compared to non-steroidal anti-inflammatory drugs (NSAIDs). The mechanism of action of APAP is mediated by its metabolite that activates transient receptor potential channels, including transient receptor potential vanilloid 1 (TRPV1) and TRP ankyrin 1 (TRPA1) or the cannabinoid receptor type 1 (CB1). However, the exact molecular mechanism and target underlying the cellular actions of APAP remain unclear. Therefore, we investigated the effect of APAP on osteoblastic differentiation and cell migration, with a particular focus on TRP channels and CB1. Effects of APAP on osteoblastic differentiation and cell migration of MC3T3-E1, a mouse pre-osteoblast cell line, were assessed by the increase in alkaline phosphatase (ALP) activity, and both wound-healing and transwell-migration assays, respectively. APAP dose-dependently inhibited osteoblastic differentiation, which was well correlated with the effects on COX activity compared with other NSAIDs. In contrast, cell migration was promoted by APAP, and this effect was not correlated with COX inhibition. None of the agonists or antagonists of TRP channels and the CB receptor affected the APAP-induced cell migration, while the effect of APAP on cell migration was abolished by down-regulating TRPV4 gene expression. APAP inhibited osteoblastic differentiation via COX inactivation while it promoted cell migration independently of previously known targets such as COX, TRPV1, TRPA1 channels, and CB receptors, but through the mechanism involving TRPV4. APAP may have still unidentified molecular targets that modify cellular functions. Copyright © 2017 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier B.V. All rights reserved.

  13. Transcriptional profiling of murine osteoblast differentiation based on RNA-seq expression analyses.

    PubMed

    Khayal, Layal Abo; Grünhagen, Johannes; Provazník, Ivo; Mundlos, Stefan; Kornak, Uwe; Robinson, Peter N; Ott, Claus-Eric

    2018-04-11

    Osteoblastic differentiation is a multistep process characterized by osteogenic induction of mesenchymal stem cells, which then differentiate into proliferative pre-osteoblasts that produce copious amounts of extracellular matrix, followed by stiffening of the extracellular matrix, and matrix mineralization by hydroxylapatite deposition. Although these processes have been well characterized biologically, a detailed transcriptional analysis of murine primary calvaria osteoblast differentiation based on RNA sequencing (RNA-seq) analyses has not previously been reported. Here, we used RNA-seq to obtain expression values of 29,148 genes at four time points as murine primary calvaria osteoblasts differentiate in vitro until onset of mineralization was clearly detectable by microscopic inspection. Expression of marker genes confirmed osteogenic differentiation. We explored differential expression of 1386 protein-coding genes using unsupervised clustering and GO analyses. 100 differentially expressed lncRNAs were investigated by co-expression with protein-coding genes that are localized within the same topologically associated domain. Additionally, we monitored expression of 237 genes that are silent or active at distinct time points and compared differential exon usage. Our data represent an in-depth profiling of murine primary calvaria osteoblast differentiation by RNA-seq and contribute to our understanding of genetic regulation of this key process in osteoblast biology. Copyright © 2018 Elsevier Inc. All rights reserved.

  14. Liraglutide attenuates the osteoblastic differentiation of MC3T3-E1 cells by modulating AMPK/mTOR signaling

    PubMed Central

    Hu, Xiong-Ke; Yin, Xin-Hua; Zhang, Hong-Qi; Guo, Chao-Feng; Tang, Ming-Xing

    2016-01-01

    Liraglutide, a synthetic analogue of glucagon-like peptide-1, is utilized in the treatment of type 2 diabetes and obesity. Liraglutide has been previously demonstrated to prevent osteoblastic differentiation of human vascular smooth muscle cells, resulting in the slowing of arterial calcification, however, its effect on bone formation remains unclear. The present study investigated the effect of liraglutide on osteoblastic differentiation using Alizarin Red S staining, and examined the molecular mechanisms underlying the regulatory effect by western blot analysis. The present study demonstrated that protein expression levels of phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK) were downregulated in MC3T3-E1 cells during osteoblastic differentiation in commercial osteogenic differentiation medium, whereas protein expression levels of transforming growth factor-β (TGF-β) and phosphorylated mammalian target of rapamycin (p-mTOR) increased. Liraglutide was subsequently demonstrated to dose-dependently attenuate the osteoblastic differentiation of MC3T3-E1 cells, to upregulate p-AMPK, and downregulate p-mTOR and TGF-β protein expression levels. Treatment with an AMPK-specific inhibitor, Compound C, eradicated the effect of liraglutide on osteoblastic differentiation, and p-mTOR and TGF-β downregulation. An mTOR activator, MHY1485, also abolished the inhibitory effect of liraglutide on osteoblastic differentiation, and resulted in p-mTOR and TGF-β downregulation, but did not attenuate the liraglutide-induced increase in p-AMPK protein expression levels. The results of the present study demonstrate that liraglutide attenuates osteoblastic differentiation of MC3T3-E1 cells via modulation of AMPK/mTOR signaling. The present study revealed a novel function of liraglutide, which contributes to the understanding of its pharmacological and physiological effects in clinical settings. PMID:27600753

  15. Interaction of human osteoblast-like Saos-2 cells with stainless steel coated by silicalite-1 films.

    PubMed

    Jirka, Ivan; Vandrovcová, Marta; Plšek, Jan; Bouša, Milan; Brabec, Libor; Dragounová, Helena; Bačáková, Lucie

    2017-07-01

    This paper investigates the interaction of human osteoblast-like Saos-2 cells with stainless steel covered by a film of densely inter-grown silicalite-1 crystals with defined outer and inner surfaces. The chemical composition of this film, labeled as SF(RT), was tuned by heat treatment at 300°C and 500°C (labeled as SF(300) and SF(500), respectively) and characterized by X-ray photoelectron spectroscopy (XPS), water drop contact angle (WCA) measurements and scanning electron microscopy (SEM). The number, the spreading area and the activity of alkaline phosphatase of human osteoblast-like Saos-2 cells in cultures on the silicalite-1 film were affected by the chemical composition of its outer surface and by its micro-porous structure. The number and the spreading area of the adhered osteoblast-like cells on day 1 was highest on the surface of SF(RT) relative to their adhesion and spreading on a glass cover slip due to the SF(RT) topology. However, SF(300) markedly supported cell growth during days 3 and 7 after seeding. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Transient upregulation of CBFA1 in response to bone morphogenetic protein-2 and transforming growth factor beta1 in C2C12 myogenic cells coincides with suppression of the myogenic phenotype but is not sufficient for osteoblast differentiation.

    PubMed

    Lee, M H; Javed, A; Kim, H J; Shin, H I; Gutierrez, S; Choi, J Y; Rosen, V; Stein, J L; van Wijnen, A J; Stein, G S; Lian, J B; Ryoo, H M

    1999-04-01

    The bone morphogenetic protein (BMP)-2 is a potent osteoinductive signal, inducing bone formation in vivo and osteoblast differentiation from non-osseous cells in vitro. The runt domain-related protein Cbfa1/PEBP2alphaA/AML-3 is a critical component of bone formation in vivo and transcriptional regulator of osteoblast differentiation. To investigate the relationship between the extracellular BMP-2 signal, Cbfa1, and osteogenesis, we examined expression of Cbfa1 and osteoblastic genes during the BMP-2 induced osteogenic transdifferentiation of the myoblastic cell line C2C12. BMP-2 treatment completely blocked myotube formation and transiently induced expression of Cbfa1 and the bone-related homeodomain protein Msx-2 concomitant with loss of the myoblast phenotype. While induction of collagen type I and alkaline phosphatase (AP) expression coincided with Cbfa1 expression, Cbfa1 mRNA was strikingly downregulated at the onset of expression of osteopontin (OPN) and osteocalcin (OCN) genes, reflecting the mature osteoblast phenotype. TGF-beta1 treatment effectively suppressed myogenesis and induced Cbfa1 expression but was insufficient to support osteoblast differentiation reflected by the absence of ALP, OPN, and OCN. We addressed whether induction of Cbfa1 in response to BMP-2 results in the transcriptional activation of the OC promoter which contains three enhancer Cbfa1 elements. Transfection studies show BMP-2 suppresses OC promoter activity in C2C12, but not in osteoblastic ROS 17/2.8 cells. Maximal suppression of OC promoter activity in response to BMP-2 requires sequences in the proximal promoter (up to nt -365) and may occur independent of the three Cbfa sites. Taken together, our results demonstrate a dissociation of Cbfa1 expression from development of the osteoblast phenotype. Our findings suggest that Cbfal may function transiently to divert a committed myoblast to a potentially osteogenic cell. However, other factors induced by BMP-2 appear to be necessary

  17. Hypoxia inhibits the growth, differentiation and bone-forming capacity of rat osteoblasts

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Utting, J.C.; Robins, S.P.; Brandao-Burch, A.

    2006-06-10

    We investigated the effect of hypoxia on rat osteoblast function in long-term primary cultures. Reduction of pO{sub 2} from 20% to 5% and 2% decreased formation of mineralized bone nodules 1.7-fold and 11-fold, respectively. When pO{sub 2} was reduced further to 0.2%, bone nodule formation was almost abolished. The inhibitory effect of hypoxia on bone formation was partly due to decreased osteoblast proliferation, as measured by {sup 3}H-thymidine incorporation. Hypoxia also sharply reduced osteoblast alkaline phosphatase (ALP) activity and expression of mRNAs for ALP and osteocalcin, suggesting inhibition of differentiation to the osteogenic phenotype. Hypoxia did not increase the apoptosismore » of osteoblasts but induced a reversible state of quiescence. Transmission electron microscopy revealed that collagen fibrils deposited by osteoblasts cultured in 2% O{sub 2} were less organized and much less abundant than in 20% O{sub 2} cultures. Furthermore, collagen produced by hypoxic osteoblasts contained a lower percentage of hydroxylysine residues and exhibited an increased sensitivity to pepsin degradation. These data demonstrate the absolute oxygen requirement of osteoblasts for successful bone formation and emphasize the importance of the vasculature in maintaining bone health. We recently showed that hypoxia also acts in a reciprocal manner as a powerful stimulator of osteoclast formation. Considered together, our results help to explain the bone loss that occurs at the sites of fracture, tumors, inflammation and infection, and in individuals with vascular disease or anemia.« less

  18. Icariside II activates EGFR-Akt-Nrf2 signaling and protects osteoblasts from dexamethasone.

    PubMed

    Liu, Weidong; Mao, Li; Ji, Feng; Chen, Fengli; Wang, Shouguo; Xie, Yue

    2017-01-10

    The potential effect of icariside II on dexamethasone-induced osteoblast cell damages was evaluated here. In MC3T3-E1 osteoblastic cells and the primary murine osteoblasts, co-treatment with icariside II dramatically attenuated dexamethasone- induced cell death and apoptosis. Icariside II activated Akt signaling, which is required for its actions in osteoblasts. Akt inhibitors (LY294002, perifosine and MK-2206) almost abolished icariside II-induced osteoblast cytoprotection against dexamethasone. Further studies showed that icariside II activated Nrf2 signaling, downstream of Akt, to inhibit dexamethasone-induced reactive oxygen species (ROS) production in MC3T3-E1 cells and primary osteoblasts. On the other hand, Nrf2 shRNA knockdown inhibited icariside II-induced anti-dexamethasone cytoprotection in MC3T3-E1 cells. Finally, we showed that icariside II induced heparin-binding EGF (HB-EGF) production and EGFR trans-activation in MC3T3-E1 cells. EGFR inhibition, via anti-HB-EGF antibody, EGFR inhibitor AG1478 or EGFR shRNA knockdown, almost blocked icariside II-induced Akt-Nrf2 activation in MC3T3-E1 cells. Collectively, we conclude that icariside II activates EGFR-Akt-Nrf2 signaling and protects osteoblasts from dexamethasone. Icariside II might have translational value for the treatment of dexamethasone-associated osteoporosis/osteonecrosis.

  19. Relative binding and biochemical effects of heterodimeric and homodimeric isoforms of platelet-derived growth factor in osteoblast-enriched cultures from fetal rat bone

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Centrella, M.; McCarthy, T.L.; Kusmik, W.F.

    1991-06-01

    Platelet-derived growth factor (PDGF) exists as a homodimer or a heterodimer comprising either PDGF-A or PDGF-B subunits, and each isoform occurs in various tissues, including bone. Although the stimulatory effects of PDGF-BB have been studied in cultures of bone cells and intact bone fragments, the influence of other isoforms that may arise locally or systematically in vivo, has not been reported. Therefore recombinant human PDGF-BB, PDGF-AB, and PDGF-AA were evaluated in osteoblast-enriched cultures from fetal rat bone. Within 24 hours these factors produced a graded response in bone cell DNA and protein synthesis, with half-maximal effects at approximately 0.6, 2.1,more » and 4.8 nM PDGF-BB, PDGF-AB, and PDGF-AA, respectively. Increases in collagen and noncollagen protein synthesis were abrogated when DNA synthesis was blocked with hydroxyurea. Furthermore, each factor reduced alkaline phosphatase activity, PDGF-BB being the most inhibitory. Binding studies with 125I-PDGF-BB or 125I-PDGF-AA and each unlabeled PDGF isoform produced discrete ligand binding and displacement patterns: 125I-PDGF-BB binding was preferentially displaced by PDGF-BB (Ki approximately 0.7 nM), less by PDGF-AB (Ki approximately 2.3 nM) and poorly by PDGF-AA. In contrast, 125I-PDGF-AA binding was measurably reduced by PDGF-AA (Ki approximately 4.0 nM), but was more effectively displaced by PDGF-BB or PDGF-AB (each with Ki approximately 0.7 nM). These studies indicate that each PDGF isoform produces biochemical effects proportional to binding site occupancy and suggest that receptors that favor PDGF-B subunit binding preferentially mediate these results in osteoblast-enriched bone cell cultures.« less

  20. Effects of orbital spaceflight on human osteoblastic cell physiology and gene expression

    NASA Technical Reports Server (NTRS)

    Harris, S. A.; Zhang, M.; Kidder, L. S.; Evans, G. L.; Spelsberg, T. C.; Turner, R. T.

    2000-01-01

    During long-term spaceflight, astronauts lose bone, in part due to a reduction in bone formation. It is not clear, however, whether the force imparted by gravity has direct effects on bone cells. To examine the response of bone forming cells to weightlessness, human fetal osteoblastic (hFOB) cells were cultured during the 17 day STS-80 space shuttle mission. Fractions of conditioned media were collected during flight and shortly after landing for analyses of glucose utilization and accumulation of type I collagen and prostaglandin E(2) (PGE(2)). Total cellular RNA was isolated from flight and ground control cultures after landing. Measurement of glucose levels in conditioned media indicated that glucose utilization occurred at a similar rate in flight and ground control cultures. Furthermore, the levels of type I collagen and PGE(2) accumulation in the flight and control conditioned media were indistinguishable. The steady-state levels of osteonectin, alkaline phosphatase, and osteocalcin messenger RNA (mRNA) were not significantly changed following spaceflight. Gene-specific reductions in mRNA levels for cytokines and skeletal growth factors were detected in the flight cultures using RNase protection assays. Steady-state mRNA levels for interleukin (IL)-1alpha and IL-6 were decreased 8 h following the flight and returned to control levels at 24 h postflight. Also, transforming growth factor (TGF)-beta(2) and TGF-beta(1) message levels were modestly reduced at 8 h and 24 h postflight, although the change was not statistically significant at 8 h. These data suggest that spaceflight did not significantly affect hFOB cell proliferation, expression of type I collagen, or PGE(2) production, further suggesting that the removal of osteoblastic cells from the context of the bone tissue results in a reduced ability to respond to weightlessness. However, spaceflight followed by return to earth significantly impacted the expression of cytokines and skeletal growth factors

  1. MiR-17 Partly Promotes Hematopoietic Cell Expansion through Augmenting HIF-1α in Osteoblasts

    PubMed Central

    Yang, Yuxia; Ma, Wei; Wu, Dan; Huang, Yu; Li, Hongge; Zou, Junhua; Zhang, Yanju; Feng, Meifu; Luo, Jianyuan

    2013-01-01

    Background Hematopoietic stem cell (HSC) regulation is highly dependent on interactions with the marrow microenvironment, of which osteogenic cells play a crucial role. While evidence is accumulating for an important role of intrinsic miR-17 in regulating HSCs and HPCs, whether miR-17 signaling pathways are also necessary in the cell-extrinsic control of hematopoiesis hereto remains poorly understood. Methodology/Principal Findings Using the immortalized clone with the characteristics of osteoblasts, FBMOB-hTERT, in vitro expansion, long-term culture initiating cell (LTC-IC) and non-obese diabetic/severe combined immunodeficient disease (NOD/SCID) mice repopulating cell (SRC) assay revealed that the ectopic expression of miR-17 partly promoted the ability of FBMOB-hTERT to support human cord blood (CB) CD34+ cell expansion and maintain their multipotency. It also seemed that osteoblastic miR-17 was prone to cause a specific expansion of the erythroid lineage. Conversely, deficient expression of miR-17 partly inhibited the hematopoietic supporting ability of FBMOB-hTERT. We further identified that HIF-1α is responsible for, at least in part, the promoted hematopoietic supporting ability of FBMOB-hTERT caused by miR-17. HIF-1α expression is markedly enhanced in miR-17 overexpressed FBMOB-hTERT upon interaction with CB CD34+ cells compared to other niche associated factors. More interestingly, the specific erythroid lineage expansion of CB CD34+ cells caused by osteoblastic miR-17 was abrogated by HIF-1α knock down. Conclusion/Significance Our data demonstrated that CB CD34+ cell expansion can be partly promoted by osteoblastic miR-17, and in particular, ectopic miR-17 can cause a specific expansion of the erythroid lineage through augmenting HIF-1α in osteoblasts. PMID:23936170

  2. In the trail of a new bio-sensor for measuring strain in bone: osteoblastic biocompatibility.

    PubMed

    Carvalho, Lídia; Alberto, Nélia J; Gomes, Pedro S; Nogueira, Rogério N; Pinto, João L; Fernandes, Maria H

    2011-06-15

    Fibre Bragg Grating (FBG) is an optical sensor recorded within the core of a standard optical fibre, which responds faithfully to strain and temperature. FBG sensors are a promising alternative to other sensing methodologies to assess bone mechanics in vivo. However, response of bone cells/bone tissue to FBGs and its sensing capability in this environment have not been recorded yet. The present study addressed these issues in long-term human osteoblastic cell cultures. Results showed that osteoblastic cells were able to adhere and proliferate over the fibre and, also, the protective polymer coating. RT-PCR analysis showed the expression of Col I, ALP, BMP-2, M-CSF, RANKL and OPG. In addition, cultures presented high ALP activity and the formation of a calcium phosphate mineralized extracellular matrix. Cell behavior over the fibre without and with the coating polymer was similar to that found in cultures grown in standard tissue culture plates (control). In addition to the excellent osteoblastic cytocompatibility, FBGs maintained the physical integrity and functionality, as its sensing capability was not affected through the culture period. Results suggest the possibility of in vivo osseointegration of the optical fibre/FBGs anticipating a variety of applications in bone mechanical dynamics. Copyright © 2011 Elsevier B.V. All rights reserved.

  3. Jagged1 is essential for osteoblast development during maxillary ossification

    PubMed Central

    Hill, Cynthia R.; Yuasa, Masato; Schoenecker, Jonathan; Goudy, Steven L.

    2015-01-01

    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; Jagged1f/f (Jag1CKO) led to maxillary hypoplasia characterized by intrinsic differences in bone morphology and density using μCT evaluation. Jag1CKO maxillas had 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

  4. Expression Profiles of TGF-β and TLR Pathways in Porphyromonas gingivalis and Prevotella intermedia Challenged Osteoblasts

    PubMed Central

    Aydin, Kubra; Ekinci, Fatma Yesim; Korachi, May

    2015-01-01

    Background: The presence of certain oral pathogens at implant sites can hinder the osseointegration process. However, it is unclear how and by what microorganisms it happens. Objectives: This study investigated whether the presence of oral pathogens of Porphyromonas gingivalis and Prevotella intermedia individually, play a role in the failure of bone formation by determining the expression profiles of Transforming Growth Factor Beta (TGF-β/Bone Morphogenic Protein (BMP) and Toll-Like Receptor (TLR) pathways in challenged osteoblasts. Materials and Methods: Cell viability of P. gingivalis and P. intermedia challenged osteoblasts were determined by WST assay. Changes in osteoblast morphology and inhibition of mineralization were observed by Scanning Electron Microscopy (SEM) and Von Kossa staining, respectively. Expression of TGF-β and TLR pathway genes on challenged cells were identified by RT profiler array. Both P. gingivalis and P. intermedia challenges resulted in reduced viability and mineralization of osteoblasts. Results: Viability was reduced to 56.8% (P. gingivalis) and 52.75% (P. intermedia) at 1000 multiplicity. Amongst 48 genes examined, expressions of BMPER, SMAD1, IL8 and NFRKB were found to be highly upregulated by both bacterial challenges (Fold Change > 4). Conclusions: P. gingivalis and P. intermedia could play a role in implant failure by changing the expression profiles of genes related to bone formation and resorption. PMID:26034550

  5. Strong and rapid induction of osteoblast differentiation by Cbfa1/Til-1 overexpression for bone regeneration.

    PubMed

    Kojima, Hiroko; Uemura, Toshimasa

    2005-01-28

    Core binding factor alpha-1 (Cbfa1), known as an essential transcription factor for osteogenic lineage, has two major N-terminal isoforms: Pebp2alphaA and Til-1. To study the roles of these isoforms in bone regeneration, we applied an adenoviral vector carrying their genes to transduce primary osteoprogenitor cells in vitro and in vivo. Overexpression of the two isoforms induced rapid and marked osteoblast differentiation, with Til-1 being more effective in vitro, by examination of the alkaline phosphatase activity, calcium content, and Alizarin red staining. Til-1 overexpressing cells/porous ceramic composites were transplanted into subcutaneous and bone defect sites in Fischer rats (cultured bone transplantation model) and markedly affected in vivo bone formation and osteoblast markers. The results demonstrated that the reconstitution of bone tissues, such as cortical bone and trabecular bone was accelerated by implantation of Til-1 overexpressing cells/porous ceramic composites. Moreover, the new bone formation by Til-1 overexpression appeared to reflect replacement of new bone within the implant boundaries. To ascertain whether implanted Cbfa1 overexpressing cells could differentiate into osteogenic cells to create bone or whether it stimulated the surrounding recipient tissue to regenerate bone, implanted male donor cells were visualized by fluorescent in situ hybridization analysis. The proportion of implanted cells in the presumptive bone forming region was over 80% and did not change throughout from 3 days to 8 weeks after implantation. These findings suggested that the newly formed bone in the porous area of the scaffold is mostly produced by the implanted donor cells or their derived cells, effectively by Til-1 overexpression.

  6. Enhanced Osteoblast Response to Porosity and Resolution of Additively Manufactured Ti-6Al-4V Constructs with Trabeculae-Inspired Porosity

    PubMed Central

    Cheng, Alice; Humayun, Aiza; Schwartz, Zvi

    2016-01-01

    Abstract The addition of porosity to the traditionally used solid titanium metal implants has been suggested to more closely mimic the natural mechanical properties of bone and increase osseointegration in dental and orthopedic implants. The objective of this study was to evaluate cellular response to three-dimensional (3D) porous Ti-6Al-4V constructs fabricated by additive manufacturing using laser sintering with low porosity (LP), medium porosity (MP), and high porosity (HP) with low resolution (LR) and high resolution (HR) based on a computed tomography scan of human trabecular bone. After surface processing, construct porosity ranged from 41.0% to 76.1%, but all possessed micro-/nanoscale surface roughness and similar surface chemistry containing mostly Ti, O, and C. Biological responses (osteoblast differentiation, maturation, and local factor production) by MG63 osteoblast-like cells and normal human osteoblasts favored 3D than two-dimensional (2D) solid constructs. First, MG63 cells were used to assess differences in cell response to 2D compared to LR and HR porous 3D constructs. MG63 cells were sensitive to porosity resolution and exhibited increased osteocalcin (OCN), vascular endothelial growth factor (VEGF), osteoprotegerin (OPG), and bone morphogenetic protein 2 (BMP2) on HR 3D constructs than on 2D and LR 3D constructs. MG63 cells also exhibited porosity-dependent responses on HR constructs, with up to a 6.9-fold increase in factor production on LP-HR and MP-HR constructs than on HP-HR constructs. NHOsts were then used to validate biological response on HR constructs. NHOsts exhibited decreased DNA content and alkaline phosphatase activity and up to a 2.9-fold increase in OCN, OPG, VEGF, BMP2, and BMP4 on 3D HR constructs than on 2D controls. These results indicate that osteoblasts prefer a 3D architecture than a 2D surface and that osteoblasts are sensitive to the resolution of trabecular detail and porosity parameters of laser-sintered 3D Ti-6Al-4V

  7. Enhanced Osteoblast Response to Porosity and Resolution of Additively Manufactured Ti-6Al-4V Constructs with Trabeculae-Inspired Porosity.

    PubMed

    Cheng, Alice; Humayun, Aiza; Boyan, Barbara D; Schwartz, Zvi

    2016-03-01

    The addition of porosity to the traditionally used solid titanium metal implants has been suggested to more closely mimic the natural mechanical properties of bone and increase osseointegration in dental and orthopedic implants. The objective of this study was to evaluate cellular response to three-dimensional (3D) porous Ti-6Al-4V constructs fabricated by additive manufacturing using laser sintering with low porosity (LP), medium porosity (MP), and high porosity (HP) with low resolution (LR) and high resolution (HR) based on a computed tomography scan of human trabecular bone. After surface processing, construct porosity ranged from 41.0% to 76.1%, but all possessed micro-/nanoscale surface roughness and similar surface chemistry containing mostly Ti, O, and C. Biological responses (osteoblast differentiation, maturation, and local factor production) by MG63 osteoblast-like cells and normal human osteoblasts favored 3D than two-dimensional (2D) solid constructs. First, MG63 cells were used to assess differences in cell response to 2D compared to LR and HR porous 3D constructs. MG63 cells were sensitive to porosity resolution and exhibited increased osteocalcin (OCN), vascular endothelial growth factor (VEGF), osteoprotegerin (OPG), and bone morphogenetic protein 2 (BMP2) on HR 3D constructs than on 2D and LR 3D constructs. MG63 cells also exhibited porosity-dependent responses on HR constructs, with up to a 6.9-fold increase in factor production on LP-HR and MP-HR constructs than on HP-HR constructs. NHOsts were then used to validate biological response on HR constructs. NHOsts exhibited decreased DNA content and alkaline phosphatase activity and up to a 2.9-fold increase in OCN, OPG, VEGF, BMP2, and BMP4 on 3D HR constructs than on 2D controls. These results indicate that osteoblasts prefer a 3D architecture than a 2D surface and that osteoblasts are sensitive to the resolution of trabecular detail and porosity parameters of laser-sintered 3D Ti-6Al-4V constructs.

  8. Indocyanine green-mediated photobiomodulation on human osteoblast cells.

    PubMed

    Ateş, Gamze Bölükbaşı; Ak, Ayşe; Garipcan, Bora; Gülsoy, Murat

    2018-05-09

    Photobiomodulation (PBM) and photodynamic therapy (PDT) share similar mechanisms but have opposite aims. Increased levels of reactive oxygen species (ROS) in the target tissue in response to light combined photosensitizer (PS) application may lead to cell proliferation or oxidative damage depending on the ROS amount. The purpose of the present study is to investigate the effect of indocyanine green (ICG)-mediated PBM on osteoblast cells by measuring cell viability, proliferation, alkaline phosphatase (ALP) activity, mineralization, and gene expressions of three phenotypic osteoblast markers. A diode laser irradiating at 809 nm (10 W output power, 50 mW/cm 2 power density) was used at 0.5, 1, and 2 J/cm 2 energy densities (10, 20, and 40 s respectively) was applied following ICG incubation. No inhibitory effect was observed in cell viability and proliferation according to the (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and Alamar Blue assays. ICG-mediated PBM did not alter cell viability but increased ALP activity and enhanced mineralization of existing osteoblasts. These results were also confirmed by real-time polymerase chain reaction (RT-PCR) analysis of osteoblastic markers. PS can be combined to PBM not only to damage the malignant cells as aimed in PDT studies, but also to promote cellular activity. The findings of this in vitro study may contribute to in vivo studies and ICG-mediated PBM can have promising outcomes in bone healing and regeneration therapies in future.

  9. Nanostructured magnesium has fewer detrimental effects on osteoblast function.

    PubMed

    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.

  10. 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

  11. Osteogenic differentiation of immature osteoblasts: Interplay of cell culture media and supplements.

    PubMed

    Brauer, A; Pohlemann, T; Metzger, W

    2016-01-01

    Differentiation of immature osteoblasts to mature osteoblasts in vitro initially was induced by supplementing the medium with β-gylcerophosphate and dexamethasone. Later, ascorbic acid, vitamin D3, vitamin K3 and TGFβ1 were used in varying concentrations as supplements to generate a mature osteoblast phenotype. We tested the effects of several combinations of cell culture media, seeding protocols and osteogenic supplements on osteogenic differentiation of human primary osteoblasts. Osteogenic differentiation was analyzed by staining alkaline phosphatase (ALP) with 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (BCIP/NBT) and by von Kossa staining of deposited calcium phosphate. The combinations of culture media and supplements significantly influenced osteogenic differentiation, but the seeding protocol did not. Staining of ALP and calcium phosphate could be achieved only if our own mix of osteogenic supplements was used in combination with Dulbecco's modified Eagle medium or if a commercial mix of osteogenic supplements was used in combination with osteoblast growth medium. Especially for von Kossa, we observed great variations in the staining intensity. Because osteogenic differentiation is a complex process, the origin of the osteoblasts, cell culture media and osteogenic supplements should be established by preliminary experiments to achieve optimal differentiation. Staining of ALP or deposited calcium phosphate should be supplemented with qRT-PCR studies to learn more about the influence of specific supplements on osteogenic markers.

  12. Arctigenin Inhibits Osteoclast Differentiation and Function by Suppressing Both Calcineurin-Dependent and Osteoblastic Cell-Dependent NFATc1 Pathways

    PubMed Central

    Yamashita, Teruhito; Uehara, Shunsuke; Udagawa, Nobuyuki; Li, Feng; Kadota, Shigetoshi; Esumi, Hiroyasu; Kobayashi, Yasuhiro; Takahashi, Naoyuki

    2014-01-01

    Arctigenin, a lignan-derived compound, is a constituent of the seeds of Arctium lappa. Arctigenin was previously shown to inhibit osteoclastogenesis; however, this inhibitory mechanism has yet to be elucidated. Here, we showed that arctigenin inhibited the action of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a key transcription factor for osteoclastogenesis. NFATc1 in osteoclast precursors was activated through two distinct pathways: the calcineurin-dependent and osteoblastic cell-dependent pathways. Among the several lignan-derived compounds examined, arctigenin most strongly inhibited receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast-like cell formation in mouse bone marrow macrophage (BMM) cultures, in which the calcineurin-dependent NFATc1 pathway was activated. Arctigenin suppressed neither the activation of nuclear factor κB and mitogen-activated protein kinases nor the up-regulation of c-Fos expression in BMMs treated with RANKL. However, arctigenin suppressed RANKL-induced NFATc1 expression. Interestingly, the treatment of osteoclast-like cells with arctigenin converted NFATc1 into a lower molecular weight species, which was translocated into the nucleus even in the absence of RANKL. Nevertheless, arctigenin as well as cyclosporin A (CsA), a calcineurin inhibitor, suppressed the NFAT-luciferase reporter activity induced by ionomycin and phorbol 12-myristate 13-acetate in BMMs. Chromatin immunoprecipitation analysis confirmed that arctigenin inhibited the recruitment of NFATc1 to the promoter region of the NFATc1 target gene. Arctigenin, but not CsA suppressed osteoclast-like cell formation in co-cultures of osteoblastic cells and bone marrow cells, in which the osteoblastic cell-dependent NFATc1 pathway was activated. The forced expression of constitutively active NFATc1 rescued osteoclastogenesis in BMM cultures treated with CsA, but not that treated with arctigenin. Arctigenin also suppressed the pit

  13. Arctigenin inhibits osteoclast differentiation and function by suppressing both calcineurin-dependent and osteoblastic cell-dependent NFATc1 pathways.

    PubMed

    Yamashita, Teruhito; Uehara, Shunsuke; Udagawa, Nobuyuki; Li, Feng; Kadota, Shigetoshi; Esumi, Hiroyasu; Kobayashi, Yasuhiro; Takahashi, Naoyuki

    2014-01-01

    Arctigenin, a lignan-derived compound, is a constituent of the seeds of Arctium lappa. Arctigenin was previously shown to inhibit osteoclastogenesis; however, this inhibitory mechanism has yet to be elucidated. Here, we showed that arctigenin inhibited the action of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a key transcription factor for osteoclastogenesis. NFATc1 in osteoclast precursors was activated through two distinct pathways: the calcineurin-dependent and osteoblastic cell-dependent pathways. Among the several lignan-derived compounds examined, arctigenin most strongly inhibited receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast-like cell formation in mouse bone marrow macrophage (BMM) cultures, in which the calcineurin-dependent NFATc1 pathway was activated. Arctigenin suppressed neither the activation of nuclear factor κB and mitogen-activated protein kinases nor the up-regulation of c-Fos expression in BMMs treated with RANKL. However, arctigenin suppressed RANKL-induced NFATc1 expression. Interestingly, the treatment of osteoclast-like cells with arctigenin converted NFATc1 into a lower molecular weight species, which was translocated into the nucleus even in the absence of RANKL. Nevertheless, arctigenin as well as cyclosporin A (CsA), a calcineurin inhibitor, suppressed the NFAT-luciferase reporter activity induced by ionomycin and phorbol 12-myristate 13-acetate in BMMs. Chromatin immunoprecipitation analysis confirmed that arctigenin inhibited the recruitment of NFATc1 to the promoter region of the NFATc1 target gene. Arctigenin, but not CsA suppressed osteoclast-like cell formation in co-cultures of osteoblastic cells and bone marrow cells, in which the osteoblastic cell-dependent NFATc1 pathway was activated. The forced expression of constitutively active NFATc1 rescued osteoclastogenesis in BMM cultures treated with CsA, but not that treated with arctigenin. Arctigenin also suppressed the pit

  14. 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.

  15. Factors affecting sign retroreflectivity

    DOT National Transportation Integrated Search

    2001-01-01

    This study was undertaken to better understand the factors that may affect road sign retroreflectivity, specifically age and physical orientation. A better understanding of these factors could provide guidance to ODOT in managing its inventory of roa...

  16. Connexin43 Potentiates Osteoblast Responsiveness to Fibroblast Growth Factor 2 via a Protein Kinase C-Delta/Runx2–dependent Mechanism

    PubMed Central

    Lima, Florence; Niger, Corinne; Hebert, Carla

    2009-01-01

    In this study, we examine the role of the gap junction protein, connexin43 (Cx43), in the transcriptional response of osteocalcin to fibroblast growth factor 2 (FGF2) in MC3T3 osteoblasts. By luciferase reporter assays, we identify that the osteocalcin transcriptional response to FGF2 is markedly increased by overexpression of Cx43, an effect that is mediated by Runx2 via its OSE2 cognate element, but not by a previously identified connexin-responsive Sp1/Sp3-binding element. Furthermore, disruption of Cx43 function with Cx43 siRNAs or overexpression of connexin45 markedly attenuates the response to FGF2. Inhibition of protein kinase C delta (PKCδ) with rottlerin or siRNA-mediated knockdown abrogates the osteocalcin response to FGF2. Additionally, we show that upon treatment with FGF2, PKCδ translocates to the nucleus, PKCδ and Runx2 are phosphorylated and these events are enhanced by Cx43 overexpression, suggesting that the degree of activation is enhanced by increased Cx43 levels. Indeed, chromatin immunoprecipitations of the osteocalcin proximal promoter with antibodies against Runx2 demonstrate that the recruitment of Runx2 to the osteocalcin promoter in response to FGF2 treatment is dramatically enhanced by Cx43 overexpression. Thus, Cx43 plays a critical role in regulating the ability of osteoblasts to respond to FGF2 by impacting PKCδ and Runx2 function. PMID:19339281

  17. Implant materials generate different peri-implant inflammatory factors: poly-ether-ether-ketone promotes fibrosis and microtextured titanium promotes osteogenic factors.

    PubMed

    Olivares-Navarrete, Rene; Hyzy, Sharon L; Slosar, Paul J; Schneider, Jennifer M; Schwartz, Zvi; Boyan, Barbara D

    2015-03-15

    An in vitro study examining factors produced by human mesenchymal stem cells on spine implant materials. The aim of this study was to examine whether the inflammatory microenvironment generated by cells on titanium-aluminum-vanadium (Ti-alloy, TiAlV) surfaces is affected by surface microtexture and whether it differs from that generated on poly-ether-ether-ketone (PEEK). Histologically, implants fabricated from PEEK have a fibrous connective tissue surface interface whereas Ti-alloy implants demonstrate close approximation with surrounding bone. Ti-alloy surfaces with complex micron/submicron scale roughness promote osteoblastic differentiation and foster a specific cellular environment that favors bone formation whereas PEEK favors fibrous tissue formation. Human mesenchymal stem cells were cultured on tissue culture polystyrene, PEEK, smooth TiAlV, or macro-/micro-/nano-textured rough TiAlV (mmnTiAlV) disks. Osteoblastic differentiation and secreted inflammatory interleukins were assessed after 7 days. Fold changes in mRNAs for inflammation, necrosis, DNA damage, or apoptosis with respect to tissue culture polystyrene were measured by low-density polymerase chain reaction array. Data were analyzed by analysis of variance, followed by Bonferroni's correction of Student's t-test. Cells on PEEK upregulated mRNAs for chemokine ligand-2, interleukin (IL) 1β, IL6, IL8, and tumor necrosis factor. Cells grown on the mmnTiAlV had an 8-fold reduction in mRNAs for toll-like receptor-4. Cells grown on mmnTiAlV had reduced levels of proinflammatory interleukins. Cells on PEEK had higher mRNAs for factors strongly associated with cell death/apoptosis, whereas cells on mmnTiAlV exhibited reduced cytokine factor levels. All results were significant (P < 0.05). These results suggest that fibrous tissue around PEEK implants may be due to several factors: reduced osteoblastic differentiation of progenitor cells and production of an inflammatory environment that favors cell death

  18. Sirtuin 1 regulates matrix metalloproteinase-13 expression induced by Porphyromonas endodontalis lipopolysaccharide via targeting nuclear factor-κB in osteoblasts.

    PubMed

    Qu, Liu; Yu, Yaqiong; Qiu, Lihong; Yang, Di; Yan, Lu; Guo, Jiajie; Jahan, Rabita

    2017-01-01

    Porphyromonas endodontalis lipopolysaccharide (P.e LPS) is an important initiating factor for periapical inflammation and bone destruction. Matrix metalloproteinase-13 (MMP-13) has been shown to participate in the formation and diffusion of periapical bone lesion in chronic apical periodontitis. Sirtuin 1 (SIRT1) is a key regulator of inflammation in mammalian cells which suppresses the release of inflammatory mediators. This study aimed to explore the role of SIRT1 in regulating MMP-13 expression induced by P.e LPS in osteoblasts. P.e LPS stimulated MMP-13 expression in MC3T3-E1 cells. Knockdown of SIRT1 reinforced the increase of MMP-13mRNA expression induced by P.e LPS. SIRT1 activator resveratrol significantly reduced the expression of MMP-13 and SIRT1 inhibitor EX-527 enhanced the expression of MMP-13. Moreover, SIRT1 activation with resveratrol inhibited acetylation of NF-κB p65 and NF-κB transcriptional activity, which were enhanced by P.e LPS. In addition, NF-κB p65 was involved in P.e LPS-induced MMP-13 expression via directly binding to the MMP-13 promoter. However, SIRT1 activation significantly interfered with this binding. These findings strongly suggest that P.e LPS induces MMP-13 expression in osteoblasts, and SIRT1 suppresses this expression of MMP-13 through targeting NF-κB p65. This provides new insights into understanding the actions of SIRT1 on anti-inflammatory and anti-bone resorption activity.

  19. Histochemical assessment for osteoblastic activity coupled with dysfunctional osteoclasts in c-src deficient mice.

    PubMed

    Toray, Hisashi; Hasegawa, Tomoka; Sakagami, Naoko; Tsuchiya, Erika; Kudo, Ai; Zhao, Shen; Moritani, Yasuhito; Abe, Miki; Yoshida, Taiji; Yamamoto, Tomomaya; Yamamoto, Tsuneyuki; Oda, Kimimitsu; Udagawa, Nobuyuki; Luiz de Freitas, Paulo Henrique; Li, Minqi

    2017-01-01

    Since osteoblastic activities are believed to be coupled with osteoclasts, we have attempted to histologically verify which of the distinct cellular circumstances, the presence of osteoclasts themselves or bone resorption by osteoclasts, is essential for coupled osteoblastic activity, by examining c-fos -/- or c-src -/- mice. Osteopetrotic c-fos deficient (c-fos -/- ) mice have no osteoclasts, while c-src deficient (c-src -/- ) mice, another osteopetrotic model, develop dysfunctional osteoclasts due to a lack of ruffled borders. c-fos -/- mice possessed no tartrate-resistant acid phosphatase (TRAPase)-reactive osteoclasts, and showed very weak tissue nonspecific alkaline phosphatase (TNALPase)-reactive mature osteoblasts. In contrast, c-src -/- mice had many TNALPase-positive osteoblasts and TRAPase-reactive osteoclasts. Interestingly, the parallel layers of TRAPase-reactive/osteopontin-positive cement lines were observed in the superficial region of c-src -/- bone matrix. This indicates the possibility that in c-src -/- mice, osteoblasts were activated to deposit new bone matrices on the surfaces that osteoclasts previously passed along, even without bone resorption. Transmission electron microscopy demonstrated cell-to-cell contacts between mature osteoblasts and neighboring ruffled border-less osteoclasts, and osteoid including many mineralized nodules in c-src -/- mice. Thus, it seems likely that osteoblastic activities would be maintained in the presence of osteoclasts, even if they are dysfunctional.

  20. High glucose impaired estrogen receptor alpha signaling via β-catenin in osteoblastic MC3T3-E1.

    PubMed

    Wang, Rui; Gao, Dong; Zhou, Yin; Chen, Lu; Luo, Bin; Yu, Yanrong; Li, Hao; Hu, Jiawei; Huang, Qiren; He, Ming; Peng, Weijie; Luo, Dan

    2017-11-01

    Diabetic Mellitus is a risk factor for osteoporosis. It has been suggested that altered estrogen or estrogen receptor α/β (ERα/β) signaling may be involved in diabetic osteoporosis. The present study is to investigate the effects of high glucose on ERα/β signaling in osteoblastic MC3T3-E1 and how the altered signaling of ERα/β affect osteoblastic bone formation. ERα/β signaling was demonstrated as ERα/β protein expression (Western Blotting) and ER transcription activity (Luciferase Reporter assays). Proliferation (WSK-1 assaying), differentiation (ALP staining) and mineralization (Alizalard Red staining) of MC3T3-E1 were examined to evaluate bone formation function. It has been found that high glucose increased ERα/β expression dose-dependently and time-dependently, but high glucose (33mM) decreased ERα transcription activity. 17β-estradiol increased the ERα/β expression dose-dependently in normal medium, but decreased the ERα/β expression dose-dependently in medium with high glucose (33mM). High glucose decreased bone formation and also decreased the osteogenic effects of 17β-estradiol (10 -8 M). High glucose decreased β-catenin expression dose-dependently and time-dependently. LiCl, an inhibitor of β-catenin degradation, decreased ERα expression but increased ERα transcription activity. When compared with high glucose treatment, LiCl (5mM) increased ALP activity and calcified nodes. Besides, high glucose also decreased the protein expression PI-3K, pAKT/AKT, GSK-3β. In conclusion, the present study suggested that high glucose may impair ERα transcription activity by inhibiting β-catenin signaling in osteoblastic MC3T3-E1, leading decreased bone formation ligand-dependently or ligand-independently. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Immortalized Mouse Floxed Fam20c Dental Papillar Mesenchymal and Osteoblast Cell Lines Retain Their Primary Characteristics

    PubMed Central

    Liu, Chao; Wang, Xiaofang; Zhang, Hua; Xie, Xiaohua; Liu, Peihong; Liu, Ying; Jani, Priyam H.; Lu, Yongbo; Chen, Shuo; Qin, Chunlin

    2016-01-01

    Fam20c is essential for the normal mineralization of dentin and bone. The generation of odontoblast and osteoblast cell lines carrying floxed Fam20c allele can offer valuable tools for the study of the roles of Fam20c in the mineralization of dentin and bone. The limited capability of the primary odontoblasts and osteoblasts to proliferate necessitates the development of odontoblast and osteoblast cell lines serving as substitutes for the study of differentiation and mineralization of the odontoblasts and osteoblasts. In this study, we established and characterized immortalized mouse floxed Fam20c dental papilla mesenchymal and osteoblast cell lines. The isolated primary mouse floxed Fam20c dental papilla mesenchymal cells and osteoblasts were immortalized by the infection of lentivirus containing Simian Virus 40 T-antigen (SV40 T-Ag). The immortalization of floxed Fam20c dental papilla mesenchymal cells and osteoblasts was verified by the long-term passages and genomic integration of SV40 T-Ag. The immortalized floxed Fam20c dental papilla mesenchymal and osteoblast cell lines not only proliferated at a high rate and retained the morphology of their primary counterparts, but also preserved the dentin and bone specific gene expression as the primary dental papilla mesenchymal cells and osteoblasts did. Consistently, the capability of the primary floxed Fam20c dental papilla mesenchymal cells and osteoblasts to mineralize was also inherited by the immortalized dental papilla mesenchymal and osteoblast cell lines. Thus, we have successfully generated the immortalized mouse floxed Fam20c dental papilla mesenchymal and osteoblast cell lines. PMID:25833681

  2. Suppression of osteoblastic phenotypes and modulation of pro- and anti-apoptotic features in normal human osteoblastic cells under a vector-averaged gravity condition.

    PubMed

    Nakamura, Hiroshi; Kumei, Yasuhiro; Morita, Sadao; Shimokawa, Hitoyata; Ohya, Keiichi; Shinomiya, Kenichi

    2003-06-01

    Spaceflight and bed rest induce loss of bone mass. A number of in vivo and in vitro studies have been conducted to clarify the mechanisms, however, the results have been conflicting. The purpose of this study was to investigate the effects of gravity unloading on proliferation, phenotypes, and apoptosis of normal human osteoblastic cells in the presence of 1alpha,25-dihydroxyvitamin D3. We used a vector-averaged gravity condition generated by clinostat rotation to simulate gravity unloading. Clinostat rotation did not affect the cell proliferation. On the first day, the mRNA levels for osteocalcin, ALP, CBFA1, VDR, RANKL, and OPG were reduced by clinostat rotation to 21%, 65%, 62%, 52%, 43%, and 54% of control, respectively. ALP activity was decreased to 75% of control. On the second day, the mRNA levels for osteocalcin and RANKL were reduced to 77% and 61% of control, respectively. The decreased VDR mRNA level might be responsible for the reduction for mRNA levels for osteocalcin, RANKL, and OPG. Clinostat rotation increased the pro-apoptotic index (Bax/Bcl-2 ratio) but did not induce apoptosis due to the simultaneous upregulation of the anti-apoptotic XIAP. Reduction of osteoblast responsiveness to 1alpha,25-dihydroxyvitamin D3 might be involved in osteopenia that is induced by gravity unloading.

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

    PubMed

    Kaneko, Keiko; Ito, Masako; Naoe, Yoshinori; Lacy-Hulbert, Adam; Ikeda, Kyoji

    2014-05-02

    Although osteoblast lineage cells, especially osteocytes, are thought to be a primary mechanosensory cell in bone, the identity of the mechano-receptor and downstream mechano-signaling pathways remain largely unknown. Here we show using osteoblastic cell model of mechanical stimulation with fluid shear stress that in the absence of integrin αv, phosphorylation of the Src substrate p130Cas and JNK was impaired, culminating in an inhibition of nuclear translocation of YAP/TAZ and subsequent transcriptional activation of target genes. Targeted deletion of the integrin αv in osteoblast lineage cells results in an attenuated response to mechanical loading in terms of Sost gene expression, indicative of a role for integrin αv in mechanoreception in vivo. Thus, integrin αv may be integral to a mechanosensing machinery in osteoblastic cells and involved in activation of a Src-JNK-YAP/TAZ pathway in response to mechanical stimulation. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Porphyromonas endodontalis lipopolysaccharides induce RANKL by mouse osteoblast in a way different from that of Escherichia coli lipopolysaccharide.

    PubMed

    Tang, Yin; Sun, Feifei; Li, Xiaoting; Zhou, Yuan; Yin, Shihai; Zhou, Xuedong

    2011-12-01

    Porphyromonas endodontalis lipopolysaccharide (LPS) has been shown to have a high positive rate in infected root canals and symptomatic apical periodontitis. It may play an integral role as a potent stimulator of inflammatory cytokines involved in apical lesions. The receptor activator of nuclear factor-κB ligand (RANKL) has been proven to be the key regulator of bone remodeling. This study investigated P. endodontalis LPS-induced RANKL production and LPS signaling in mouse osteoblasts. LPS-induced RANKL production in mouse osteoblast MC3T3-E1 cells was measured by Western blot and real-time polymerase chain reaction, and the Toll-like receptors (TLRs) were determined by the blocking test using anti-TLRs antibodies. In addition, specific inhibitors were used to analyze the intracellular signaling pathways. Escherichia coli LPS was used as the control. Both of the anti-TLR2 and anti-TLR4 antibodies significantly (P < .05) inhibited the expression of RANKL from osteoblasts stimulated with P. endodontalis LPS; only anti-TLR2 antibody had a significant (P < .05) inhibitory effect on E. coli LPS signaling. SP600125 (c-Jun N-terminal kinase [JNK] inhibitor) prevented the up-regulation of RANKL expression in P. endodontalis LPS-infected osteoblasts (P < .05). The inhibitory effect of wortmannin (phosphatidylinositol 3-kinase inhibitor) and PD98059 (mitogen-activated protein kinase [MAPK]/extracellular signal-regulated kinase [ERK] kinase-1/2 [MEK 1/2] inhibitor) were observed in E. coli LPS-treated mouse osteoblasts (P < .05). Results from this study showed that P. endodontalis LPS has the ability to promote the expression of RANKL in mouse osteoblasts, and this induction was mainly through the TLR2/4-JNK signaling pathway, a situation quite different from that of typical bacterial endotoxin (E. coli LPS). Copyright © 2011 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  5. Effect of Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) on the expression of EphA2 in osteoblasts and osteoclasts.

    PubMed

    Gao, Aichao; Wang, Xichao; Yu, Haiyan; Li, Na; Hou, Yubo; Yu, Weixian

    2016-02-01

    Porphyromonas gingivalis (Pg) as the major pathogenic bacterium of chronic periodontitis can cause alveolar bone resorption. Lipopolysaccharide (LPS) is its main virulence factor. The Eph family plays an important role in maintaining bone homeostasis. In this study, the effects of P. gingivalis lipopolysaccharide (Pg-LPS) on the expression of EphA2 in osteoblasts and osteoclasts were investigated. MC3T3-E1 cells and RAW264.7 cells were separately cultured in osteoblast-conditioned medium and osteoclast-conditioned medium to induce their differentiation into osteoblasts and osteoclasts, respectively. MC3T3-E1 cells were treated with 1 μg/mL of Pg-LPS 3, 7, and 14 d later, while RAW264.7 cells were treated with 10 μg/mL of Pg-LPS 1, 3, and 5 d later. The results have shown that Pg-LPS increased the expression of EphA2 both in osteoblasts and osteoclasts, decreased the expression of osteogenic-related genes (ALP, Sp7), and increased the expression of osteoclast-related genes (MMP9, c-fos, ACP5, CtsK, and NFATc1). Tartrate-resistant acid phosphatase (TRAP) staining illustrated that Pg-LPS promoted osteoclast differentiation and decreased the activity of alkaline phosphatase. Therefore, analysis indicates that, when treated with Pg-LPS, the expression of EphA2 is upregulated while the activity of osteoblasts and osteoclasts was reduced and increased, respectively. Our data suggest that EphA2 is closely related to the formation of osteoblasts and resorption of osteoclast and is likely to play an role in bone resorption induced in chronic periodontitis. These findings may provide information on new targets for prevention and treatment of chronic periodontitis.

  6. Phosphoproteome analysis reveals a critical role for hedgehog signalling in osteoblast morphological transitions.

    PubMed

    Marumoto, Ariane; Milani, Renato; da Silva, Rodrigo A; da Costa Fernandes, Célio Junior; Granjeiro, José Mauro; Ferreira, Carmen V; Peppelenbosch, Maikel P; Zambuzzi, Willian F

    2017-10-01

    The reciprocal and adaptive interactions between cells and substrates governing morphological transitions in the osteoblast compartment remain largely obscure. Here we show that osteoblast cultured in basement membrane matrix (Matrigel™) exhibits significant morphological changes after ten days of culture, and we decided to exploit this situation to investigate the molecular mechanisms responsible for guiding osteoblast morphological transitions. As almost all aspects of cellular physiology are under control of kinases, we generated more or less comprehensive cellular kinome profiles employing PepChip peptide arrays that contain over 1000 consensus substrates of kinase peptide. The results obtained were used to construct interactomes, and these revealed an important role for FoxO in mediating morphological changes of osteoblast, which was validated by Western blot technology when FoxO was significantly up-expressed in response to Matrigel™. As FoxO is a critical protein in canonical hedgehog signalling, we decided to explore the possible involvement of hedgehog signalling during osteoblast morphological changes. It appeared that osteoblast culture in Matrigel™ stimulates release of a substantial amounts Shh while concomitantly inducing upregulation of the expression of the bona fide hedgehog target genes Gli-1 and Patched. Functional confirmation of the relevance of these results for osteoblast morphological transitions came from experiments in which Shh hedgehog signalling was inhibited using the well-established pathway inhibitor cyclopamine (Cyc). In the presence of Cyc, culture of osteoblasts in Matrigel™ is not capable of inducing morphological changes but appears to provoke a proliferative response as evident from the upregulation of Cyclin D3 and cdk4. The most straightforward interpretation of our results is that hedgehog signalling is both necessary and sufficient for membrane matrix-based morphological transitions. Copyright © 2017 Elsevier Inc

  7. Repression of Osteoblast Maturation by ERRα Accounts for Bone Loss Induced by Estrogen Deficiency

    PubMed Central

    Gallet, Marlène; Saïdi, Soraya; Haÿ, Eric; Photsavang, Johann; Marty, Caroline; Sailland, Juliette; Carnesecchi, Julie; Tribollet, Violaine; Barenton, Bruno; Forcet, Christelle; Birling, Marie-Christine; Sorg, Tania; Chassande, Olivier; Cohen-Solal, Martine; Vanacker, Jean-Marc

    2013-01-01

    ERRα is an orphan member of the nuclear receptor family, the complete inactivation of which confers resistance to bone loss induced by ageing and estrogen withdrawal to female mice in correlation with increased bone formation in vivo. Furthermore ERRα negatively regulates the commitment of mesenchymal cells to the osteoblast lineage ex vivo as well as later steps of osteoblast maturation. We searched to determine whether the activities of ERRα on osteoblast maturation are responsible for one or both types of in vivo induced bone loss. To this end we have generated conditional knock out mice in which the receptor is normally present during early osteoblast differentiation but inactivated upon osteoblast maturation. Bone ageing in these animals was similar to that observed for control animals. In contrast conditional ERRαKO mice were completely resistant to bone loss induced by ovariectomy. We conclude that the late (maturation), but not early (commitment), negative effects of ERRα on the osteoblast lineage contribute to the reduced bone mineral density observed upon estrogen deficiency. PMID:23359549

  8. Repression of osteoblast maturation by ERRα accounts for bone loss induced by estrogen deficiency.

    PubMed

    Gallet, Marlène; Saïdi, Soraya; Haÿ, Eric; Photsavang, Johann; Marty, Caroline; Sailland, Juliette; Carnesecchi, Julie; Tribollet, Violaine; Barenton, Bruno; Forcet, Christelle; Birling, Marie-Christine; Sorg, Tania; Chassande, Olivier; Cohen-Solal, Martine; Vanacker, Jean-Marc

    2013-01-01

    ERRα is an orphan member of the nuclear receptor family, the complete inactivation of which confers resistance to bone loss induced by ageing and estrogen withdrawal to female mice in correlation with increased bone formation in vivo. Furthermore ERRα negatively regulates the commitment of mesenchymal cells to the osteoblast lineage ex vivo as well as later steps of osteoblast maturation. We searched to determine whether the activities of ERRα on osteoblast maturation are responsible for one or both types of in vivo induced bone loss. To this end we have generated conditional knock out mice in which the receptor is normally present during early osteoblast differentiation but inactivated upon osteoblast maturation. Bone ageing in these animals was similar to that observed for control animals. In contrast conditional ERRαKO mice were completely resistant to bone loss induced by ovariectomy. We conclude that the late (maturation), but not early (commitment), negative effects of ERRα on the osteoblast lineage contribute to the reduced bone mineral density observed upon estrogen deficiency.

  9. Role of biphasic calcium phosphate ceramic-mediated secretion of signaling molecules by macrophages in migration and osteoblastic differentiation of MSCs.

    PubMed

    Wang, Jing; Liu, Dan; Guo, Bo; Yang, Xiao; Chen, Xuening; Zhu, Xiangdong; Fan, Yujiang; Zhang, Xingdong

    2017-03-15

    The inflammatory reaction initiates fracture healing and could play a role in the osteoinductive effect of calcium phosphate (CaP) ceramics, which has been widely confirmed; however, the underlying mechanism has not been fully elucidated. In this study, various signaling molecules from macrophages under the stimulation of osteoinductive biphasic calcium phosphate (BCP) ceramic and its degradation products were examined and evaluated for their influence on the migration and osteoblastic differentiation of mesenchymal stem cells (MSCs). The results of cellular experiments confirmed that the gene expression of most inflammatory factors (IL-1, IL-6 and MCP-1) and growth factors (VEGF, PDGF and EGF) by macrophages were up-regulated to varying degrees by BCP ceramic and its degradation products. Cell migration tests demonstrated that the conditioned media (CMs), which contained abundant signaling molecules secreted by macrophages cultured on BCP ceramic and its degradation products, promoted the migration of MSCs. qRT-PCR analysis indicated that CMs promoted the gene expression of osteogenic markers (ALP, COL-I, OSX, BSP and OPN) in MSCs. ALP activity and mineralization staining further confirmed that CMs promoted the osteoblastic differentiation of MSCs. The present study confirmed the correlation between the inflammatory reaction and osteoinductive capacity of BCP ceramic. The ceramic itself and its degradation products can induce macrophages to express and secrete various signaling molecules, which then recruit and promote the MSCs to differentiate into osteoblasts. Compared with BCP conditioned media, degradation particles played a more substantial role in this process. Thus, inflammation initiated by BCP ceramic and its degradation products could be necessary for osteoinduction by the ceramic. It is known that the inflammatory reaction initiates fracture healing. The aim of this study was to examine whether osteoinductive BCP ceramics could cause macrophages to

  10. Immortalized Mouse Floxed Fam20c Dental Papillar Mesenchymal and Osteoblast Cell Lines Retain Their Primary Characteristics.

    PubMed

    Liu, Chao; Wang, Xiaofang; Zhang, Hua; Xie, Xiaohua; Liu, Peihong; Liu, Ying; Jani, Priyam H; Lu, Yongbo; Chen, Shuo; Qin, Chunlin

    2015-11-01

    Fam20c is essential for the normal mineralization of dentin and bone. The generation of odontoblast and osteoblast cell lines carrying floxed Fam20c allele can offer valuable tools for the study of the roles of Fam20c in the mineralization of dentin and bone. The limited capability of the primary odontoblasts and osteoblasts to proliferate necessitates the development of odontoblast and osteoblast cell lines serving as substitutes for the study of differentiation and mineralization of the odontoblasts and osteoblasts. In this study, we established and characterized immortalized mouse floxed Fam20c dental papilla mesenchymal and osteoblast cell lines. The isolated primary mouse floxed Fam20c dental papilla mesenchymal cells and osteoblasts were immortalized by the infection of lentivirus containing Simian Virus 40 T-antigen (SV40 T-Ag). The immortalization of floxed Fam20c dental papilla mesenchymal cells and osteoblasts was verified by the long-term passages and genomic integration of SV40 T-Ag. The immortalized floxed Fam20c dental papilla mesenchymal and osteoblast cell lines not only proliferated at a high rate and retained the morphology of their primary counterparts, but also preserved the dentin and bone specific gene expression as the primary dental papilla mesenchymal cells and osteoblasts did. Consistently, the capability of the primary floxed Fam20c dental papilla mesenchymal cells and osteoblasts to mineralize was also inherited by the immortalized dental papilla mesenchymal and osteoblast cell lines. Thus, we have successfully generated the immortalized mouse floxed Fam20c dental papilla mesenchymal and osteoblast cell lines. © 2015 Wiley Periodicals, Inc.

  11. Nuclear factor I-C reciprocally regulates adipocyte and osteoblast differentiation via control of canonical Wnt signaling.

    PubMed

    Zhou, Jie; Wang, Shan; Qi, Qi; Yang, Xiaoyue; Zhu, Endong; Yuan, Hairui; Li, Xuemei; Liu, Ying; Li, Xiaoxia; Wang, Baoli

    2017-05-01

    Nuclear factor I-C (NFIC) has recently been identified as an important player in osteogenesis and bone homeostasis in vivo However, the molecular mechanisms involved have yet to be defined. In the current study, Nfic expression was altered in primary marrow stromal cells and established progenitor lines after adipogenic and osteogenic treatment. Overexpression of Nfic in stromal cells ST2, mesenchymal cells C3H10T1/2, and primary marrow stromal cells inhibited adipogenic differentiation, whereas it promoted osteogenic differentiation. Conversely, silencing of endogenous Nfic in the cell lines enhanced adipogenic differentiation, whereas it blocked osteogenic differentiation. Mechanism investigations revealed that Nfic overexpression promoted nuclear translocation of β-catenin and increased nuclear protein levels of β-catenin and transcription factor 7-like 2 (TCF7L2). Promoter studies and the chromatin immunoprecipitation (ChIP) assay revealed that NFIC directly binds to the promoter of low-density lipoprotein receptor-related protein 5 (Lrp5) and thereafter transactivates the promoter. Finally, inactivation of canonical Wnt signaling in ST2 attenuated the inhibition of adipogenic differentiation and stimulation of osteogenic differentiation by NFIC. Our study suggests that NFIC balances adipogenic and osteogenic differentiation from progenitor cells through controlling canonical Wnt signaling and highlights the potential of NFIC as a target for new therapies to control metabolic disorders like osteoporosis and obesity.-Zhou, J., Wang, S., Qi, Q., Yang, X., Zhu, E., Yuan, H., Li, X., Liu, Y., Li, X., Wang, B. Nuclear factor I-C reciprocally regulates adipocyte and osteoblast differentiation via control of canonical Wnt signaling. © FASEB.

  12. miR-203 and miR-320 Regulate Bone Morphogenetic Protein-2-Induced Osteoblast Differentiation by Targeting Distal-Less Homeobox 5 (Dlx5).

    PubMed

    Laxman, Navya; Mallmin, Hans; Nilsson, Olle; Kindmark, Andreas

    2016-12-23

    MicroRNAs (miRNAs) are a family of small, non-coding RNAs (17-24 nucleotides), which regulate gene expression either by the degradation of the target mRNAs or inhibiting the translation of genes. Recent studies have indicated that miRNA plays an important role in regulating osteoblast differentiation. In this study, we identified miR-203 and miR-320b as important miRNAs modulating osteoblast differentiation. We identified Dlx5 as potential common target by prediction algorithms and confirmed this by knock-down and over expression of the miRNAs and assessing Dlx5 at mRNA and protein levels and specificity was verified by luciferase reporter assays. We examined the effect of miR-203 and miR-320b on osteoblast differentiation by transfecting with pre- and anti-miRs. Over-expression of miR-203 and miR-320b inhibited osteoblast differentiation, whereas inhibition of miR-203 and miR-320b stimulated alkaline phosphatase activity and matrix mineralization. We show that miR-203 and miR-320b negatively regulate BMP-2-induced osteoblast differentiation by suppressing Dlx5 , which in turn suppresses the downstream osteogenic master transcription factor Runx2 and Osx and together they suppress osteoblast differentiation. Taken together, we propose a role for miR-203 and miR-320b in modulating bone metabolism.

  13. miR-203 and miR-320 Regulate Bone Morphogenetic Protein-2-Induced Osteoblast Differentiation by Targeting Distal-Less Homeobox 5 (Dlx5)

    PubMed Central

    Laxman, Navya; Mallmin, Hans; Nilsson, Olle; Kindmark, Andreas

    2016-01-01

    MicroRNAs (miRNAs) are a family of small, non-coding RNAs (17–24 nucleotides), which regulate gene expression either by the degradation of the target mRNAs or inhibiting the translation of genes. Recent studies have indicated that miRNA plays an important role in regulating osteoblast differentiation. In this study, we identified miR-203 and miR-320b as important miRNAs modulating osteoblast differentiation. We identified Dlx5 as potential common target by prediction algorithms and confirmed this by knock-down and over expression of the miRNAs and assessing Dlx5 at mRNA and protein levels and specificity was verified by luciferase reporter assays. We examined the effect of miR-203 and miR-320b on osteoblast differentiation by transfecting with pre- and anti-miRs. Over-expression of miR-203 and miR-320b inhibited osteoblast differentiation, whereas inhibition of miR-203 and miR-320b stimulated alkaline phosphatase activity and matrix mineralization. We show that miR-203 and miR-320b negatively regulate BMP-2-induced osteoblast differentiation by suppressing Dlx5, which in turn suppresses the downstream osteogenic master transcription factor Runx2 and Osx and together they suppress osteoblast differentiation. Taken together, we propose a role for miR-203 and miR-320b in modulating bone metabolism. PMID:28025541

  14. 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.

  15. Factors that affect postnatal bone growth retardation in the twitcher murine model of Krabbe disease.

    PubMed

    Contreras, Miguel Agustin; Ries, William Louis; Shanmugarajan, Srinivasan; Arboleda, Gonzalo; Singh, Inderjit; Singh, Avtar Kaur

    2010-01-01

    Krabbe disease is an inherited lysosomal disorder in which galactosylsphingosine (psychosine) accumulates mainly in the central nervous system. To gain insight into the possible mechanism(s) that may be participating in the inhibition of the postnatal somatic growth described in the animal model of this disease (twitcher mouse, twi), we studied their femora. This study reports that twi femora are smaller than of those of wild type (wt), and present with abnormality of marrow cellularity, bone deposition (osteoblastic function), and osteoclastic activity. Furthermore, lipidomic analysis indicates altered sphingolipid homeostasis, but without significant changes in the levels of sphingolipid-derived intermediates of cell death (ceramide) or the levels of the osteoclast-osteoblast coupling factor (sphingosine-1-phosphate). However, there was significant accumulation of psychosine in the femora of adult twi animals as compared to wt, without induction of tumor necrosis factor-alpha or interleukin-6. Analysis of insulin-like growth factor-1 (IGF-1) plasma levels, a liver secreted hormone known to play a role in bone growth, indicated a drastic reduction in twi animals when compared to wt. To identify the cause of the decrease, we examined the IGF-1 mRNA expression and protein levels in the liver. The results indicated a significant reduction of IGF-1 mRNA as well as protein levels in the liver from twi as compared to wt littermates. Our data suggest that a combination of endogenous (psychosine) and endocrine (IGF-1) factors play a role in the inhibition of postnatal bone growth in twi mice; and further suggest that derangements of liver function may be contributing, at least in part, to this alteration. Copyright 2010 Elsevier B.V. All rights reserved.

  16. Activation of the mitogen-activated protein kinase pathway by bone sialoprotein regulates osteoblast differentiation.

    PubMed

    Gordon, Jonathan A R; Hunter, Graeme K; Goldberg, Harvey A

    2009-01-01

    Bone sialoprotein (BSP) is an abundant protein in the extracellular matrix of bone that has been suggested to have several different physiological functions, including the nucleation of hydroxyapatite (HA), promotion of cell attachment and binding of collagen. Studies in our lab have demonstrated that increased expression of BSP in osteoblast cells can increase expression of the osteoblast-related genes Runx2 and Osx as well as alkaline phosphatase and osteocalcin and increase matrix mineralization. To determine the molecular mechanisms responsible for the BSP-mediated increase in osteoblastic differentiation, several functional domain mutants of BSP were expressed in primary rat bone osteoblastic cells, including the contiguous glutamic acid sequences (polyGlu) and the arginine-glycine-aspartic acid (RGD) motif. Markers of osteoblast differentiation, including matrix mineralization and alkaline phosphatase staining, were increased in cells expressing BSP mutants of the polyGlu sequences but not in cells expressing RGD-mutated BSP. We also determined the dependence on integrin-associated pathways in promoting BSP-mediated differentiation responses in osteoblasts by demonstrating the activation of focal adhesion kinase, MAP kinase-associated proteins ERK1/2, ribosomal s6 kinase 2 and the AP-1 protein cFos. Thus, the mechanism regulating osteoblast differentiation by BSP was determined to be dependent on integrin-mediated intracellular signaling pathways. Copyright 2008 S. Karger AG, Basel.

  17. 5,6-Dehydrokawain from Alpinia zerumbet promotes osteoblastic MC3T3-E1 cell differentiation.

    PubMed

    Kumagai, Momochika; Mishima, Takashi; Watanabe, Akio; Harada, Teppei; Yoshida, Izumi; Fujita, Kazuhiro; Watai, Masatoshi; Tawata, Shinkichi; Nishikawa, Keisuke; Morimoto, Yoshiki

    2016-07-01

    Bone homeostasis is maintained by balancing bone formation and bone resorption, but an imbalance between them is associated with various bone-related diseases such as osteoporosis and rheumatoid arthritis. We found that 5,6-dehydrokawain (DK) and dihydro-5,6-dehydrokawain (DDK), which were isolated as promising compounds from Alpinia zerumbet rhizomes, promote differentiation of osteoblastic MC3T3-E1 cells. DK and DDK increased the alkaline phosphatase activity and matrix mineralization of MC3T3-E1 cells. DK exerts larger effects than DDK. The gene expression of runt-related transcription factor 2 and osterix, which are essential transcription factors in the early period of osteoblast differentiation, was significantly increased by DK treatment. The mRNA level of distal-less homeobox 5 was also enhanced by DK treatment, and DK activated the p38 mitogen-activated protein kinase pathway. Therefore, DK may have clinical potential for preventing osteoporosis, and could be considered as a potential anabolic therapeutic agent.

  18. 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

  19. Low proliferation and high apoptosis of osteoblastic cells on hydrophobic surface are associated with defective Ras signaling

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chang, Eun-Ju; Kim, Hong-Hee; Huh, Jung-Eun

    2005-02-01

    The hydrophobic (HPB) nature of most polymeric biomaterials has been a major obstacle in using those materials in vivo due to low compatibility with cells. However, there is little knowledge of the molecular detail to explain how surface hydrophobicity affects cell responses. In this study, we compared the proliferation and apoptosis of human osteoblastic MG63 cells adhered to hydrophilic (HPL) and hydrophobic surfaces. On the hydrophobic surface, less formation of focal contacts and actin stress fibers, a delay in cell cycle progression, and an increase in apoptosis were observed. By using fibroblast growth factor 1 (FGF1) as a model growthmore » factor, we also investigated intracellular signaling pathways on hydrophilic and hydrophobic surfaces. The activation of Ras, Akt, and ERK by FGF1 was impaired in MG63 cells on the hydrophobic surface. The overexpression of constitutively active form of Ras and Akt rescued those cells from apoptosis and recovered cell cycle progression. Furthermore, their overexpression also restored the actin cytoskeletal organization on the hydrophobic surface. Finally, the proliferative, antiapoptotic, and cytoskeletal effects of constitutively active Ras in MG63 cells on the hydrophobic surface were blocked by wortmannin and PD98059 that inhibit Akt and ERK activation, respectively. Therefore, our results suggest that the activation of Ras and its downstream molecules Akt and ERK to an appropriate level is one of crucial elements in the determination of osteoblast cell responses. The Ras pathway may represent a cell biological target that should be considered for successful surface modification of biomaterials to induce adequate cell responses in the bone tissue.« less

  20. Evaluate the growth and adhesion of osteoblast cells on nanocomposite scaffold of hydroxyapatite/titania coated with poly hydroxybutyrate.

    PubMed

    Pourmollaabbassi, Babak; Karbasi, Saeed; Hashemibeni, Batool

    2016-01-01

    The generation of bioartificial bone tissues may help to overcome the problems related to donor site morbidity and size limitations. In this paper, hydroxyapatite (HA) powder was made out of bovine bone by thermal analysis at 900°C and first, and then, porous HA (50 weight percentage) was produced by polyurethane sponge replication method. In order to improve the scaffold mechanical properties, they have been coated with poly hydroxybutyrate. In terms of phase studies, morphology, and specifying agent groups, the specific characterization devices such as X-ray diffraction and Fourier transform infrared, were employed. To compare the behavior of cellular scaffolds, they were divided into four groups of scaffolds. The osteoblast cells were cultured. To perform phase studies, analysis of Methylthiazole tetrazolium (MTT) and Trypan blue were carried out for the viability and attachment on the surface of the scaffold, and the specification of Scanning electron microscopy was employed for the morphology of the cells. The results of MTT analysis performed on four groups of scaffolds have shown that Titanium oxide (Tio 2 ) had no effect on cell growth alone and HA was the main factor of growth and cell osteoblast adhesion on the scaffold. Moreover, the results showed that the use of coating with poly-3-hydroxybutyrate saved the factors and placed the osteoblasts within the pore. Since the main part of bone consists of HA, the TiO 2 accelerates the formation of apatite crystals at the scaffold surface which is the evidence for bone tissue regeneration. It is likely that the relation between HA and TiO 2 leads to an increase in osteoblast adhesion and growth of cells on the scaffold surface.

  1. 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.

  2. Effects of Arsenic on Osteoblast Differentiation in Vitro and on Bone Mineral Density and Microstructure in Rats

    PubMed Central

    Wu, Cheng-Tien; Lu, Tung-Ying; Chan, Ding-Cheng; Tsai, Keh-Sung; Yang, Rong-Sen

    2014-01-01

    Background: Arsenic is a ubiquitous toxic element and is known to contaminate drinking water in many countries. Several epidemiological studies have shown that arsenic exposure augments the risk of bone disorders. However, the detailed effect and mechanism of inorganic arsenic on osteoblast differentiation of bone marrow stromal cells and bone loss still remain unclear. Objectives: We investigated the effects and mechanism of arsenic on osteoblast differentiation in vitro and evaluated bone mineral density (BMD) and bone microstructure in rats at doses relevant to human exposure from drinking water. Methods: We used a cell model of rat primary bone marrow stromal cells (BMSCs) and a rat model of long-term exposure with arsenic-contaminated drinking water, and determined bone microstructure and BMD in rats by microcomputed tomography (μCT). Results: We observed significant attenuation of osteoblast differentiation after exposure of BMSCs to arsenic trioxide (0.5 or 1 μM). After arsenic treatment during differentiation, expression of runt-related transcription factor-2 (Runx2), bone morphogenetic protein-2 (BMP-2), and osteocalcin in BMSCs was inhibited and phosphorylation of enhanced extracellular signal-regulated kinase (ERK) was increased. These altered differentiation-related molecules could be reversed by the ERK inhibitor PD98059. Exposure of rats to arsenic trioxide (0.05 or 0.5 ppm) in drinking water for 12 weeks altered BMD and microstructure, decreased Runx2 expression, and increased ERK phosphorylation in bones. In BMSCs isolated from arsenic-treated rats, osteoblast differentiation was inhibited. Conclusions: Our results suggest that arsenic is capable of inhibiting osteoblast differentiation of BMSCs via an ERK-dependent signaling pathway and thus increasing bone loss. Citation: Wu CT, Lu TY, Chan DC, Tsai KS, Yang RS, Liu SH. 2014. Effects of arsenic on osteoblast differentiation in vitro and on bone mineral density and microstructure in rats. Environ

  3. Autophagy mediated CoCrMo particle-induced peri-implant osteolysis by promoting osteoblast apoptosis

    PubMed Central

    Wang, Zhenheng; Liu, Naicheng; Liu, Kang; Zhou, Gang; Gan, Jingjing; Wang, Zhenzhen; Shi, Tongguo; He, Wei; Wang, Lintao; Guo, Ting; Bao, Nirong; Wang, Rui; Huang, Zhen; Chen, Jiangning; Dong, Lei; Zhao, Jianning; Zhang, Junfeng

    2015-01-01

    Wear particle-induced osteolysis is the leading cause of aseptic loosening, which is the most common reason for THA (total hip arthroplasty) failure and revision surgery. Although existing studies suggest that osteoblast apoptosis induced by wear debris is involved in aseptic loosening, the underlying mechanism linking wear particles to osteoblast apoptosis remains almost totally unknown. In the present study, we investigated the effect of autophagy on osteoblast apoptosis induced by CoCrMo metal particles (CoPs) in vitro and in a calvarial resorption animal model. Our study demonstrated that CoPs stimulated autophagy in osteoblasts and PIO (particle-induced osteolysis) animal models. Both autophagy inhibitor 3-MA (3-methyladenine) and siRNA of Atg5 could dramatically reduce CoPs-induced apoptosis in osteoblasts. Further, inhibition of autophagy with 3-MA ameliorated the severity of osteolysis in PIO animal models. Moreover, 3-MA also prevented osteoblast apoptosis in an antiautophagic way when tested in PIO model. Collectively, these results suggest that autophagy plays a key role in CoPs-induced osteolysis and that targeting autophagy-related pathways may represent a potential therapeutic approach for treating particle-induced peri-implant osteolysis. PMID:26566231

  4. Osteoblast differentiation and skeletal development are regulated by Mdm2–p53 signaling

    PubMed Central

    Lengner, Christopher J.; Steinman, Heather A.; Gagnon, James; Smith, Thomas W.; Henderson, Janet E.; Kream, Barbara E.; Stein, Gary S.; Lian, Jane B.; Jones, Stephen N.

    2006-01-01

    Mdm2 is required to negatively regulate p53 activity at the peri-implantation stage of early mouse development. However, the absolute requirement for Mdm2 throughout embryogenesis and in organogenesis is unknown. To explore Mdm2–p53 signaling in osteogenesis, Mdm2-conditional mice were bred with Col3.6-Cre–transgenic mice that express Cre recombinase in osteoblast lineage cells. Mdm2-conditional Col3.6-Cre mice die at birth and display multiple skeletal defects. Osteoblast progenitor cells deleted for Mdm2 have elevated p53 activity, reduced proliferation, reduced levels of the master osteoblast transcriptional regulator Runx2, and reduced differentiation. In contrast, p53-null osteoprogenitor cells have increased proliferation, increased expression of Runx2, increased osteoblast maturation, and increased tumorigenic potential, as mice specifically deleted for p53 in osteoblasts develop osteosarcomas. These results demonstrate that p53 plays a critical role in bone organogenesis and homeostasis by negatively regulating bone development and growth and by suppressing bone neoplasia and that Mdm2-mediated inhibition of p53 function is a prerequisite for Runx2 activation, osteoblast differentiation, and proper skeletal formation. PMID:16533949

  5. Osteoblasts are target cells for transformation in c-fos transgenic mice

    PubMed Central

    1993-01-01

    We have generated transgenic mice expressing the proto-oncogene c-fos from an H-2Kb class I MHC promoter as a tool to identify and isolate cell populations which are sensitive to altered levels of Fos protein. All homozygous H2-c-fosLTR mice develop osteosarcomas with a short latency period. This phenotype is specific for c-fos as transgenic mice expressing the fos- and jun-related genes, fosB and c-jun, from the same regulatory elements do not develop any pathology despite high expression in bone tissues. The c-fos transgene is not expressed during embryogenesis but is expressed after birth in bone tissues before the onset of tumor formation, specifically in putative preosteoblasts, bone- forming osteoblasts, osteocytes, as well as in osteoblastic cells present within the tumors. Primary and clonal cell lines established from c-fos-induced tumors expressed high levels of exogenous c-fos as well as the bone cell marker genes, type I collagen, alkaline phosphatase, and osteopontin/2ar. In contrast, osteocalcin/BGP expression was either low or absent. All cell lines were tumorigenic in vivo, some of which gave rise to osteosarcomas, expressing exogenous c- fos mRNA, and Fos protein in osteoblastic cells. Detailed analysis of one osteogenic cell line, P1, and several P1-derived clonal cell lines indicated that bone-forming osteoblastic cells were transformed by Fos. The regulation of osteocalcin/BGP and alkaline phosphatase gene expression by 1,25-dihydroxyvitamin D3 was abrogated in P1-derived clonal cells, whereas glucocorticoid responsiveness was unaltered. These results suggest that high levels of Fos perturb the normal growth control of osteoblastic cells and exert specific effects on the expression of the osteoblast phenotype. PMID:8335693

  6. Factors affecting construction performance: exploratory factor analysis

    NASA Astrophysics Data System (ADS)

    Soewin, E.; Chinda, T.

    2018-04-01

    The present work attempts to develop a multidimensional performance evaluation framework for a construction company by considering all relevant measures of performance. Based on the previous studies, this study hypothesizes nine key factors, with a total of 57 associated items. The hypothesized factors, with their associated items, are then used to develop questionnaire survey to gather data. The exploratory factor analysis (EFA) was applied to the collected data which gave rise 10 factors with 57 items affecting construction performance. The findings further reveal that the items constituting ten key performance factors (KPIs) namely; 1) Time, 2) Cost, 3) Quality, 4) Safety & Health, 5) Internal Stakeholder, 6) External Stakeholder, 7) Client Satisfaction, 8) Financial Performance, 9) Environment, and 10) Information, Technology & Innovation. The analysis helps to develop multi-dimensional performance evaluation framework for an effective measurement of the construction performance. The 10 key performance factors can be broadly categorized into economic aspect, social aspect, environmental aspect, and technology aspects. It is important to understand a multi-dimension performance evaluation framework by including all key factors affecting the construction performance of a company, so that the management level can effectively plan to implement an effective performance development plan to match with the mission and vision of the company.

  7. Fibronectins containing extradomain A or B enhance osteoblast differentiation via distinct integrins

    PubMed Central

    Sens, Carla; Huck, Katrin; Pettera, Stefan; Uebel, Stephan; Wabnitz, Guido; Moser, Markus; Nakchbandi, Inaam A.

    2017-01-01

    Fibronectin is a multidomain protein secreted by various cell types. It forms a network of fibers within the extracellular matrix and impacts intracellular processes by binding to various molecules, primarily integrin receptors on the cells. Both the presence of several isoforms and the ability of the various domains and isoforms to bind to a variety of integrins result in a wide range of effects. In vivo findings suggest that fibronectin isoforms produced by the osteoblasts enhance their differentiation. Here we report that the isoform characterized by the presence of extradomain A activates α4β1 integrin and augments osteoblast differentiation. In addition, the isoform containing extradomain B enhances the binding of fibronectin through the RGD sequence to β3-containing integrin, resulting in increased mineralization by and differentiation of osteoblasts. Our study thus reveals novel functions for two fibronectin isoforms and the mediating receptors in osteoblast differentiation. PMID:28325836

  8. 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.

  9. Developmental regulation of collagenase-3 mRNA in normal, differentiating osteoblasts through the activator protein-1 and the runt domain binding sites

    NASA Technical Reports Server (NTRS)

    Winchester, S. K.; Selvamurugan, N.; D'Alonzo, R. C.; Partridge, N. C.

    2000-01-01

    Collagenase-3 mRNA is initially detectable when osteoblasts cease proliferation, increasing during differentiation and mineralization. We showed that this developmental expression is due to an increase in collagenase-3 gene transcription. Mutation of either the activator protein-1 or the runt domain binding site decreased collagenase-3 promoter activity, demonstrating that these sites are responsible for collagenase-3 gene transcription. The activator protein-1 and runt domain binding sites bind members of the activator protein-1 and core-binding factor family of transcription factors, respectively. We identified core-binding factor a1 binding to the runt domain binding site and JunD in addition to a Fos-related antigen binding to the activator protein-1 site. Overexpression of both c-Fos and c-Jun in osteoblasts or core-binding factor a1 increased collagenase-3 promoter activity. Furthermore, overexpression of c-Fos, c-Jun, and core-binding factor a1 synergistically increased collagenase-3 promoter activity. Mutation of either the activator protein-1 or the runt domain binding site resulted in the inability of c-Fos and c-Jun or core-binding factor a1 to increase collagenase-3 promoter activity, suggesting that there is cooperative interaction between the sites and the proteins. Overexpression of Fra-2 and JunD repressed core-binding factor a1-induced collagenase-3 promoter activity. Our results suggest that members of the activator protein-1 and core-binding factor families, binding to the activator protein-1 and runt domain binding sites are responsible for the developmental regulation of collagenase-3 gene expression in osteoblasts.

  10. Steroid and xenobiotic receptor-mediated effects of bisphenol A on human osteoblasts.

    PubMed

    Miki, Yasuhiro; Hata, Shuko; Nagasaki, Shuji; Suzuki, Takashi; Ito, Kiyoshi; Kumamoto, Hiroyuki; Sasano, Hironobu

    2016-06-15

    Bisphenol A, one of the industrial chemicals used in plastics and in the coating of dishes and medical equipment, behaves as an endocrine disruptor in the human body. Bisphenol A can bind directly to several types of nuclear receptors, including steroid and xenobiotic receptor (SXR). SXR plays an important role in bone metabolism through the activation of osteoblasts in vitro, but SXR protein localization has not been reported in bone tissues. Additionally, it is not known whether bisphenol A acts on osteoblasts through SXR activation. Therefore, in this study, we first examined the immunolocalization of the SXR protein in human adult and fetal bone tissues. We then examined the effects of bisphenol A on human osteoblasts in vitro. SXR immunoreactivity was detected in osteoblasts, but not in osteoclasts, of both adult and fetal bone tissues. In fetal bone tissues, the mesenchymal cells or fetal connective tissue were also positive for SXR immunoreactivity. Expression of SXR target genes (tsukushi, matrilin-2, and CYP3A4) and SXR response element-luciferase activity were increased by bisphenol A treatment in normal osteoblasts transfected with SXR (hFOB/SXR) and in osteoblast-like cells (MG-63). Bisphenol A also stimulated cell proliferation and collagen accumulation in hFOB/SXR cells. These results suggest that, as in other tissues, SXR plays important roles in bone metabolism and fetal bone development and that bisphenol A may disturb bone homeostasis in both adult and fetus through SXR. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Osteoblast-Specific Loss of IGF1R Signaling Results in Impaired Endochondral Bone Formation During Fracture Healing.

    PubMed

    Wang, Tao; Wang, Yongmei; Menendez, Alicia; Fong, Chak; Babey, Muriel; Tahimic, Candice G T; Cheng, Zhiqiang; Li, Alfred; Chang, Wenhan; Bikle, Daniel D

    2015-09-01

    Insulin-like growth factors (IGFs) are important local regulators during fracture healing. Although IGF1 deficiency is known to increase the risk of delayed union or non-union fractures in the elderly population, the underlying mechanisms that contribute to this defect remains unclear. In this study, IGF1 signaling during fracture healing was investigated in an osteoblast-specific IGF1 receptor (IGF1R) conditional knockout (KO) mouse model. A closed tibial fracture was induced in IGF1R(flox/flox) /2.3-kb α1(1)-collagen-Cre (KO) and IGF1R(flox/flox) (control) mice aged 12 weeks. Fracture callus samples and nonfractured tibial diaphysis were collected and analyzed by μCT, histology, immunohistochemistry, histomorphometry, and gene expression analysis at 10, 15, 21, and 28 days after fracture. A smaller size callus, lower bone volume accompanied by a defect in mineralization, bone microarchitectural abnormalities, and a higher cartilage volume were observed in the callus of these KO mice. The levels of osteoblast differentiation markers (osteocalcin, alkaline phosphatase, collagen 1α1) were significantly reduced, but the early osteoblast transcription factor runx2, as well as chondrocyte differentiation markers (collagen 2α1 and collagen 10α1) were significantly increased in the KO callus. Moreover, increased numbers of osteoclasts and impaired angiogenesis were observed during the first 15 days of fracture repair, but decreased numbers of osteoclasts were found in the later stages of fracture repair in the KO mice. Although baseline nonfractured tibias of KO mice had decreased trabecular and cortical bone compared to control mice, subsequent studies with mice expressing the 2.3-kb α1(1)-collagen-Cre ERT2 construct and given tamoxifen at the time of fracture and so starting with comparable bone levels showed similar impairment in fracture repair at least initially. Our data indicate that not only is the IGF1R in osteoblasts involved in osteoblast differentiation

  12. Osteoblast-Specific Loss of IGF1R Signaling Results in Impaired Endochondral Bone Formation During Fracture Healing

    PubMed Central

    Wang, Tao; Wang, Yongmei; Menendez, Alicia; Fong, Chak; Babey, Muriel; Tahimic, Candice GT; Cheng, Zhiqiang; Li, Alfred; Chang, Wenhan; Bikle, Daniel D.

    2017-01-01

    Insulin-like growth factors (IGFs) are important local regulators during fracture healing. Although IGF1 deficiency is known to increase the risk of delayed union or non-union fractures in the elderly population, the underlying mechanisms that contribute to this defect remains unclear. In this study, IGF1 signaling during fracture healing was investigated in an osteoblast-specific IGF1 receptor (IGF1R) conditional knockout (KO) mouse model. A closed tibial fracture was induced in IGF1Rflox/flox/2.3-kb α1(1)-collagen-Cre (KO) and IGF1Rflox/flox (control) mice aged 12 weeks. Fracture callus samples and nonfractured tibial diaphysis were collected and analyzed by μCT, histology, immunohistochemistry, histomorphometry, and gene expression analysis at 10, 15, 21, and 28 days after fracture. A smaller size callus, lower bone volume accompanied by a defect in mineralization, bone microarchitectural abnormalities, and a higher cartilage volume were observed in the callus of these KO mice. The levels of osteoblast differentiation markers (osteocalcin, alkaline phosphatase, collagen 1α1) were significantly reduced, but the early osteoblast transcription factor runx2, as well as chondrocyte differentiation markers (collagen 2α1 and collagen 10α1) were significantly increased in the KO callus. Moreover, increased numbers of osteoclasts and impaired angiogenesis were observed during the first 15 days of fracture repair, but decreased numbers of osteoclasts were found in the later stages of fracture repair in the KO mice. Although baseline nonfractured tibias of KO mice had decreased trabecular and cortical bone compared to control mice, subsequent studies with mice expressing the 2.3-kb α1(1)-collagen-Cre ERT2 construct and given tamoxifen at the time of fracture and so starting with comparable bone levels showed similar impairment in fracture repair at least initially. Our data indicate that not only is the IGF1R in osteoblasts involved in osteoblast differentiation

  13. [Effect of osthole on proliferation of neonatal rat osteoblast and the relative mechanism research].

    PubMed

    Li, Ling-Hui; Ding, Dao-Fang; Du, Guo-Qing; Gong, Hao; Wang, Hui-Hao; Zhan, Hong-Sheng

    2013-05-01

    To investigate the effects of osthole on proliferation of neonatal rat osteoblast and the mechanism. Ten 24 hours old SD rats were executed by dislocating. The cranium of rats were removed and cut into blocks of 1 mm x 1 mm size. After digested by trypsin for 15 min, the cranium were digested by type I collagenase for one hour two times. The mixed cells were cultured in thermostat incubator with 5% CO2 under the condition of 37 degrees C. To identify the cells, ALP staining and alizarin red staining were performed after cultured 48 h and 28 d. The osteoblasts were randomly divided into five groups. Cells were treated with osthole at concentrations of 100, 50, 25, 12.5, 0 micromol/L. CCK-8 method was used to evaluate the proliferation after 24 h,48 h and 72 h. The expression of PCNA and beta-catenin protein were detected through the method of Western Blot after one week. The cells had irregular shapes and showed typical features of osteoblast. The results of ALP staining and alizarin red staining were both positive. CCK-8 detection showed that the osthole with final concentration of 100 micromol/L inhibited the proliferation of osteoblast after 24 h, while the osthole with final concentrations of 50 micromol/L and 25 micromol/L displayed the inhibition effect after 48 h. The osthole of 12.5 micromol/L had no obvious influence on the proliferation of osteoblast. The result of Western Blot showed that osthole reduced the expression of PCNA and beta-catenin protein in a dose-dependent manner. The osthole with final concentrations of 100, 50, 25 micromol/L inhibited the proliferation of osteoblast (P < 0.05). The osthole with final concentrations of 12.5 micromol/L had no obvious influence on the proliferation of osteoblast (P > 0.05). These findings demonstrate that osthole may inhibit the proliferation of osteoblast by regulating the Wnt/beta-catenin signaling in osteoblast.

  14. The role of intracellular calcium phosphate in osteoblast-mediated bone apatite formation

    PubMed Central

    Boonrungsiman, Suwimon; Gentleman, Eileen; Carzaniga, Raffaella; Evans, Nicholas D.; McComb, David W.; Porter, Alexandra E.; Stevens, Molly M.

    2012-01-01

    Mineralization is a ubiquitous process in the animal kingdom and is fundamental to human development and health. Dysfunctional or aberrant mineralization leads to a variety of medical problems, and so an understanding of these processes is essential to their mitigation. Osteoblasts create the nano-composite structure of bone by secreting a collagenous extracellular matrix (ECM) on which apatite crystals subsequently form. However, despite their requisite function in building bone and decades of observations describing intracellular calcium phosphate, the precise role osteoblasts play in mediating bone apatite formation remains largely unknown. To better understand the relationship between intracellular and extracellular mineralization, we combined a sample-preparation method that simultaneously preserved mineral, ions, and ECM with nano-analytical electron microscopy techniques to examine osteoblasts in an in vitro model of bone formation. We identified calcium phosphate both within osteoblast mitochondrial granules and intracellular vesicles that transported material to the ECM. Moreover, we observed calcium-containing vesicles conjoining mitochondria, which also contained calcium, suggesting a storage and transport mechanism. Our observations further highlight the important relationship between intracellular calcium phosphate in osteoblasts and their role in mineralizing the ECM. These observations may have important implications in deciphering both how normal bone forms and in understanding pathological mineralization. PMID:22879397

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-03-18

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

  17. Biocompatibility evaluation of HDPE-UHMWPE reinforced β-TCP nanocomposites using highly purified human osteoblast cells.

    PubMed

    Shokrgozar, M A; Farokhi, M; Rajaei, F; Bagheri, M H A; Azari, Sh; Ghasemi, I; Mottaghitalab, F; Azadmanesh, K; Radfar, J

    2010-12-15

    Biocompatibility of β-TCP/HDPE-UHMWPE nanocomposite as a new bone substitute material was evaluated by using highly purified human osteoblast cells. Human osteoblast cells were isolated from bone tissue and characterized by immunofluorescence Staining before and after purification using magnetic bead system. Moreover, proliferation, alkaline phosphatase production, cell attachment, calcium deposition, gene expression, and morphology of osteoblast cells on β-TCP/HDPE-UHMWPE nanocomposites were evaluated. The results have shown that the human osteoblast cells were successfully purified and were suitable for subsequent cell culturing process. The high proliferation rate of osteoblast cells on β-TCP/HDPE-UHMWPE nanocomposite confirmed the great biocompatibility of the scaffold. Expression of bone-specific genes was taken place after the cells were incubated in composite extract solutions. Furthermore, osteoblast cells were able to mineralize the matrix next to composite samples. Scanning electron microscopy demonstrated that cells had normal morphology on the scaffold. Thus, these results indicated that the nanosized β-TCP/HDPE-UHMWPE blend composites could be potential scaffold, which is used in bone tissue engineering. Copyright © 2010 Wiley Periodicals, Inc.

  18. Factors Affecting Medical Service Quality.

    PubMed

    Mosadeghrad, Ali Mohammad

    2014-02-01

    A better understanding of factors influencing quality of medical service can pinpoint better strategies for quality assurance in medical services. This study aimed to identify factors affecting the quality of medical services provided by Iranian physicians. Exploratory in-depth individual interviews were conducted with sixty-four physicians working in various medical institutions in Iran. Individual, organizational and environmental factors enhance or inhibit the quality of medical services. Quality of medical services depends on the personal factors of the physician and patient, and factors pertaining to the healthcare setting and the broader environment. Differences in internal and external factors such as availability of resources, patient cooperation and collaboration among providers affect the quality of medical services and patient outcomes. Supportive leadership, proper planning, education and training and effective management of resources and processes improve the quality of medical services. This article contributes to healthcare theory and practice by developing a conceptual framework for understanding factors that influence medical services quality.

  19. Oxidized low-density lipoprotein acts synergistically with beta-glycerophosphate to induce osteoblast differentiation in primary cultures of vascular smooth muscle cells.

    PubMed

    Bear, Mackenzie; Butcher, Martin; Shaughnessy, Stephen G

    2008-09-01

    Previous studies have localized osteoblast specific markers to sites of calcified atherosclerotic lesions. We therefore decided to use an established in vitro model of vascular calcification in order to confirm earlier reports of oxidized low-density lipoprotein (oxLDL) promoting the osteogenic differentiation of vascular smooth muscle cells. Treatment of primary bovine aortic smooth muscle cells (BASMCs) with beta-glycerophosphate was found to induce a time-dependent increase in osteoblast differentiation. In contrast, no effect was seen when BASMCs were cultured in the presence of oxLDL alone. However, when the BASMCs were cultured in the presence of both beta-glycerophosphate and oxLDL, beta-glycerophosphate's ability to induce osteoblast differentiation was significantly enhanced. In an attempt to resolve the mechanism by which this effect was occurring, we examined the effect of beta-glycerophosphate and oxLDL on several pathways known to be critical to the differentiation of osteoblasts. Surprisingly, beta-glycerophosphate alone was found to enhance Osterix (Osx) expression by inducing both Smad 1/5/8 activation and Runx2 expression. In contrast, oxLDL did not affect either Smad 1/5/8 activation or Runx2 activation but rather, it enhanced both beta-glycerophosphate-induced Osx expression and osteoblast differentiation in an extracellular signal-regulated kinase 1 and 2 (Erk 1 and 2) -dependent manner. When taken together, these findings suggest a plausible mechanism by which oxLDL may promote osteogenic differentiation and vascular calcification in vivo. J. Cell. Biochem. 105: 185-193, 2008. (c) 2008 Wiley-Liss, Inc. (c) 2008 Wiley-Liss, Inc.

  20. Monosodium urate monohydrate crystals inhibit osteoblast viability and function: implications for development of bone erosion in gout.

    PubMed

    Chhana, Ashika; Callon, Karen E; Pool, Bregina; Naot, Dorit; Watson, Maureen; Gamble, Greg D; McQueen, Fiona M; Cornish, Jillian; Dalbeth, Nicola

    2011-09-01

    Bone erosion is a common manifestation of chronic tophaceous gout. To investigate the effects of monosodium urate monohydrate (MSU) crystals on osteoblast viability and function. The MTT assay and flow cytometry were used to assess osteoblast cell viability in the MC3T3-E1 and ST2 osteoblast-like cell lines, and primary rat and primary human osteoblasts cultured with MSU crystals. Quantitative real-time PCR and von Kossa stained mineralised bone formation assays were used to assess the effects of MSU crystals on osteoblast differentiation using MC3T3-E1 cells. The numbers of osteoblasts and bone lining cells were quantified in bone samples from patients with gout. MSU crystals rapidly reduced viability in all cell types in a dose-dependent manner. The inhibitory effect on cell viability was independent of crystal phagocytosis and was not influenced by differing crystal length or addition of serum. Long-term culture of MC3T3-E1 cells with MSU crystals showed a reduction in mineralisation and decreased mRNA expression of genes related to osteoblast differentiation such as Runx2, Sp7 (osterix), Ibsp (bone sialoprotein), and Bglap (osteocalcin). Fewer osteoblast and lining cells were present on bone directly adjacent to gouty tophus than bone unaffected by tophus in patients with gout. MSU crystals have profound inhibitory effects on osteoblast viability and differentiation. These data suggest that bone erosion in gout occurs at the tophus-bone interface through alteration of physiological bone turnover, with both excessive osteoclast formation, and reduced osteoblast differentiation from mesenchymal stem cells.

  1. Osteoblast mineralization requires β1 integrin/ICAP-1–dependent fibronectin deposition

    PubMed Central

    Brunner, Molly; Millon-Frémillon, Angélique; Chevalier, Genevieve; Nakchbandi, Inaam A.; Mosher, Deane; Block, Marc R.

    2011-01-01

    The morphogenetic and differentiation events required for bone formation are orchestrated by diffusible and insoluble factors that are localized within the extracellular matrix. In mice, the deletion of ICAP-1, a modulator of β1 integrin activation, leads to severe defects in osteoblast proliferation, differentiation, and mineralization and to a delay in bone formation. Deposition of fibronectin and maturation of fibrillar adhesions, adhesive structures that accompany fibronectin deposition, are impaired upon ICAP-1 loss, as are type I collagen deposition and mineralization. Expression of β1 integrin with a mutated binding site for ICAP-1 recapitulates the ICAP-1–null phenotype. Follow-up experiments demonstrated that ICAP-1 negatively regulates kindlin-2 recruitment onto the β1 integrin cytoplasmic domain, whereas an excess of kindlin-2 binding has a deleterious effect on fibrillar adhesion formation. These results suggest that ICAP-1 works in concert with kindlin-2 to control the dynamics of β1 integrin–containing fibrillar adhesions and, thereby, regulates fibronectin deposition and osteoblast mineralization. PMID:21768292

  2. Assessing the osteoblast transcriptome in a model of enhanced bone formation due to constitutive G{sub s}–G protein signaling in osteoblasts

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wattanachanya, Lalita, E-mail: lalita_md@yahoo.com; Division of Endocrinology and Metabolism, Department of Medicine, Faculty of Medicine, Chulalongkorn University and King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Bangkok; Wang, Liping, E-mail: lipingwang05@yahoo.com

    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 cellularmore » 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

  3. Gene expression during skeletal development in three osteopetrotic rat mutations. Evidence for osteoblast abnormalities.

    PubMed

    Shalhoub, V; Jackson, M E; Lian, J B; Stein, G S; Marks, S C

    1991-05-25

    Osteopetrosis is a group of metabolic bone diseases characterized by reductions in osteoclast development and/or function. These aspects of osteoclast biology are known to be influenced by osteoblasts and their products. To ascertain whether osteoblast dysfunction contributes to aberrations in the structural and functional properties of osteoclasts in osteopetrosis, we systematically examined gene expression as reflected by mRNA levels for a series of cell growth- and tissue-related genes associated with the osteoblast phenotype during skeletal development in normal and mutant rats of three different osteopetrotic stocks. We show that the methods used permit the reproducible isolation of undegraded total cellular RNA from bone and that mRNA levels can be reliably quantitated in these preparations. Each osteopetrotic mutation exhibits a distinct aberrant pattern of osteoblast gene expression that may be correlated with and explain some abnormalities in extracellular matrix composition, mineralization, osteoclast development, and effects of elevated serum levels of 1 alpha,25-dihydroxyvitamin D3, depending upon the mutation. Normal rats show minor variations in gene expression that reflect the genetic background (stock). This, the first comprehensive molecular analysis of osteoblast gene expression in osteopetrosis, suggests that some osteopetroses, particularly in the toothless rat, are associated with and potentially related to mechanisms associated with aberrations in osteoblast function. More generally, the present studies demonstrate alterations in gene expression as reflected by mRNA levels that are associated with functional properties of the osteoblast, particularly those contributing to the recruitment and/or differentiation of osteoclasts, thereby influencing skeletal modeling.

  4. The Effects of Orbital Spaceflight on Human Osteoblastic Cell Physiology and Gene Expression

    NASA Technical Reports Server (NTRS)

    Turner, R. T.

    1999-01-01

    The purpose of the proposed study is to establish whether changes in gravitational loading have a direct effect on osteoblasts to regulate TGF-6 expression. The effects of spaceflight and reloading on TGF-B MRNA and peptide levels will be studied in a newly developed line of immortalized human fetal osteoblasts (HFOB) transfected with an SV-40 temperature dependent mutant to generate proliferating, undifferentiated hFOB cells at 33-34 C and a non-proliferating, differentiated HFOB cells at 37-39'C. Unlike previous cell culture models, HFOB cells have unlimited proliferative capacity yet can be precisely regulated to differentiate into mature cells which express mature osteoblast function. If isolated osteoblasts respond to changes in mechanical loading in a manner similar to their response in animals, the cell system could provide a powerful model to investigate the signal transduction pathway for gravitational loading.

  5. 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

  6. Evaluate the growth and adhesion of osteoblast cells on nanocomposite scaffold of hydroxyapatite/titania coated with poly hydroxybutyrate

    PubMed Central

    Pourmollaabbassi, Babak; Karbasi, Saeed; Hashemibeni, Batool

    2016-01-01

    Background: The generation of bioartificial bone tissues may help to overcome the problems related to donor site morbidity and size limitations. Materials and Methods: In this paper, hydroxyapatite (HA) powder was made out of bovine bone by thermal analysis at 900°C and first, and then, porous HA (50 weight percentage) was produced by polyurethane sponge replication method. In order to improve the scaffold mechanical properties, they have been coated with poly hydroxybutyrate. In terms of phase studies, morphology, and specifying agent groups, the specific characterization devices such as X-ray diffraction and Fourier transform infrared, were employed. To compare the behavior of cellular scaffolds, they were divided into four groups of scaffolds. The osteoblast cells were cultured. To perform phase studies, analysis of Methylthiazole tetrazolium (MTT) and Trypan blue were carried out for the viability and attachment on the surface of the scaffold, and the specification of Scanning electron microscopy was employed for the morphology of the cells. Results: The results of MTT analysis performed on four groups of scaffolds have shown that Titanium oxide (Tio2) had no effect on cell growth alone and HA was the main factor of growth and cell osteoblast adhesion on the scaffold. Moreover, the results showed that the use of coating with poly-3-hydroxybutyrate saved the factors and placed the osteoblasts within the pore. Since the main part of bone consists of HA, the TiO2 accelerates the formation of apatite crystals at the scaffold surface which is the evidence for bone tissue regeneration. Conclusions: It is likely that the relation between HA and TiO2 leads to an increase in osteoblast adhesion and growth of cells on the scaffold surface. PMID:27761431

  7. Osteoblast Differentiation on Collagen Scaffold with Immobilized Alkaline Phosphatase.

    PubMed

    Jafary, F; Hanachi, P; Gorjipour, K

    2017-01-01

    In tissue engineering, scaffold characteristics play an important role in the biological interactions between cells and the scaffold. Cell adhesion, proliferation, and activation depend on material properties used for the fabrication of scaffolds. In the present investigation, we used collagen with proper characteristics including mechanically stability, biodegradability and low antigenicity. Optimization of the scaffold was done by immobilization of alkaline phosphatase on the collagen surface via cross-linking method, because this enzyme is one of the most important markers of osteoblast, which increases inorganic phosphate concentration and promote mineralization of bone formation. Alkaline phosphatase was immobilized on a collagen surface by 1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride, as a reagent. Then, rat mesenchymal stem cells were cultured in osteogenic medium in control and treated groups. The osteogenesis-related genes were compared between treatments (differentiated cells with immobilized alkaline phosphatase/collagen scaffold) and control groups (differentiated cells on collagen surface without alkaline phosphatase) on days 3 and 7 by quantitative real-time PCR (QIAGEN software). Several genes, including alkaline phosphatase, collagen type I and osteocalcine associated with calcium binding and mineralization, showed upregulation in expression during the first 3 days, whereas tumor necrosis factor-α, acting as an inhibitor of differentiation, was down-regulated during osteogenesis. Collagen scaffold with immobilized alkaline phosphatase can be utilized as a good candidate for enhancing the differentiation of osteoblasts from mesenchymal stem cells.

  8. Osteocyte viability and regulation of osteoblast function in a 3D trabecular bone explant under dynamic hydrostatic pressure.

    PubMed

    Takai, Erica; Mauck, Robert L; Hung, Clark T; Guo, X Edward

    2004-09-01

    A new trabecular bone explant model was used to examine osteocyte-osteoblast interactions under DHP loading. DHP loading enhanced osteocyte viability as well as osteoblast function measured by osteoid formation. However, live osteocytes were necessary for osteoblasts to form osteoids in response to DHP, which directly show osteoblast-osteocyte interactions in this in vitro culture. A trabecular bone explant model was characterized and used to examine the effect of osteocyte and osteoblast interactions and dynamic hydrostatic pressure (DHP) loading on osteocyte viability and osteoblast function in long-term culture. Trabecular bone cores obtained from metacarpals of calves were cleaned of bone marrow and trabecular surface cells and divided into six groups, (1) live cores + dynamic hydrostatic pressure (DHP), (2) live cores + sham, (3) live cores + osteoblast + DHP, (4) live cores + osteoblast + sham, (5) devitalized cores + osteoblast + DHP, and (6) devitalized cores + osteoblast + sham, with four culture durations (2, 8, 15, and 22 days; n = 4/group). Cores from groups 3-6 were seeded with osteoblasts, and cores from groups 5 and 6 were devitalized before seeding. Groups 1, 3, and 5 were subjected to daily DHP loading. Bone histomorphometry was performed to quantify osteocyte viability based on morphology and to assess osteoblast function based on osteoid surface per bone surface (Os/Bs). TUNEL staining was performed to evaluate the mode of osteocyte death under various conditions. A portion of osteocytes remained viable for the duration of culture. DHP loading significantly enhanced osteocyte viability up to day 8, whereas the presence of seeded osteoblasts significantly decreased osteocyte viability. Cores with live osteocytes showed higher Os/Bs compared with devitalized cores, which reached significant levels over a greater range of time-points when combined with DHP loading. DHP loading did not increase Os/Bs in the absence of live osteocytes. The percentage

  9. Iron overload induced death of osteoblasts in vitro: involvement of the mitochondrial apoptotic pathway

    PubMed Central

    Dai, Zhipeng; Yang, Jingjing; Zheng, Jin

    2016-01-01

    Background Iron overload is recognized as a new pathogenfor osteoporosis. Various studies demonstrated that iron overload could induce apoptosis in osteoblasts and osteoporosis in vivo. However, the exact molecular mechanisms involved in the iron overload-mediated induction of apoptosis in osteoblasts has not been explored. Purpose In this study, we attempted to determine whether the mitochondrial apoptotic pathway is involved in iron-induced osteoblastic cell death and to investigate the beneficial effect of N-acetyl-cysteine (NAC) in iron-induced cytotoxicity. Methods The MC3T3-E1 osteoblastic cell line was treated with various concentrations of ferric ion in the absence or presence of NAC, and intracellular iron, cell viability, reactive oxygen species, functionand morphology changes of mitochondria and mitochondrial apoptosis related key indicators were detected by commercial kits. In addition, to further explain potential mechanisms underlying iron overload-related osteoporosis, we also assessed cell viability, apoptosis, and osteogenic differentiation potential in bone marrow-derived mesenchymal stemcells(MSCs) by commercial kits. Results Ferric ion demonstrated concentration-dependent cytotoxic effects on osteoblasts. After incubation with iron, an elevation of intracelluar labile iron levels and a concomitant over-generation of reactive oxygen species (ROS) were detected by flow cytometry in osteoblasts. Nox4 (NADPH oxidase 4), an important ROS producer, was also evaluated by western blot. Apoptosis, which was evaluated by Annexin V/propidium iodide staining, Hoechst 33258 staining, and the activation of caspase-3, was detected after exposure to iron. Iron contributed to the permeabilizatio of mitochondria, leading to the release of cytochrome C (cyto C), which, in turn, induced mitochondrial apoptosis in osteoblasts via activation of Caspase-3, up-regulation of Bax, and down-regulation of Bcl-2. NAC could reverse iron-mediated mitochondrial dysfunction and

  10. Formation of surface reaction products on bioactive glass and their effects on the expression of the osteoblastic phenotype and the deposition of mineralized extracellular matrix.

    PubMed

    el-Ghannam, A; Ducheyne, P; Shapiro, I M

    1997-02-01

    The objective of the study was to examine the effect of alkali ion release, pH control and buffer capacity on the expression of the osteoblastic phenotype. In addition we determined the importance of modifications of the surface of porous bioactive glass (BG) on the activity of rat calvaria osteoblasts in vitro. We found that at a low tissue culture medium (TCM) volume to BG surface area (Vol/SA) ratio, the products of glass corrosion elevated the pH of the TCM to a value that adversely affected cellular activity; thus, the matrix synthesized by the cells was non-mineralized. On the other hand, when the Vol/SA was high and the buffer capacity of the medium was not exceeded, the cells generated a mineralized extracellular matrix. Addressing the second issue, we observed that modification of the composition of the BG surface markedly influenced osteoblast activity. BG that was coated with either a calcium phosphate-rich layer only or a serum protein layer changed the phenotypic characteristics of the osteoblasts. The presence of either of these surfaces lowered the alkaline phosphatase activity of the attached cells; this finding indicated that the osteoblast phenotype was not conserved. However, when the BG was coated with a bilayer of calcium phosphate and serum proteins, the alkaline phosphatase (AP) activity was elevated and the extracellular matrix contained characteristic bone markers. Our findings indicate that the calcium phosphate-rich layer promotes adsorption and concentration of proteins from the TCM, and it is utilized by the osteoblasts to form the mineralized extracellular matrix.

  11. Identification and characterization of novel IGFBP5 interacting protein: evidence IGFBP5-IP is a potential regulator of osteoblast cell proliferation

    PubMed Central

    Amaar, Yousef G.; Tapia, Blanca; Chen, Shin-Tai; Baylink, David J.; Mohan, Subburaman

    2010-01-01

    Insulin-like growth factor binding protein-5 (IGFBP5) is a multifunctional protein, which acts not only as a traditional binding protein, but also functions as a growth factor independent of IGFs to stimulate bone formation. It has been predicted that the intrinsic growth factor action of IGFBP5 involves binding of IGFBP5 to a putative receptor to induce downstream signaling pathways and/or nuclear translocation of IGFBP5 to influence transcription of genes involved in osteoblast cell proliferation/differentiation. Our study indentified proteins that bound to IGFBP5 using IGFBP5 as bait in a yeast two-hybrid screen of the U2 human osteosarcoma cell cDNA library. One of the clones that interacted strongly with the bait under high-stringency conditions corresponded to a novel IGFBP5 interacting protein (IGFBP5-IP) encoded by a gene that resides in mouse chromosome 10. The interaction between IGFBP5-IP and IGFBP5 is confirmed by in vitro coimmunoprecipitation studies that used pFlag and IGFBP5 polyclonal antibody, and cell lysates overexpressing both IGFBP5-IP and IGFBP5. Northern blot and RT-PCR analysis showed that the IGFBP-IP is expressed in both untransformed normal human osteoblasts and in osteosarcoma cell lines, which are known to produce IGFBP5. To determine the roles of IGFBP5-IP, we evaluated the effect of blocking the expression of IGFBP5-IP on osteoblast proliferation. We found that using a IGFBP5-IP-specific small interfering-hairpin plasmid resulted in a decrease in both basal and IGFBP5-induced osteoblast cell proliferation. On the basis of these findings, we predict that IGFBP5-IP may act as intracellular mediator of growth promoting actions of IGFBP5 and perhaps other osteoregulatory agents in bone cells. PMID:16269403

  12. Signal transduction pathways mediating parathyroid hormone regulation of osteoblastic gene expression

    NASA Technical Reports Server (NTRS)

    Partridge, N. C.; Bloch, S. R.; Pearman, A. T.

    1994-01-01

    Parathyroid hormone (PTH) plays a central role in regulation of calcium metabolism. For example, excessive or inappropriate production of PTH or the related hormone, parathyroid hormone related protein (PTHrP), accounts for the majority of the causes of hypercalcemia. Both hormones act through the same receptor on the osteoblast to elicit enhanced bone resorption by the osteoclast. Thus, the osteoblast mediates the effect of PTH in the resorption process. In this process, PTH causes a change in the function and phenotype of the osteoblast from a cell involved in bone formation to one directing the process of bone resorption. In response to PTH, the osteoblast decreases collagen, alkaline phosphatase, and osteopontin expression and increases production of osteocalcin, cytokines, and neutral proteases. Many of these changes have been shown to be due to effects on mRNA abundance through either transcriptional or post-transcriptional mechanisms. However, the signal transduction pathway for the hormone to cause these changes is not completely elucidated in any case. Binding of PTH and PTHrP to their common receptor has been shown to result in activation of protein kinases A and C and increases in intracellular calcium. The latter has not been implicated in any changes in mRNA of osteoblastic genes. On the other hand activation of PKA can mimic all the effects of PTH; protein kinase C may be involved in some responses. We will discuss possible mechanisms linking PKA and PKC activation to changes in gene expression, particularly at the nuclear level.

  13. Osteoinductive effect of bone bank allografts on human osteoblasts in culture.

    PubMed

    de la Piedra, Concepción; Vicario, Carlos; de Acuña, Lucrecia Rodríguez; García-Moreno, Carmen; Traba, Maria Luisa; Arlandis, Santiago; Marco, Fernando; López-Durán, Luis

    2008-02-01

    Incorporation of a human bone allograft requires osteoclast activity and growth of recipient osteoblasts. The aim of this work was to study the effects produced by autoclavated and -80 degrees C frozen bone allografts on osteoblast proliferation and synthesis of interleukin 6 (IL6), activator of bone resorption, aminoterminal propeptide of procollagen I (PINP), marker of bone matrix formation, and osteoprotegerin (OPG), inhibitor of osteoclast activity and differentiation. Allografts were obtained from human femoral heads. Human osteoblasts were cultured in the presence (problem group) or in the absence (control group) of allografts during 15 days. Allografts produced a decrease in osteoblast proliferation in the first week of the experiment, and an increase in IL6 mRNA, both at 3 h and 2 days, and an increase in the IL6 released to the culture medium the second day of the experiment. We found a decrease in OPG released to the culture on the 2nd and fourth days. These results suggest an increase in bone resorption and a decrease in bone formation in the first week of the experiment. In the second week, allografts produced an increase in osteoblast proliferation and PINP release to the culture medium, indicating an increase in bone formation; an increase in OPG released to the culture medium, which would indicate a decrease in bone resorption; and a decrease in IL6, indicating a decrease in bone resorption stimulation. These results demonstrate that autoclavated and -80 degrees C frozen bone allografts produce in bone environment changes that regulate their own incorporation to the recipient bone.

  14. Activating AMP-activated protein kinase by an α1 selective activator compound 13 attenuates dexamethasone-induced osteoblast cell death

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Guo, Shiguang; Mao, Li; Ji, Feng, E-mail: huaiaifengjidr@163.com

    Excessive glucocorticoid (GC) usage may lead to non-traumatic femoral head osteonecrosis. Dexamethasone (Dex) exerts cytotoxic effect to cultured osteoblasts. Here, we investigated the potential activity of Compound 13 (C13), a novel α1 selective AMP-activated protein kinase (AMPK) activator, against the process. Our data revealed that C13 pretreatment significantly attenuated Dex-induced apoptosis and necrosis in both osteoblastic-like MC3T3-E1 cells and primary murine osteoblasts. AMPK activation mediated C13′ cytoprotective effect in osteoblasts. The AMPK inhibitor Compound C, shRNA-mediated knockdown of AMPKα1, or dominant negative mutation of AMPKα1 (T172A) almost abolished C13-induced AMPK activation and its pro-survival effect in osteoblasts. On the othermore » hand, forced AMPK activation by adding AMPK activator A-769662 or exogenous expression a constitutively-active (ca) AMPKα1 (T172D) mimicked C13's actions and inhibited Dex-induced osteoblast cell death. Meanwhile, A-769662 or ca-AMPKα1 almost nullified C13's activity in osteoblast. Further studies showed that C13 activated AMPK-dependent nicotinamide adenine dinucleotide phosphate (NADPH) pathway to inhibit Dex-induced reactive oxygen species (ROS) production in MC3T3-E1 cells and primary murine osteoblasts. Such effects by C13 were almost reversed by Compound C or AMPKα1 depletion/mutation. Together, these results suggest that C13 alleviates Dex-induced osteoblast cell death via activating AMPK signaling pathway. - Highlights: • Compound 13 (C13) attenuates dexamethasone (Dex)-induced osteoblast cell death. • C13-induced cytoprotective effect against Dex in osteoblasts requires AMPK activation. • Forced AMPK activation protects osteoblasts from Dex, nullifying C13's activities. • C13 increases NADPH activity and inhibits Dex-induced oxidative stress in osteoblasts.« less

  15. The structurally effect of surface coated rhamnogalacturonan I on response of the osteoblast-like cell line SaOS-2.

    PubMed

    Svava, Rikke; Gurzawska, Katarzyna; Yihau, Yu; Haugshøj, Kenneth Brian; Dirscherl, Kai; Levery, Steven B; Jørgensen, Niklas Rye; Gotfredsen, Klaus; Damager, Iben; Ulvskov, Peter; Jørgensen, Bodil

    2014-06-01

    Osseointegration is important when implants are inserted into the bone and can be improved by biochemical surface coating of the implant. In this paper enzymatically modified rhamnogalacturonan I (RG-I) from apple and lupin was used for biochemical coating of aminated surfaces and the importance of the quality of RG-I, the nature of the binding, the fine structure of RG-I, and its effect on SaOS-2 cell line cultured on coated surfaces was investigated. SaOS-2 cells are osteoblast-like cells and a well-established in vitro model of bone-matrix forming osteoblasts. Purification by gel filtration could remove small fragments of galacturonic acid (GalA) and binding studies showed that the purity of the RG-I molecules was important for the quality of the coating. The structure of RG-I and osteoblast-like cells' viability were positively correlated so that high content of 1,4-linked galactose (Gal) and a low content of arabinose in the RG-I molecules favored cell viability. These results indicate that coating of implants with RG-I affect osseointegration positively. Copyright © 2013 Wiley Periodicals, Inc.

  16. 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.

  17. Blockade of Drp1 rescues oxidative stress-induced osteoblast dysfunction

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gan, Xueqi; Huang, Shengbin; Yu, Qing

    Osteoblast dysfunction, induced by oxidative stress, plays a critical role in the pathophysiology of osteoporosis. However, the underlying mechanisms remain unclarified. Imbalance of mitochondrial dynamics has been closely linked to oxidative stress. Here, we reveal an unexplored role of dynamic related protein 1(Drp1), the major regulator in mitochondrial fission, in the oxidative stress-induced osteoblast injury model. We demonstrate that levels of phosphorylation and expression of Drp1 significantly increased under oxidative stress. Blockade of Drp1, through pharmaceutical inhibitor or gene knockdown, significantly protected against H{sub 2}O{sub 2}-induced osteoblast dysfunction, as shown by increased cell viability, improved cellular alkaline phosphatase (ALP) activitymore » and mineralization and restored mitochondrial function. The protective effects of blocking Drp1 in H{sub 2}O{sub 2}-induced osteoblast dysfunction were evidenced by increased mitochondrial function and suppressed production of reactive oxygen species (ROS). These findings provide new insights into the role of the Drp1-dependent mitochondrial pathway in the pathology of osteoporosis, indicating that the Drp1 pathway may be targetable for the development of new therapeutic approaches in the prevention and the treatment of osteoporosis. - Highlights: • Oxidative stress is an early pathological event in osteoporosis. • Imbalance of mitochondrial dynamics are linked to oxidative stress in osteoporosis. • The role of the Drp1-dependent mitochondrial pathway in osteoporosis.« less

  18. Age of donor alters the effect of cyclic hydrostatic pressure on production by human macrophages and osteoblasts of sRANKL, OPG and RANK

    PubMed Central

    Evans, CE; Mylchreest, S; Andrew, JG

    2006-01-01

    Background Cyclic hydrostatic pressure within bone has been proposed both as a stimulus of aseptic implant loosening and associated bone resorption and of bone formation. We showed previously that cyclical hydrostatic pressure influenced macrophage synthesis of several factors linked to osteoclastogenesis. The osteoprotegerin/soluble receptor activator of NF-kappa β ligand /receptor activator of NF-kappa β (OPG/ RANKL/ RANK) triumvirate has been implicated in control of bone resorption under various circumstances. We studied whether cyclical pressure might affect bone turnover via effects on OPG/ sRANKL/ RANK. Methods In this study, cultures of human osteoblasts or macrophages (supplemented with osteoclastogenic factors) or co-cultures of macrophages and osteoblasts (from the same donor), were subjected to cyclic hydrostatic pressure. Secretion of OPG and sRANKL was assayed in the culture media and the cells were stained for RANK and osteoclast markers. Data were analysed by nonparametric statistics. Results In co-cultures of macrophages and osteoblasts, pressure modulated secretion of sRANKL or OPG in a variable manner. Examination of the OPG:sRANKL ratio in co cultures without pressurisation showed that the ratio was greater in donors <70 years at the time of operation (p < 0.05 Mann Whitney U) than it was in patients >70 years. However, with pressure the difference in the OPG:sRANKL ratios between young and old donors was not significant. It was striking that in some patients the OPG:sRANKL ratio increased with pressure whereas in some it decreased. The tendency was for the ratio to decrease with pressure in patients younger than 70 years, and increase in patients ≥ 70 years (Fishers exact p < 0.01). Cultures of osteoblasts alone showed a significant increase in both sRANKL and OPG with pressure, and again there was a decrease in the ratio of OPG:RANKL. Secretion of sRANKL by cultures of macrophages alone was not modulated by pressure. Only sRANKL was

  19. Differentiation of Bovine Spermatogonial Stem Cells into Osteoblasts

    PubMed Central

    Qasemi-Panahi, Babak; Tajik, Parviz; Movahedin, Mansoureh; Moghaddam, Gholamali; Barzgar, Younes; Heidari-Vala, Hamed

    2011-01-01

    Spermatogonial Stem Cell (SSC) technologies provide multiple opportunities for research in the field of biotechnology and regenerative medicine. The therapeutic use of Embryonic Stem Cells (ESCs) is restricted due to severe ethical and immunological concerns. Therefore, we need a new pluripotent cell type. Despite well-known role of germ cells in the gametogenesis, some facts apparently show their multipotentiality. In the present study, bovine SSCs were co-cultured with Sertoli cell for 7 days. Sertoli cells and SSCs were identified by Vimentin and Oct-4 immunocytochemical staining method, respectively. In order to differentiate SSCs into osteoblasts, we used consecutive inducer media without separation of the colonies. We characterized osteoblasts using Alizarin red staining. PMID:23408761

  20. Differentiation of bovine spermatogonial stem cells into osteoblasts.

    PubMed

    Qasemi-Panahi, Babak; Tajik, Parviz; Movahedin, Mansoureh; Moghaddam, Gholamali; Barzgar, Younes; Heidari-Vala, Hamed

    2011-07-01

    Spermatogonial Stem Cell (SSC) technologies provide multiple opportunities for research in the field of biotechnology and regenerative medicine. The therapeutic use of Embryonic Stem Cells (ESCs) is restricted due to severe ethical and immunological concerns. Therefore, we need a new pluripotent cell type. Despite well-known role of germ cells in the gametogenesis, some facts apparently show their multipotentiality. In the present study, bovine SSCs were co-cultured with Sertoli cell for 7 days. Sertoli cells and SSCs were identified by Vimentin and Oct-4 immunocytochemical staining method, respectively. In order to differentiate SSCs into osteoblasts, we used consecutive inducer media without separation of the colonies. We characterized osteoblasts using Alizarin red staining.

  1. DKK1 and Kremen Expression Predicts the Osteoblastic Response to Bone Metastasis.

    PubMed

    Clines, Katrina L; Clines, Gregory A

    2018-05-14

    Bone metastasis is a complication of advanced breast and prostate cancer. Tumor-secreted Dickkopf homolog 1 (DKK1), an inhibitor of canonical Wnt signaling and osteoblast differentiation, was proposed to regulate the osteoblastic response to metastatic cancer in bone. The objectives of this study were to compare DKK1 expression with the in vivo osteoblastic response in a panel of breast and prostate cancer cell lines, and to discover mechanisms that regulate cancer DKK1 expression. DKK1 expression was highest in MDA-MB-231 and PC3 cells that produce osteolytic lesions, and hence a suppressed osteoblastic response, in animal models of bone metastasis. LnCaP, C4-2B, LuCaP23.1, T47D, ZR-75-1, MCF-7, ARCaP and ARCaP M cancer cells that generate osteoblastic, mixed or no bone lesions had the lowest DKK1 expression. The cell lines with negligible expression, LnCaP, C4-2B and T47D, exhibited methylation of the DKK1 promoter. Canonical Wnt signaling activity was then determined and found in all cell lines tested, even in the MDA-MB-231 and PC3 cell lines despite sizeable amounts of DKK1 protein expression expected to block canonical Wnt signaling. A mechanism of DKK1 resistance in the osteolytic cell lines was investigated and determined to be at least partially due to down-regulation of the DKK1 receptors Kremen1 and Kremen2 in the MDA-MB-231 and PC3 cell lines. Combined DKK1 and Kremen expression in cancer cells may serve as predictive markers of the osteoblastic response of breast and prostate cancer bone metastasis. Published by Elsevier Inc.

  2. The Influence of Primary Microenvironment on Prostate Cancer Osteoblastic Bone Lesion Development

    DTIC Science & Technology

    2015-09-01

    for inhibiting PCa bone lesion development: 3a. Basic fibroblast growth factor (bFGF) in PC3 bone metastasis: bFGF was identified by cytokine...II receptor (TβRII) knockout (Tgfbr2 KO) mouse models. Col1creERT/Tgfbr2 KO (Col/Tgfbr2 KO), which have TGF-β signaling specific KO in fibroblasts ... fibroblasts and osteoblasts in the bone by Colcre/Tgfbr2 KO, or in the myeloid lineage cells, including osteoclasts in the bone by LysMcre/Tgfbr2 KO

  3. Fibronectin gene expression, synthesis and accumulation during in vitro differentiation of chicken osteoblasts

    NASA Technical Reports Server (NTRS)

    Winnard, R. G.; Gerstenfeld, L. C.; Toma, C. D.; Franceschi, R. T.; Landis, W. J. (Principal Investigator)

    1995-01-01

    A well-defined chicken osteoblast culture system(18) has been used to examine fibronectin (FN) mRNA levels, synthesis, and accumulation during in vitro differentiation and matrix mineralization. Immunofluorescent staining of cells after 6 or 18 days in culture revealed that FN was initially associated with the cell surface and in partial coalignment with cytoskeletal elements while at the latter time most FN was associated with the extracellular matrix as a ubiquitous fibrillar network. Western blot analysis of total cell-associated proteins also detected FN at all culture times. However, when results were normalized to cellular DNA, FN levels increased until 12-16 and remained relatively constant thereafter. Similarly, FN synthesis as measured by [35S]-methionine labeling, and immunoprecipitation was greatest in early cultures (culture day 3) and then declined such that synthesis decreased 60% at day 18 and 94% after 24-31 days. FN mRNA levels as measured by Northern blot analysis were well correlated with FN synthesis. These results clearly show that FN is made by primary osteoblasts during their in vitro maturation. In contrast to other osteoblast markers such as alkaline phosphatase, osteocalcin, and osteopontin, whose expression increases as cells differentiate, FN accumulates in the matrix during periods of early cell growth and attachment and then remains proportional to cell number. Results with FN differ from those obtained with collagen which continues to accumulate in the extracellular matrix during osteoblast maturation. These results are consistent with FN being important for the initial attachment of early osteoblasts or osteoblast precursors to the pericellular matrix.

  4. Low bone mass and changes in the osteocyte network in mice lacking autophagy in the osteoblast lineage

    PubMed Central

    Piemontese, Marilina; Onal, Melda; Xiong, Jinhu; Han, Li; Thostenson, Jeff D.; Almeida, Maria; O’Brien, Charles A.

    2016-01-01

    Autophagy maintains cell function and homeostasis by recycling intracellular components. This process is also required for morphological changes associated with maturation of some cell types. Osteoblasts are bone forming cells some of which become embedded in bone and differentiate into osteocytes. This transformation includes development of long cellular projections and a reduction in endoplasmic reticulum and mitochondria. We examined the role of autophagy in osteoblasts by deleting Atg7 using an Osterix1-Cre transgene, which causes recombination in osteoblast progenitors and their descendants. Mice lacking Atg7 in the entire osteoblast lineage had low bone mass and fractures associated with reduced numbers of osteoclasts and osteoblasts. Suppression of autophagy also reduced the amount of osteocyte cellular projections and led to retention of endoplasmic reticulum and mitochondria in osteocytes. These results demonstrate that autophagy in osteoblasts contributes to skeletal homeostasis and to the morphological changes associated with osteocyte formation. PMID:27064143

  5. Low bone mass and changes in the osteocyte network in mice lacking autophagy in the osteoblast lineage.

    PubMed

    Piemontese, Marilina; Onal, Melda; Xiong, Jinhu; Han, Li; Thostenson, Jeff D; Almeida, Maria; O'Brien, Charles A

    2016-04-11

    Autophagy maintains cell function and homeostasis by recycling intracellular components. This process is also required for morphological changes associated with maturation of some cell types. Osteoblasts are bone forming cells some of which become embedded in bone and differentiate into osteocytes. This transformation includes development of long cellular projections and a reduction in endoplasmic reticulum and mitochondria. We examined the role of autophagy in osteoblasts by deleting Atg7 using an Osterix1-Cre transgene, which causes recombination in osteoblast progenitors and their descendants. Mice lacking Atg7 in the entire osteoblast lineage had low bone mass and fractures associated with reduced numbers of osteoclasts and osteoblasts. Suppression of autophagy also reduced the amount of osteocyte cellular projections and led to retention of endoplasmic reticulum and mitochondria in osteocytes. These results demonstrate that autophagy in osteoblasts contributes to skeletal homeostasis and to the morphological changes associated with osteocyte formation.

  6. Biocompatibility of nano-hydroxyapatite/polyetheretherketone composite materials with osteoblasts cultured in vitro

    NASA Astrophysics Data System (ADS)

    Wang, Lin; Huang, Feijuan; Wu, Zhengzhi; Ma, Rui

    2017-04-01

    The biocompatibility of the Sprague Dawley (SD) rat osteoblasts, which were cultured on the surfaces of nano-hydroxyapatite/polyetheretherketone (n-HA/PEEK) composites were investigated in this work. The osteoblasts of 24- hour old SD rats were cultured and identified by modified enzymatic digestion in vitro. The morphology and proliferation of cells were observed in CCK-8 regent staining, inverted microscopes, and by scanning electron microscopy (SEM) respectively. The results show that n-HA/PEEK composites have good biocompatibility with SD osteoblasts and that they can promote the growth of the cells that were cultured on the surfaces of the composites. The content of HA in n- HA/PEEK composites plays an important role in cell proliferation.

  7. Human osteoblast cells: isolation, characterization, and growth on polymers for musculoskeletal tissue engineering.

    PubMed

    El-Amin, Saadiq F; Botchwey, Edward; Tuli, Richard; Kofron, Michelle D; Mesfin, Addisu; Sethuraman, Swaminathan; Tuan, Rocky S; Laurencin, Cato T

    2006-03-01

    We performed a detailed examination of the isolation, characterization, and growth of human osteoblast cells derived from trabecular bone. We further examined the morphology, phenotypic gene expression, mineralization,and growth of these human osteoblasts on polyester polymers used for musculoskeletal tissue engineering. Polylactic-co-glycolic acid [PLAGA (85:15, 50:50, 75:25)], and poly-lactic acid (L-PLA, D,L-PLA) were examined. The osteoblastic expression of key phenotypic markers osteocalcin, alkaline phosphatase, collagen, and bone sialoprotein at 4 and 8 weeks was examined. Reverse transcription-polymerase chain reaction studies revealed that trabecular-derived osteoblasts were positive for all markers evaluated with higher levels expressed over long-term culture. These cells also revealed mineralization and maturation as evidenced by energy dispersive X-ray analysis and scanning electron microscopy. Growth studies on PLAGA at 50:50,75:25, and 85:15 ratios and PLA in the L and DL isoforms revealed that human osteoblasts actively grew, with significantly higher cell numbers attached to scaffolds composed of PLAGA 50:50 in the short term and PLAGA 85:15 in the long term compared with PLA (p < 0.05). We believe human cell adhesion among these polymeric materials may be dependent on differences in cellular integrin expression and extracellular matrix protein elaboration. (c) 2005 Wiley Periodicals, Inc.

  8. Gravity affects the responsiveness of Runx2 to 1, 25-dihydroxyvitamin D3 (VD3)

    NASA Astrophysics Data System (ADS)

    Guo, Feima; Dai, Zhongquan; Wu, Feng; Liu, Zhaoxia; Tan, Yingjun; Wan, Yumin; Shang, Peng; Li, Yinghui

    2013-03-01

    Bone loss resulting from spaceflight is mainly caused by decreased bone formation, and decreased osteoblast proliferation and differentiation. Transcription factor Runx2 plays an important role in osteoblast differentiation and function by responding to microenvironment changes including cytokine and mechanical factors. The effects of 1, 25-dihydroxyvitamin D3 (VD3) on Runx2 in terms of mechanical competence is far less clear. This study describes how gravity affects the response of Runx2 to VD3. A MC3T3-6OSE2-Luc osteoblast model was constructed in which the activity of Runx2 was reflected by reporter luciferase activity identifed by bone-related cytokines. The results showed that luciferase activity in MC3T3-6OSE2-Luc cells transfected with Runx2 was twice that of the vacant vector. Alkaline phosphatase (ALP) activity was increased in MC3T3-6OSE2-Luc cells by different concentrations of IGF-I and BMP2. MC3T3-6OSE2-Luc cells were cultured under simulated microgravity or centrifuge with or without VD3. In simulated microgravity, luciferase activity was decreased after 48 h of clinorotation culture, but increased in the centrifuge culture. Luciferase activity was increased after VD3 treatment in normal conditions and simulated microgravity, the increase in luciferase activity in simulated microgravity was lower than that in the 1 g condition when simultaneously treated with VD3 and higher than that in the centrifuge condition. Co-immunoprecipitation showed that the interaction between the VD3 receptor (VDR) and Runx2 was decreased by simulated microgravity, but increased by centrifugation. From these results, we conclude that gravity affects the response of Runx2 to VD3 which results from an alteration in the interaction between VDR and Runx2 under different gravity conditions.

  9. Osteoblast dysfunctions in bone diseases: from cellular and molecular mechanisms to therapeutic strategies.

    PubMed

    Marie, Pierre J

    2015-04-01

    Several metabolic, genetic and oncogenic bone diseases are characterized by defective or excessive bone formation. These abnormalities are caused by dysfunctions in the commitment, differentiation or survival of cells of the osteoblast lineage. During the recent years, significant advances have been made in our understanding of the cellular and molecular mechanisms underlying the osteoblast dysfunctions in osteoporosis, skeletal dysplasias and primary bone tumors. This led to suggest novel therapeutic approaches to correct these abnormalities such as the modulation of WNT signaling, the pharmacological modulation of proteasome-mediated protein degradation, the induction of osteoprogenitor cell differentiation, the repression of cancer cell proliferation and the manipulation of epigenetic mechanisms. This article reviews our current understanding of the major cellular and molecular mechanisms inducing osteoblastic cell abnormalities in age-related bone loss, genetic skeletal dysplasias and primary bone tumors, and discusses emerging therapeutic strategies to counteract the osteoblast abnormalities in these disorders of bone formation.

  10. 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.

  11. 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

  12. Human Immunodeficiency Virus Envelope Protein Gp120 Induces Proliferation but Not Apoptosis in Osteoblasts at Physiologic Concentrations

    PubMed Central

    Cummins, Nathan W.; Klicpera, Anna; Sainski, Amy M.; Bren, Gary D.; Khosla, Sundeep; Westendorf, Jennifer J.; Badley, Andrew D.

    2011-01-01

    Patients with HIV infection have decreased numbers of osteoblasts, decreased bone mineral density and increased risk of fracture compared to uninfected patients; however, the molecular mechanisms behind these associations remain unclear. We questioned whether Gp120, a component of the envelope protein of HIV capable of inducing apoptosis in many cell types, is able to induce cell death in bone-forming osteoblasts. We show that treatment of immortalized osteoblast-like cells and primary human osteoblasts with exogenous Gp120 in vitro at physiologic concentrations does not result in apoptosis. Instead, in the osteoblast-like U2OS cell line, cells expressing CXCR4, a receptor for Gp120, had increased proliferation when treated with Gp120 compared to control (P<0.05), which was inhibited by pretreatment with a CXCR4 inhibitor and a G-protein inhibitor. This suggests that Gp120 is not an inducer of apoptosis in human osteoblasts and likely does not directly contribute to osteoporosis in infected patients by this mechanism. PMID:21931863

  13. Hematopoiesis is severely altered in mice with an induced osteoblast deficiency.

    PubMed

    Visnjic, Dora; Kalajzic, Zana; Rowe, David W; Katavic, Vedran; Lorenzo, Joseph; Aguila, Hector L

    2004-05-01

    We previously reported a transgenic mouse model expressing herpesvirus thymidine kinase (TK) gene under the control of a 2.3-kilobase fragment of the rat collagen alpha1 type I promoter (Col2.3 Delta TK). This construct confers lineage-specific expression in developing osteoblasts, allowing the conditional ablation of osteoblast lineage after treatment with ganciclovir (GCV). After GCV treatment these mice have profound alterations on bone formation leading to a progressive bone loss. In addition, treated animals also lose bone marrow cellularity. In this report we characterized hematopoietic parameters in GCV-treated Col2.3 Delta TK mice, and we show that after treatment transgenic animals lose lymphoid, erythroid, and myeloid progenitors in the bone marrow, followed by decreases in the number of hematopoietic stem cells (HSCs). Together with the decrease in bone marrow hematopoiesis, active extramedullary hematopoiesis was observed in the spleen and liver, as measured by an increase in peripheral HSCs and active primary in vitro hematopoiesis. After withdrawal of GCV, osteoblasts reappeared in the bone compartment together with a recovery of medullary and decrease in extramedullary hematopoiesis. These observations directly demonstrate the role of osteoblasts in hematopoiesis and provide a model to study the interactions between the mesenchymal and hematopoietic compartments in the marrow.

  14. Phenotype discovery by gene expression profiling: mapping of biological processes linked to BMP-2-mediated osteoblast differentiation.

    PubMed

    Balint, Eva; Lapointe, David; Drissi, Hicham; van der Meijden, Caroline; Young, Daniel W; van Wijnen, Andre J; Stein, Janet L; Stein, Gary S; Lian, Jane B

    2003-05-15

    Understanding physiological control of osteoblast differentiation necessitates characterization of the regulatory signals that initiate the events directing a cell to lineage commitment and establishing competency for bone formation. The bone morphogenetic protein, BMP-2, a member of the TGFbeta superfamily, induces osteoblast differentiation and functions through the Smad signal transduction pathway during in vivo bone formation. However, the molecular targets of BMP-mediated gene transcription during the process of osteoblast differentiation have not been comprehensively identified. In the present study, BMP-2 responsive factors involved in the early stages of commitment and differentiation to the osteoblast phenotype were analyzed by microarray gene expression profiling in samples ranging from 1 to 24 h following BMP-2 dependent differentiation of C2C12 premyoblasts into the osteogenic lineage. A total of 1,800 genes were responsive to BMP-2 and expression was modulated from 3- to 14-fold for less than 100 genes during the time course. Approximately 50% of these 100 genes are either up- or downregulated. Major events associated with phenotypic changes towards the osteogenic lineage were identified from hierarchical and functional clustering analyses. BMP-2 immediately responsive genes (1-4 h), which exhibited either transient or sustained expression, reflect activation and repression of non-osseous BMP-2 developmental systems. This initial response was followed by waves of expression of nuclear proteins and developmental regulatory factors including inhibitors of DNA binding, Runx2, C/EBP, Zn finger binding proteins, forkhead, and numerous homeobox proteins (e.g., CDP/cut, paired, distaless, Hox) which are expressed at characterized stages during osteoblast differentiation. A sequential profile of genes mediating changes in cell morphology, cell growth, and basement membrane formation is observed as a secondary transient early response (2-8 h). Commitment to the

  15. Nanocrystallinity effects on osteoblast and osteoclast response to silicon substituted hydroxyapatite.

    PubMed

    Casarrubios, Laura; Matesanz, María Concepción; Sánchez-Salcedo, Sandra; Arcos, Daniel; Vallet-Regí, María; Portolés, María Teresa

    2016-11-15

    Silicon substituted hydroxyapatites (SiHA) are highly crystalline bioceramics treated at high temperatures (about 1200°C) which have been approved for clinical use with spinal, orthopedic, periodontal, oral and craniomaxillofacial applications. The preparation of SiHA with lower temperature methods (about 700°C) provides nanocrystalline SiHA (nano-SiHA) with enhanced bioreactivity due to higher surface area and smaller crystal size. The aim of this study has been to know the nanocrystallinity effects on the response of both osteoblasts and osteoclasts (the two main cell types involved in bone remodelling) to silicon substituted hydroxyapatite. Saos-2 osteoblasts and osteoclast-like cells (differentiated from RAW-264.7 macrophages) have been cultured on the surface of nano-SiHA and SiHA disks and different cell parameters have been evaluated: cell adhesion, proliferation, viability, intracellular content of reactive oxygen species, cell cycle phases, apoptosis, cell morphology, osteoclast-like cell differentiation and resorptive activity. This comparative in vitro study evidences that nanocrystallinity of SiHA affects the cell/biomaterial interface inducing bone cell apoptosis by loss of cell anchorage (anoikis), delaying osteoclast-like cell differentiation and decreasing the resorptive activity of this cell type. These results suggest the potential use of nano-SiHA biomaterial for preventing bone resorption in treatment of osteoporotic bone. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Mechanism of chlorogenic acid treatment on femoral head necrosis and its protection of osteoblasts.

    PubMed

    Zhang, Mingjuan; Hu, Xianda

    2016-07-01

    The aim of the present study was to investigate the therapeutic effect of chlorogenic acid on hormonal femoral head necrosis and its protection of osteoblasts. The study established a femoral head necrosis model in Wistar rats using Escherichia coli endotoxin and prednisolone acetate. The rats were divided into five groups and were treated with different concentrations of chlorogenic acid (1, 10 and 20 mg/kg). The main detected indicators were the blood rheology, bone mineral density, and the hydroxyproline and hexosamine (HOM) contents. At a cellular level, osteoblasts were cultured and treated by drug-containing serum. Subsequently, cell proliferation and the osteoblast cycle were measured using flow cytometry, and the protein expression levels of Bax and B-cell lymphoma 2 (Bcl-2) were detected using western blotting. Chlorogenic acid at a concentration of 20 mg/kg (high-dose) enhanced the bone mineral density of the femoral head and femoral neck following ischemia. Simultaneously, blood flow following the injection of prednisolone acetate was significantly improved, and the HOM contents of the high-dose chlorogenic acid group were significantly different. The results from the flow cytometry analysis indicated that chlorogenic acid can efficiently ameliorate hormone-induced necrosis. The osteoblasts were isolated and cultured. The MTT colorimetric assay showed that chlorogenic acid at different densities can increase the proliferation capabilities of osteoblasts and accelerate the transition process of G 0 /G 1 phase to S phase, as well as enhance mitosis and the regeneration of osteoblasts. Western blotting detection indicated that chlorogenic acid may prohibit the decrease of Bcl-2 and the increase of Bax during apoptosis, thereby inhibiting osteoblast apoptosis and preventing the deterioration of femoral head necrosis. In conclusion, chlorogenic acid at the density of 20 mg/kg is effective in the treatment of hormonal femoral head necrosis, which may be

  17. Osteoblast and osteoclast behaviors in the turnover of attachment bones during medaka tooth replacement.

    PubMed

    Mantoku, Akiko; Chatani, Masahiro; Aono, Kazushi; Inohaya, Keiji; Kudo, Akira

    2016-01-15

    Tooth replacement in polyphyodont is a well-organized system for maintenance of homeostasis of teeth, containing the dynamic structural change in skeletal tissues such as the attachment bone, which is the supporting element of teeth. Histological analyses have revealed the character of tooth replacement, however, the cellular mechanism of how skeletal tissues are modified during tooth replacement is largely unknown. Here, we showed the important role of osteoblasts for controlling osteoclasts to modify the attachment bone during tooth replacement in medaka pharyngeal teeth, coupled with an osterix-DsRed/TRAP-GFP transgenic line to visualize osteoblasts and osteoclasts. In the turnover of the row of attachment bones, these bones were resorbed at the posterior side where most developed functional teeth were located, and generated at the anterior side where teeth were newly erupted, which caused continuous tooth replacement. In the cellular analysis, osteoclasts and osteoblasts were located at attachment bones separately, since mature osteoclasts were localized at the resorbing side and osteoblasts gathered at the generating side. To demonstrate the role of osteoclasts in tooth replacement, we established medaka made deficient in c-fms-a by TALEN. c-fms-a deficient medaka showed hyperplasia of attachment bones along with reduced bone resorption accompanied by a low number of TRAP-positive osteoclasts, indicating an important role of osteoclasts in the turnover of attachment bones. Furthermore, nitroreductase-mediated osteoblast-specific ablation induced disappearance of osteoclasts, indicating that osteoblasts were essential for maintenance of osteoclasts for the proper turnover. Taken together, our results suggested that the medaka attachment bone provides the model to understand the cellular mechanism for tooth replacement, and that osteoblasts act in the coordination of bone morphology by supporting osteoclasts. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. A Conditional Knockout Mouse Model Reveals a Critical Role of PKD1 in Osteoblast Differentiation and Bone Development

    PubMed Central

    Li, Shao; Xu, Wanfu; Xing, Zhe; Qian, Jiabi; Chen, Liping; Gu, Ruonan; Guo, Wenjing; Lai, Xiaoju; Zhao, Wanlu; Li, Songyu; Wang, Yaodong; Wang, Q. Jane; Deng, Fan

    2017-01-01

    The protein kinase D family of serine/threonine kinases, particularly PKD1, has been implicated in the regulation of a complex array of fundamental biological processes. However, its function and mechanism underlying PKD1-mediated the bone development and osteoblast differentiation are not fully understood. Here we demonstrate that loss of PKD1 function led to impaired bone development and osteoblast differentiation through STAT3 and p38 MAPK signaling using in vitro and in vivo bone-specific conditional PKD1-knockout (PKD1-KO) mice models. These mice developed markedly craniofacial dysplasia, scapula dysplasia, long bone length shortage and body weight decrease compared with wild-type littermates. Moreover, deletion of PKD1 in vivo reduced trabecular development and activity of osteoblast development, confirmed by Micro-CT and histological staining as well as expression of osteoblastic marker (OPN, Runx2 and OSX). Mechanistically, loss of PKD1 mediated the downregulation of osteoblast markers and impaired osteoblast differentiation through STAT3 and p38 MAPK signaling pathways. Taken together, these results demonstrated that PKD1 contributes to the osteoblast differentiation and bone development via elevation of osteoblast markers through activation of STAT3 and p38 MAPK signaling pathways. PMID:28084409

  19. Bone morphogenetic protein type IA receptor signaling regulates postnatal osteoblast function and bone remodeling.

    PubMed

    Mishina, Yuji; Starbuck, Michael W; Gentile, Michael A; Fukuda, Tomokazu; Kasparcova, Viera; Seedor, J Gregory; Hanks, Mark C; Amling, Michael; Pinero, Gerald J; Harada, Shun-ichi; Behringer, Richard R

    2004-06-25

    Bone morphogenetic proteins (BMPs) function during various aspects of embryonic development including skeletogenesis. However, their biological functions after birth are less understood. To investigate the role of BMPs during bone remodeling, we generated a postnatal osteoblast-specific disruption of Bmpr1a that encodes the type IA receptor for BMPs in mice. Mutant mice were smaller than controls up to 6 months after birth. Irregular calcification and low bone mass were observed, but there were normal numbers of osteoblasts. The ability of the mutant osteoblasts to form mineralized nodules in culture was severely reduced. Interestingly, bone mass was increased in aged mutant mice due to reduced bone resorption evidenced by reduced bone turnover. The mutant mice lost more bone after ovariectomy likely resulting from decreased osteoblast function which could not overcome ovariectomy-induced bone resorption. In organ culture of bones from aged mice, ablation of the Bmpr1a gene by adenoviral Cre recombinase abolished the stimulatory effects of BMP4 on the expression of lysosomal enzymes essential for osteoclastic bone resorption. These results demonstrate essential and age-dependent roles for BMP signaling mediated by BMPRIA (a type IA receptor for BMP) in osteoblasts for bone remodeling.

  20. Protein kinase Cα (PKCα) regulates bone architecture and osteoblast activity.

    PubMed

    Galea, Gabriel L; Meakin, Lee B; Williams, Christopher M; Hulin-Curtis, Sarah L; Lanyon, Lance E; Poole, Alastair W; Price, Joanna S

    2014-09-12

    Bones' strength is achieved and maintained through adaptation to load bearing. The role of the protein kinase PKCα in this process has not been previously reported. However, we observed a phenotype in the long bones of Prkca(-/-) female but not male mice, in which bone tissue progressively invades the medullary cavity in the mid-diaphysis. This bone deposition progresses with age and is prevented by disuse but unaffected by ovariectomy. Castration of male Prkca(-/-) but not WT mice results in the formation of small amounts of intramedullary bone. Osteoblast differentiation markers and Wnt target gene expression were up-regulated in osteoblast-like cells derived from cortical bone of female Prkca(-/-) mice compared with WT. Additionally, although osteoblastic cells derived from WT proliferate following exposure to estradiol or mechanical strain, those from Prkca(-/-) mice do not. Female Prkca(-/-) mice develop splenomegaly and reduced marrow GBA1 expression reminiscent of Gaucher disease, in which PKC involvement has been suggested previously. From these data, we infer that in female mice, PKCα normally serves to prevent endosteal bone formation stimulated by load bearing. This phenotype appears to be suppressed by testicular hormones in male Prkca(-/-) mice. Within osteoblastic cells, PKCα enhances proliferation and suppresses differentiation, and this regulation involves the Wnt pathway. These findings implicate PKCα as a target gene for therapeutic approaches in low bone mass conditions. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Lysophosphatidic acid induces chemotaxis in MC3T3-E1 osteoblastic cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Masiello, Lisa M.; Fotos, Joseph S.; Galileo, Deni S.

    2006-07-01

    Lysophosphatidic acid (LPA) is a bioactive lipid that has pleiotropic effects on a variety of cell types and enhances the migration of endothelial and cancer cells, but it is not known if this lipid can alter osteoblast motility. We performed transwell migration assays using MC3T3-E1 osteoblastic cells and found LPA to be a potent chemotactic agent. Quantitative time-lapse video analysis of osteoblast migration after wounds were introduced into cell monolayers indicated that LPA stimulated both migration velocity and the average migration distance per cell. LPA also elicited substantial changes in cell shape and actin cytoskeletal structure; lipid-treated cells contained fewermore » stress fibers and displayed long membrane processes that were enriched in F-actin. Quantitative RT-PCR analysis showed that MC3T3-E1 cells express all four known LPA-specific G protein-coupled receptors (LPA1-LPA4) with a relative mRNA abundance of LPA1 > LPA4 > LPA2 >> LPA3. LPA-induced changes in osteoblast motility and morphology were antagonized by both pertussis toxin and Ki16425, a subtype-specific blocker of LPA1 and LPA3 receptor function. Cell migration in many cell types is linked to changes in intracellular Ca2+. Ki16425 also inhibited LPA-induced Ca2+ signaling in a dose-dependent manner, suggesting a link between LPA-induced Ca2+ transients and osteoblast chemotaxis. Our data show that LPA stimulates MC3T3-E1 osteoblast motility via a mechanism that is linked primarily to the G protein-coupled receptor LPA1.« less

  2. Transient changes in oxygen tension inhibit osteogenic differentiation and Runx2 expression in osteoblasts.

    PubMed

    Salim, Ali; Nacamuli, Randall P; Morgan, Elise F; Giaccia, Amato J; Longaker, Michael T

    2004-09-17

    Vascular disruption following bony injury results in a hypoxic gradient within the wound microenvironment. Nevertheless, the effects of low oxygen tension on osteogenic precursors remain to be fully elucidated. In the present study, we investigated in vitro osteoblast and mesenchymal stem cell differentiation following exposure to 21% O(2) (ambient oxygen), 2% O(2) (hypoxia), and <0.02% O(2) (anoxia). Hypoxia had little effect on osteogenic differentiation. In contrast, short-term anoxic treatment of primary osteoblasts and mesenchymal precursors inhibited in vitro bone nodule formation and extracellular calcium deposition. Cell viability assays revealed that this effect was not caused by immediate or delayed cell death. Microarray profiling implicated down-regulation of the key osteogenic transcription factor Runx2 as a potential mechanism for the anoxic inhibition of differentiation. Subsequent analysis revealed not only a short-term differential regulation of Runx2 and its targets by anoxia and hypoxia, but a long-term inhibition of Runx2 transcriptional and protein levels after only 12-24 h of anoxic insult. Furthermore, we present evidence that Runx2 inhibition may, at least in part, be because of anoxic repression of BMP2, and that restoring Runx2 levels during anoxia by pretreatment with recombinant BMP2 rescued the anoxic inhibition of differentiation. Taken together, our findings indicate that brief exposure to anoxia (but not 2% hypoxia) down-regulated BMP2 and Runx2 expression, thus inhibiting critical steps in the osteogenic differentiation of pluripotent mesenchymal precursors and committed osteoblasts.

  3. Effects of Coating a Titanium Alloy with Fibronectin on the Expression of Osteoblast Gene Markers in the MC3T3 Osteoprogenitor Cell Line

    PubMed Central

    Rapuano, Bruce E.; Hackshaw, Kyle M.; Schniepp, Hannes C.; MacDonald, Daniel E.

    2013-01-01

    Purpose A number of environmental and patient-related factors contribute to implant failure. A significant fraction of these failures can be attributed to limited osseointegration resulting from poor bone healing responses. The overall goal of this study was to determine whether surface treatment of a titanium-aluminum-vanadium alloy (Ti-6Al-4V) implant material with a biomimetic protein coating could promote the differentiation of attached osteoblastic cells. The specific aims of the study were to investigate whether osteoprogenitor cells cultured on a rigorously cleaned implant specimen showed a normal pattern of differentiation and whether preadsorbed fibronectin accelerated or enhanced osteoblast differentiation. Materials and Methods Ti-6Al-4V disks were rigorously cleaned, passivated in nitric acid, and dry heat–sterilized; some of the disks were then coated with 1 nmol/L fibronectin. MC3T3 osteoprogenitor cells were then cultured on the pretreated disks for several weeks. Quantitative real-time polymerase chain reaction was performed to measure changes over time in the mRNA levels of osteoblast genes. Results Fibronectin increased the peak expression of all analyzed osteoblast gene markers. “Early” genes that normally mark the proliferative phase (0 to 10 days) of osteoblastic development showed peak expression within the first 10 days after cell attachment to the titanium alloy. In contrast, “late” genes that normally mark the differentiation (10 to 20 days) and mineralization (20 to 36 days) phases of osteoblastogenesis achieved peak expression only after approximately 3 to 4 weeks of culture. Conclusions Osteoprogenitors cultured on a rigorously cleaned Ti-6Al-4V alloy were found to demonstrate a normal pattern of osteoblast differentiation. Preadsorbed fibronectin was observed to stimulate osteoblast differentiation during the mineralization phase of osteoblastogenesis. PMID:23057020

  4. Differential expression of miR-672-5p and miR-146a-5p in osteoblasts in rats after steroid intervention.

    PubMed

    Li, Pengfei; Sun, Nan; Zeng, Jianchun; Zeng, Yirong; Fan, Yueguang; Feng, Wenjun; Li, Jie

    2016-10-10

    Apoptosis of osteoblasts and osteocytes is one cause of steroid-induced osteonecrosis of the femoral head; however, the molecular mechanism of steroid affecting osteoblasts at the genetic level is unclear. The aim of the present work is to examine differential expression of osteoblasts in rats after steroid intervention and to verify expression by real-time polymerase chain reaction (RT-PCR). Primary culture, passaging and identification of osteoblasts of SD neonatal rats were conducted; osteoblasts were divided into two groups, the control group, and the steroid group. Total RNA was extracted separately, and quality control was performed; by means of RNA labeling and microarray hybridization, data were collected and then standardized to ascertain differences in miRNA expression between the two groups. The gene expression spectrum was analyzed. Obvious differential expression of miR-672-5p and miR-146a-5p was verified by RT-PCR. Miranda, microcosm and mirdb bioinformatics software were used to predict target genes. Compared with the control group, morphologically, the osteoblasts in the steroid group were more irregular and showed various shapes. The number of miRNAs (fold change >2) in the steroid group was six. Four miRNAs were upregulated and two miRNAs were downregulated. In particular, upregulated miR-672-5p expression and downregulated miR-146a-5p expression were significant. RT-PCR results showed that the 2(-△△) CT value of miR-672-5p in the steroid group was 3.743-fold of that in the control group, and the 2(-△△) CT value of miR-146a-5p in the steroid group was 0.322-fold of that in the control group. Angptl4, Ccdc51, Ssbp3 and RGD1306991 were predicted as the target gene of miR-672-5p, while Hrp12 was that of miR-146a-5p. Expression profiles of miR-672-5p and miR-146a-5p had the most significant changes in the osteoblasts of rats with steroid intervention, which may provide a new viewpoint to pathogenesis of osteonecrosis of the femoral head

  5. Fluid flow stimulates rapid and continuous release of nitric oxide in osteoblasts

    NASA Technical Reports Server (NTRS)

    Johnson, D. L.; McAllister, T. N.; Frangos, J. A.

    1996-01-01

    Interstitial fluid flow may mediate skeletal remodeling in response to mechanical loading. Because nitric oxide (NO) has been shown to be an osteoblast mitogen and inhibitor of osteoclastic resorption, we investigated and characterized the role of fluid shear on the release of NO in osteoblasts. Rat calvarial cells in a stationary culture produced undetectable levels of NO. Fluid shear stress (6 dyn/cm2) rapidly increased NO release rate to 9.8 nmol.h-1.mg protein-1 and sustained this production for 12 h of exposure to flow. Cytokine treatment also induced NO synthesis after a 12-h lag phase of zero production, followed by a production rate of 0.6 nmol.h-1.mg protein-1. Flow-induced NO production was blocked by the NO synthase (NOS) inhibitor NG-amino-L-arginine, but not by dexamethasone, which suggests that the flow stimulated a constitutive NOS isoform. This is the first time that a functional constitutively present NOS isoform has been identified in osteoblasts. Moreover, fluid flow represents the most potent stimulus of NO release in osteoblasts reported to date. Fluid flow-induced NO production may therefore play a primary role in bone maintenance and remodeling.

  6. Loss of TGF-β signaling in osteoblasts increases basic-FGF and promotes prostate cancer bone metastasis.

    PubMed

    Meng, Xiangqi; Vander Ark, Alexandra; Daft, Paul; Woodford, Erica; Wang, Jie; Madaj, Zachary; Li, Xiaohong

    2018-04-01

    TGF-β plays a central role in prostate cancer (PCa) bone metastasis, and it is crucial to understand the bone cell-specific role of TGF-β signaling in this process. Thus, we used knockout (KO) mouse models having deletion of the Tgfbr2 gene specifically in osteoblasts (Tgfbr2 Col1CreERT KO) or in osteoclasts (Tgfbr2 LysMCre KO). We found that PCa-induced bone lesion development was promoted in the Tgfbr2 Col1CreERT KO mice, but was inhibited in the Tgfbr2 LysMCre KO mice, relative to their respective control Tgfbr2 FloxE2 littermates. Since metastatic PCa cells attach to osteoblasts when colonized in the bone microenvironment, we focused on the mechanistic studies using the Tgfbr2 Col1CreERT KO mouse model. We found that bFGF was upregulated in osteoblasts from PC3-injected tibiae of Tgfbr2 Col1CreERT KO mice and correlated with increased tumor cell proliferation, angiogenesis, amounts of cancer-associated fibroblasts and osteoclasts. In vitro studies showed that osteoblastogenesis was inhibited, osteoclastogenesis was stimulated, but PC3 viability was not affected, by bFGF treatments. Lastly, the increased PC3-induced bone lesions in Tgfbr2 Col1CreERT KO mice were significantly attenuated by blocking bFGF using neutralizing antibody, suggesting bFGF is a promising target inhibiting bone metastasis. Copyright © 2018 Elsevier B.V. All rights reserved.

  7. Optimisation of the differing conditions required for bone formation in vitro by primary osteoblasts from mice and rats

    PubMed Central

    ORRISS, ISABEL R.; HAJJAWI, MARK O.R.; HUESA, CARMEN; MACRAE, VICKY E.; ARNETT, TIMOTHY R.

    2014-01-01

    The in vitro culture of calvarial osteoblasts from neonatal rodents remains an important method for studying the regulation of bone formation. The widespread use of transgenic mice has created a particular need for a reliable, simple method that allows the differentiation and bone-forming activity of murine osteoblasts to be studied. In the present study, we established such a method and identified key differences in optimal culture conditions between mouse and rat osteoblasts. Cells isolated from neonatal rodent calvariae by collagenase digestion were cultured for 14–28 days before staining for tissue non-specific alkaline phosphatase (TNAP) and bone mineralisation (alizarin red). The reliable differentiation of mouse osteoblasts, resulting in abundant TNAP expression and the formation of mineralised ‘trabecular-shaped’ bone nodules, occurred only following culture in α minimum essential medium (αMEM) and took 21–28 days. Dexamethasone (10 nM) inhibited bone mineralisation in the mouse osteoblasts. By contrast, TNAP expression and bone formation by rat osteoblasts were observed following culture in both αMEM and Dulbecco’s modified Eagle’s medium (DMEM) after approximately 14 days (although ~3-fold more effectively in αMEM) and was strongly dependent on dexamethasone. Both the mouse and rat osteoblasts required ascorbate (50 μg/ml) for osteogenic differentiation and β-glycerophosphate (2 mM) for mineralisation. The rat and mouse osteoblasts showed similar sensitivity to the well-established inhibitors of mineralisation, inorganic pyrophosphate (PPi) and adenosine triphosphate (ATP; 1–100 μM). The high efficiency of osteogenic differentiation observed following culture in αMEM, compared with culture in DMEM possibly reflects the richer formulation of the former. These findings offer a reliable technique for inducing mouse osteoblasts to form bone in vitro and a more effective method for culturing bone-forming rat osteoblasts. PMID:25200658

  8. Osteoblast response to porous titanium and biomimetic surface: In vitro analysis.

    PubMed

    Prado, Renata Falchete do; de Oliveira, Fernanda Saraiva; Nascimento, Rodrigo Dias; de Vasconcellos, Luana Marotta Reis; Carvalho, Yasmin Rodarte; Cairo, Carlos Alberto Alves

    2015-01-01

    This study analyzed the behavior of human osteoblasts cultured on porous titanium specimens, with and without biomimetic treatment, compared to dense titanium. The experiment had seven groups: Group 1: cells cultured on polystyrene of culture plate wells; Group 2: cells cultured on dense titanium specimen; Group 3: specimen with 33.79% of pores; Group 4: 41.79% of pores; Groups 5, 6 and 7: specimens similar to groups 2, 3 and 4, yet with biomimetic treatment. Real time-polymerase chain reaction with reverse transcription of the following genes was performed: prostaglandin E2 synthase, integrin β1, osterix, Runx2, Interleukin 6, macrophage colony stimulating factor, apolipoprotein E and others. The study achieved data on cell adhesion, growth and viability, total protein content, alkaline phosphatase activity and quantity of mineralized nodule formations. Data were statistically evaluated. Adherent cells and alkaline phosphatase activity were similar in titanium specimens, regardless of the groups. Biomimetic treatment reduced the total protein activity and the viability of tested cells. Most tested genes had statistically similar expression in all groups. The tested porosities did not cause alterations in osteoblast behavior and the biomimetic treatment impaired the biocompatibility of titanium causing cytotoxicity. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Temporal Profiling and Pulsed SILAC Labeling Identify Novel Secreted Proteins During Ex Vivo Osteoblast Differentiation of Human Stromal Stem Cells*

    PubMed Central

    Kristensen, Lars P.; Chen, Li; Nielsen, Maria Overbeck; Qanie, Diyako W.; Kratchmarova, Irina; Kassem, Moustapha; Andersen, Jens S.

    2012-01-01

    It is well established that bone forming cells (osteoblasts) secrete proteins with autocrine, paracrine, and endocrine function. However, the identity and functional role for the majority of these secreted and differentially expressed proteins during the osteoblast (OB) differentiation process, is not fully established. To address these questions, we quantified the temporal dynamics of the human stromal (mesenchymal, skeletal) stem cell (hMSC) secretome during ex vivo OB differentiation using stable isotope labeling by amino acids in cell culture (SILAC). In addition, we employed pulsed SILAC labeling to distinguish genuine secreted proteins from intracellular contaminants. We identified 466 potentially secreted proteins that were quantified at 5 time-points during 14-days ex vivo OB differentiation including 41 proteins known to be involved in OB functions. Among these, 315 proteins exhibited more than 2-fold up or down-regulation. The pulsed SILAC method revealed a strong correlation between the fraction of isotope labeling and the subset of proteins known to be secreted and involved in OB differentiation. We verified SILAC data using qRT-PCR analysis of 9 identified potential novel regulators of OB differentiation. Furthermore, we studied the biological effects of one of these proteins, the hormone stanniocalcin 2 (STC2) and demonstrated its autocrine effects in enhancing osteoblastic differentiation of hMSC. In conclusion, combining complete and pulsed SILAC labeling facilitated the identification of novel factors produced by hMSC with potential role in OB differentiation. Our study demonstrates that the secretome of osteoblastic cells is more complex than previously reported and supports the emerging evidence that osteoblastic cells secrete proteins with endocrine functions and regulate cellular processes beyond bone formation. PMID:22801418

  10. Comparison of Two Porcine Collagen Membranes Combined with rhBMP-2 and rhBMP-9 on Osteoblast Behavior In Vitro.

    PubMed

    Fujioka-Kobayashi, Masako; Schaler, Benoit; Shirakata, Yoshinori; Nakamura, Toshiaki; Noguchi, Kazuyuki; Zhang, Yufeng; Miron, Richard J

    To investigate the bone-inducing properties of two types of collagen membranes in combination with recombinant human bone morphogenetic protein (rhBMP)-2 and rhBMP-9 on osteoblast behavior. Porcine pericardium collagen membranes (PPCM) and porcine dermis-derived collagen membranes (PDCM) were coated with either rhBMP-2 or rhBMP-9. The adsorption and release abilities were first investigated via enzyme-linked immunosorbent assay up to 10 days. Moreover, murine bone stromal ST2 cell adhesion, proliferation, and osteoblast differentiation were assessed by MTS assay; real-time polymerase chain reaction for genes encoding runt-related transcription factor 2 (Runx2); alkaline phosphatase (ALP); and osteocalcin, ALP assay, and alizarin red staining. Both rhBMP-2 and rhBMP-9 adsorbed to collagen membranes and were gradually released over time up to 10 days. PPCM showed significantly less cell attachment, whereas PDCM demonstrated comparable cell attachment with the control tissue culture plastic at 8 hours. While both rhBMPs were shown not to affect cell proliferation, collagen membranes combined with rhBMP-9 significantly increased ALP activity at 7 days and ALP mRNA levels at either 3 or 14 days compared with the control tissue culture plastic. Furthermore, rhBMP-9 increased osteocalcin mRNA levels and alizarin red staining at 14 days compared with the control tissue culture plastic. The results from this study suggest that both porcine-derived collagen membranes combined with rhBMP-9 accelerated the osteopromotive potential of ST2 cells. Interestingly, rhBMP-9 demonstrated additional osteogenic differentiation compared with rhBMP-2 and may serve as a suitable growth factor for future clinical use.

  11. Bioactive treatment promotes osteoblast differentiation on titanium materials fabricated by selective laser melting technology.

    PubMed

    Tsukanaka, Masako; Fujibayashi, Shunsuke; Takemoto, Mitsuru; Matsushita, Tomiharu; Kokubo, Tadashi; Nakamura, Takashi; Sasaki, Kiyoyuki; Matsuda, Shuichi

    2016-01-01

    Selective laser melting (SLM) technology is useful for the fabrication of porous titanium implants with complex shapes and structures. The materials fabricated by SLM characteristically have a very rough surface (average surface roughness, Ra=24.58 µm). In this study, we evaluated morphologically and biochemically the specific effects of this very rough surface and the additional effects of a bioactive treatment on osteoblast proliferation and differentiation. Flat-rolled titanium materials (Ra=1.02 µm) were used as the controls. On the treated materials fabricated by SLM, we observed enhanced osteoblast differentiation compared with the flat-rolled materials and the untreated materials fabricated by SLM. No significant differences were observed between the flat-rolled materials and the untreated materials fabricated by SLM in their effects on osteoblast differentiation. We concluded that the very rough surface fabricated by SLM had to undergo a bioactive treatment to obtain a positive effect on osteoblast differentiation.

  12. Early effects of extracorporeal shock wave treatment on osteoblast-like cells: a comparative study between electromagnetic and electrohydraulic devices.

    PubMed

    Martini, Lucia; Giavaresi, Gianluca; Fini, Milena; Borsari, Veronica; Torricelli, Paola; Giardino, Roberto

    2006-11-01

    Extracorporeal shockwave therapy (ESWT) has been increasingly applied to treat orthopedic and musculoskeletal pathologies. ESWT involves mechanical perturbations that, as with other physical therapies, can result in mechanical stimuli to a large number of cells, including bone cells. The aim of this study was to evaluate the effects of shock waves on osteoblast-like cells (MG63) when using two different generators of shock waves (electrohydraulic and electromagnetic devices), in terms of cell damage, cell viability, osteogenic phenotype expression, and cytokine production. MG63 cells were suspended in 1.5 mL screw-cap cryotubes (1 x 10 cells/mL), containing phosphate buffer solution (PBS), which were maintained at 37 degrees C during all the experimental times. Two levels of energy flux density (EFD) were evaluated for each device: 0.15 to 0.18 mJ/mm2 and 0.40 mJ/mm2. Cells were then cultivated for 72 hours starting from a concentration of 1 x 10 cells/mL, and biological activity and viability were evaluated 24 and 72 hours after treatment. The results obtained demonstrate that the factors most affecting osteoblast activity involve both the device and the level of EFD selected, and they must be considered all together. The use of the electromagnetic device and a level of EFD lower than 0.40 mJ/mm2 would appear to induce fewer immediate cytodestructive effects and better stimulate subsequent proliferation and the synthetic activity of MG63.

  13. Factors Affecting Tocopherol Concentrations in Soybean Seeds.

    PubMed

    Carrera, Constanza S; Seguin, Philippe

    2016-12-21

    Soybean seeds contain several health-beneficial compounds, including tocopherols, which are used by the nutraceutical and functional food industries. Soybean tocopherol concentrations are, however, highly variable. Large differences observed in tocopherol concentrations among soybean genotypes together with the relatively simple biosynthetic pathway involving few genes support the feasibility of selecting for high-tocopherol soybean. Tocopherol concentrations are also highly influenced by environmental factors and field management. Temperature during seed filling and soil moisture appear to be the main factors affecting tocopherol concentrations; other factors such as soil fertility and solar radiation also affect concentrations and composition. Field management decisions including seeding date, row spacing, irrigation, and fertilization also affect tocopherols. Knowledge of factors affecting soybean tocopherols is essential to develop management strategies that will lead to the production of seeds with consistent target concentrations that will meet the needs of the nutraceutical and functional food industries.

  14. The Bone-specific Expression of Runx2 Oscillates during the Cell Cycle to Support a G1-related Antiproliferative Function in Osteoblasts*

    PubMed Central

    Galindo, Mario; Pratap, Jitesh; Young, Daniel W.; Hovhannisyan, Hayk; Im, Hee-Jeong; Choi, Je-Yong; Lian, Jane B.; Stein, Janet L.; Stein, Gary S.; van Wijnen, Andre J.

    2010-01-01

    The Runx2 (CBFA1/AML3/PEBP2αA) transcription factor promotes skeletal cell differentiation, but it also has a novel cell growth regulatory activity in osteoblasts. We addressed here whether Runx2 activity is functionally linked to cell cycle-related mechanisms that control normal osteoblast proliferation and differentiation. We found that the levels of Runx2 gene transcription, mRNA and protein, are each up-regulated with cessation of cell growth (i.e. G0/G1 transition) in preconfluent MC3T3 osteoblastic cells that do not yet express mature bone phenotypic gene expression. Cell growth regulation of Runx2 is also observed in primary calvarial osteoblasts and other osteoblastic cells with relatively normal cell growth characteristics, but not in osteosarcoma cells (e.g. SAOS-2 and ROS17/2.8). Runx2 levels are cell cycle-regulated in MC3T3 cells with respect to the G1/S and M/G1 transitions: expression oscillates from maximal levels during early G1 to minimal levels during early S phase and mitosis. However, in normal or immortalized (e.g. ATDC5) chondrocytic cells, Runx2 expression is suppressed during quiescence, and Runx2 levels are not regulated during G1 and S phase in ATDC5 cells. Antisense or small interfering RNA-mediated reduction of the low physiological levels of Runx2 in proliferating MC3T3 cells does not accelerate cell cycle progression. However, forced expression of Runx2 suppresses proliferation of MC3T3 preosteoblasts or C2C12 mesenchymal cells which have osteogenic potential. Forced elevation of Runx2 in synchronized MC3T3 cells causes a delay in G1. We propose that Runx2 levels and function are biologically linked to a cell growth-related G1 transition in osteoblastic cells. PMID:15781466

  15. [Comparison of the activity and yield rate of osteoblast obtained by different digestion methods].

    PubMed

    Li, Ling-hui; Ding, Dao-Fang; Du, Guo-Qing; Wang, Hui-Hao; Zhan, Hong-Sheng

    2013-04-01

    To compared the activity and yield rate of osteoblast obtained by different collagenase digestion methods, to find a better way to extract osteoblast for the experimental researches of osteoporosis. Ten 24-hour-old SD rats were were euthanized. The cranium of rats were removed and cuted into blocks of 1 mm x 1 mm size. After digested by trypsin for 15 min, all the cranium were divided into two equal parts, and randomly divided into two groups which would be digested by type I collagenase and type II collagenase separately for two times. The rat cells of the two groups were cultured in thermostat incubator with 5% CO2 under the condition of 37 degrees C. The primary culture osteoblasts were counted by using a haemacytometer after digestion and 72 hours later. The second generation osteoblasts cultured 48 h were dyed by NBT/BCIP staining solution, and were detected by quantitative measurement with PNPP. The cells had irregular shapes. The results of cell counting showed that the cell number of type I group was larger than type 11 group. Alkaline phosphatase dyeing were positive. Detecting of alkaline phosphatase using the method of PNPP showed that the absorbance value in type I group were higher than type II group (P<0.05). Two types of collagenase are both suitable for the in vitro culture of rat osteoblasts. The activity and yield rate of osteoblasts in type I group are higher which could provide more stable seed cells for the treatment of osteoporosis.

  16. 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.

  17. Porous titanium and Ti-35Nb alloy: effects on gene expression of osteoblastic cells derived from human alveolar bone.

    PubMed

    do Prado, Renata Falchete; Rabêlo, Sylvia Bicalho; de Andrade, Dennia Perez; Nascimento, Rodrigo Dias; Henriques, Vinicius André Rodrigues; Carvalho, Yasmin Rodarte; Cairo, Carlos Alberto Alves; de Vasconcellos, Luana Marotta Reis

    2015-11-01

    Tests on titanium alloys that possess low elastic modulus, corrosion resistance and minimal potential toxicity are ongoing. This study aimed to evaluate the behavior of human osteoblastic cells cultured on dense and porous Titanium (Ti) samples comparing to dense and porous Ti-35 Niobium (Ti-35Nb) samples, using gene expression analysis. Scanning electronic microscopy confirmed surface porosity and pore interconnectivity and X-ray diffraction showed titanium beta-phase stabilization in Ti-35Nb alloy. There were no differences in expression of transforming growth factor-β, integrin-β1, alkaline phosphatase, osteopontin, macrophage colony stimulating factor, prostaglandin E synthase, and apolipoprotein E regarding the type of alloy, porosity and experimental period. The experimental period was a significant factor for the markers: bone sialoprotein II and interleukin 6, with expression increasing over time. Porosity diminished Runt-related transcription factor-2 (Runx-2) expression. Cells adhering to the Ti-35Nb alloy showed statistically similar expression to those adhering to commercially pure Ti grade II, for all the markers tested. In conclusion, the molecular mechanisms of interaction between human osteoblasts and the Ti-35Nb alloy follow the principal routes of osseointegration of commercially pure Ti grade II. Porosity impaired the route of transcription factor Runx-2.

  18. Thiazide diuretics directly induce osteoblast differentiation and mineralized nodule formation by targeting a NaCl cotransporter in bone

    PubMed Central

    Dvorak, Melita M; De Joussineau, Cyrille; Carter, D Howard; Pisitkun, Trairak; Knepper, Mark A; Gamba, Gerardo; Kemp, Paul J; Riccardi, Daniela

    2008-01-01

    Thiazide diuretics are used, worldwide, as the first-choice drug for patients with uncomplicated hypertension. In addition to their anti-hypertensive actions, they increase bone mineral density and reduce the prevalence of fractures, indicating that thiazides may have a role in the management of postmenopausal osteoporosis. Traditionally, the bone-protective effects of thiazides have been attributed to an increase in renal calcium reabsorption, secondary to the inhibition of the sodium chloride cotransporter, NCC, expressed in the kidney distal tubule. Whether thiazides exert a direct osteoanabolic effect independently of their renal action is controversial. Here we demonstrate that freshly frozen sections of human and rat bone express NCC, principally in bone-forming cells, the osteoblasts. In primary and established culture models of osteoblasts, fetal rat calvarial (FRC) and human MG63 cells, NCC protein is virtually absent in proliferating cells while its expression is dramatically increased during differentiation. Thiazides directly stimulate the production of osteoblast markers, runt-related transcription factor 2 (runx2) and osteopontin, in the absence of a proliferative effect. Using overexpression/knockdown studies in FRC cells, we show that thiazides, but not loop diuretics, increase mineralized nodule formation acting on NCC. Overall, our study demonstrates that thiazides stimulate osteoblast differentiation and bone mineral formation independently of their renal actions. In addition to their use as part of a therapeutic treatment plan for elderly, hypertensive individuals, our discovery opens up the possibility that bone-specific drug targeting by thiazides may be developed for the prevention and treatment of osteoporosis in the patient population as a whole. PMID:17656470

  19. [Effects of kidney-tonifying Chinese herbal drugs on human osteoblast Ca2+ intake and mineralization in vitro].

    PubMed

    Li, Juan; Wu, Wei-kang; Sun, Wei; Yu, Ke-qiang

    2004-12-01

    To study the effects of kidney-tonifying Chinese herbal drugs on Ca2+ intake and mineralization of human osteoblasts in vitro. Human osteoblasts were isolated from the iliac trabecular bone followed by purification and culture at 37 degrees Celsius with 5% CO2. The cells were identified by cell morphology, calcium nodule formation and alkaline phosphatase (ALP) activity assay. The third passage of the cultured osteoblasts were treated with 10% scrum from rat fed with the decoction of the kidney-tonifying Chinese herbal drugs of different concentrations for 30 min, 3 d and 28 d, respectively. The cells treated with 10% rat serum without the drugs served as the control. Flow cytometry was used to observe the changes in cell proliferation and intracellular Ca2+ concentration, and von Kossa staining employed for quantification of the mineral nodules. The osteoblasts obtained were positive for ALP staining and could form calcium nodules in vitro. Flow cytometry showed that the drugs at different concentrations all increased Ca2+ influx, as compared with the control cells. The drugs also increased the relative proliferation index of the osteoblasts, and high concentration of the drugs resulted in greater number of the mineral nodules in the osteoblasts (P<0.05). The kidney-tonifying Chinese herbal drugs may increase Ca2+ influx and stimulate proliferation and differentiation of adult osteoblasts in vitro.

  20. Characterization of cadmium uptake and cytotoxicity in human osteoblast-like MG-63 cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Levesque, Martine; Martineau, Corine; Jumarie, Catherine

    Since bone mass is maintained constant by the balance between osteoclastic bone resorption and osteoblastic bone formation, alterations in osteoblast proliferation and differentiation may disturb the equilibrium of bone remodeling. Exposure to cadmium (Cd) has been associated with the alteration of bone metabolism and the development of osteoporosis. Because little information is available about the direct effects of Cd on osteoblastic cells, we have characterized in vitro the cellular accumulation and cytotoxicity of Cd in human osteoblastic cells. Incubation of osteoblast-like MG-63 cells with increasing concentrations of Cd in serum-free culture medium reduced cell viability in a time- and concentration-dependentmore » manner, suggesting that Cd accumulates in osteoblasts. Consequently, an uptake time-course could be characterized for the cellular accumulation of {sup 109}Cd in serum-free culture medium. In order to characterize the mechanisms of Cd uptake, experiments have been conducted under well-defined metal speciation conditions in chloride and nitrate transport media. The results revealed a preferential uptake of Cd{sup 2+} species. The cellular accumulation and cytotoxicity of Cd increased in the absence of extracellular calcium (Ca), suggesting that Cd may enter the cells in part through Ca channels. However, neither the cellular accumulation nor the cytotoxicity of Cd was modified by voltage-dependent Ca channel (VDCC) modulators or potassium-induced depolarization. Moreover, exposure conditions activating or inhibiting capacitative Ca entry (CCE) failed to modify the cellular accumulation and cytotoxicity of Cd, which excludes the involvement of canonical transient receptor potential (TRPC) channels. The cellular accumulation and cytotoxicity of Cd were reduced by 2-APB, a known inhibitor of the Mg and Ca channel TRPM7 and were increased in the absence of extracellular magnesium (Mg). The inhibition of Cd uptake by Mg and Ca was not additive

  1. 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

  2. Osteoblast-specific deletion of Hrpt2/Cdc73 results in high bone mass and increased bone turnover.

    PubMed

    Droscha, Casey J; Diegel, Cassandra R; Ethen, Nicole J; Burgers, Travis A; McDonald, Mitchell J; Maupin, Kevin A; Naidu, Agni S; Wang, PengFei; Teh, Bin T; Williams, Bart O

    2017-05-01

    Inactivating mutations that lead to loss of heterozygosity within the HRPT2/Cdc73 gene are directly linked to the development of primary hyperparathyroidism, parathyroid adenomas, and ossifying fibromas of the jaw (HPT-JT). The protein product of the Cdc73 gene, parafibromin, is a core member of the polymerase-associated factors (PAF) complex, which coordinates epigenetic modifiers and transcriptional machinery to control gene expression. We conditionally deleted Cdc73 within mesenchymal progenitors or within mature osteoblasts and osteocytes to determine the consequences of parafibromin loss within the mesenchymal lineage. Homozygous deletion of Cdc73 via the Dermo1-Cre driver resulted in embryos which lacked mesenchymal organ development of internal organs, including the heart and fetal liver. Immunohistochemical detection of cleaved caspase-3 revealed extensive apoptosis within the progenitor pools of developing organs. Unexpectedly, when Cdc73 was homozygously deleted within mature osteoblasts and osteocytes (via the Ocn-Cre driver), the mice had a normal life span but increased cortical and trabecular bone. OCN-Cre;Cdc73 flox/flox bones displayed large cortical pores actively undergoing bone remodeling. Additionally the cortical bone of OCN-Cre;Cdc73 flox/flox femurs contained osteocytes with marked amounts of cytoplasmic RNA and a high rate of apoptosis. Transcriptional analysis via RNA-seq within OCN-Cre;Cdc73 flox/flox osteoblasts showed that loss of Cdc73 led to a derepression of osteoblast-specific genes, specifically those for collagen and other bone matrix proteins. These results aid in our understanding of the role parafibromin plays within transcriptional regulation, terminal differentiation, and bone homeostasis. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. [Experimental research on the effect of nanophase ceramics on osteoblasts functions].

    PubMed

    Wen, Bo; Chen, Zhiqing; Jiang, Yinshan; Yang, Zhengwen; Xu, Yongzhong

    2005-06-01

    In order to study the cytocompatibility of nanophase hydroxyapatite ceramic in vitro, we prepared hydroxyapatite by use of the wet chemistry techniques. The grain size of hydroxyapatite of interest to the present study was determined by scanning electron microscopy and atomic force microscopy with image analysis software. Primary culture of osteoblast from rat calvaria was established. Protein content, synthesis of alkaline phosphatase and deposition of calcium-containing mineral by osteoblasts cultured on nanophase hydroxyapatite ceramics and on conventional hydroxyapatite ceramics for 7, 14, 21 and 28 days were examined. The results showed that the average surface grain size of the nanophase and that of the conventional HA compact formulations was 55 (nanophase) and 780 (conventional) nm, respectively. More importantly, compared to the synthesis of alkaline phosphatase and deposition of calcium-containing mineral by osteoblasts cultured on nanophase was significantly greater than that on conventional ceramics after 21 and 28 days. The cytocompatibility was significantly greater on nanophase HA than on conventional formulations of the same ceramic.

  4. Galectin-3 as a novel regulator of osteoblast-osteoclast interaction and bone homeostasis.

    PubMed

    Simon, Dominic; Derer, Anja; Andes, Fabian T; Lezuo, Patrick; Bozec, Aline; Schett, Georg; Herrmann, Martin; Harre, Ulrike

    2017-12-01

    Bone tissue undergoes permanent and lifelong remodeling with a concerted action of bone-building osteoblasts and bone-resorbing osteoclasts. A precise cooperation between those two cell types is critical in the complex process of bone renewal. Galectin-3 is a member of the β-galactoside-binding lectin family playing multiple roles in cell growth, differentiation and aggregation. As it has been described to be expressed in bone, galectin-3 might influence bone homeostasis by regulating the function and/or interplay of osteoblasts and osteoclasts. Here, we investigated the role of galectin-3 in osteoclastogenesis and osteoblast-osteoclast interactions. Bone histomorphometric analysis and μCT measurements revealed a decreased trabecular bone volume and an increased osteoclast number in 12weeks old male galectin-3 knockout mice compared to wildtype littermates. Galectin-3 deficient bone marrow cells displayed a higher osteoclastogenic capacity in ex vivo differentiation assays, associated with elevated TRAF6 mRNA levels, suggesting an intrinsic inhibition of osteoclastogenesis by galectin-3 interfering with RANKL-mediated signaling. Furthermore, the addition of extracellular galectin-3 to murine or human osteoclastogenesis assays inhibited osteoclast formation and osteoclast numbers were higher in co-culture assays with galectin-3 deficient osteoblasts. In conclusion, our data suggest the secretion of galectin-3 as a novel mechanism for osteoblasts to control osteoclastogenesis and to maintain trabecular bone homeostasis independently of the RANKL/OPG-axis. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Effect of natural uranium on the UMR-106 osteoblastic cell line: impairment of the autophagic process as an underlying mechanism of uranium toxicity.

    PubMed

    Pierrefite-Carle, Valérie; Santucci-Darmanin, Sabine; Breuil, Véronique; Gritsaenko, Tatiana; Vidaud, Claude; Creff, Gaelle; Solari, Pier Lorenzo; Pagnotta, Sophie; Al-Sahlanee, Rasha; Auwer, Christophe Den; Carle, Georges F

    2017-04-01

    Natural uranium (U), which is present in our environment, exerts a chemical toxicity, particularly in bone where it accumulates. Generally, U is found at oxidation state +VI in its oxocationic form [Formula: see text] in aqueous media. Although U(VI) has been reported to induce cell death in osteoblasts, the cells in charge of bone formation, the molecular mechanism for U(VI) effects in these cells remains poorly understood. The objective of our study was to explore U(VI) effect at doses ranging from 5 to 600 µM, on mineralization and autophagy induction in the UMR-106 model osteoblastic cell line and to determine U(VI) speciation after cellular uptake. Our results indicate that U(VI) affects mineralization function, even at subtoxic concentrations (<100 µM). The combination of thermodynamic modeling of U with EXAFS data in the culture medium and in the cells clearly indicates the biotransformation of U(VI) carbonate species into a meta-autunite phase upon uptake by osteoblasts. We next assessed U(VI) effect at 100 and 300 µM on autophagy, a survival process triggered by various stresses such as metal exposure. We observed that U(VI) was able to rapidly activate autophagy but an inhibition of the autophagic flux was observed after 24 h. Thus, our results indicate that U(VI) perturbs osteoblastic functions by reducing mineralization capacity. Our study identifies for the first time U(VI) in the form of meta-autunite in mammalian cells. In addition, U(VI)-mediated inhibition of the autophagic flux may be one of the underlying mechanisms leading to the decreased mineralization and the toxicity observed in osteoblasts.

  6. Spontaneous Osteoblastic Osteosarcoma in a Mongolian Gerbil (Meriones unguiculatus)

    PubMed Central

    Salyards, Gregory W; Blas-Machado, Uriel; Mishra, Sasmita; Harvey, Stephen B; Butler, Abigail M

    2013-01-01

    Spontaneous neoplasms in Mongolian gerbils have an incidence of 20% to 26.8%, but osteosarcomas occur at a much lower rate. Here we report a 1-y-old Mongolian gerbil with a spontaneous osteosarcoma at the level of the proximal tibia, with metastases to the pectoral muscles and lungs. Grossly, the tibial mass obliterated the tibia and adjacent muscles, and an axillary mass with a bloody, cavitary center expanded the pectoral muscles. Microscopically, the tibial mass was an infiltrative, osteoblastic mesenchymal neoplasm, and the axillary mass was an anaplastic mesenchymal neoplasm with hemorrhage. The lung contained multiple metastatic foci. Immunohistochemistry for osteonectin was strongly positive in the tibial, axillary, and pulmonary metastases. Although osteosarcoma is the most common primary malignant bone neoplasm that occurs spontaneously in all laboratory and domestic animal species and humans, it arises less frequently than does other neoplasms. The current case of spontaneous osteoblastic osteosarcoma of the proximal tibia and metastases to the pectoral muscles and lung in a Mongolian gerbil is similar in presentation, histology, and predilection site of both osteoblastic and telangiectatic osteosarcomas in humans. In addition, this case is an unusual manifestation of osteosarcoma in the appendicular skeleton of a Mongolian gerbil. PMID:23561939

  7. 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.

  8. Rictor/mTORC2 loss in osteoblasts impairs bone mass and strength.

    PubMed

    Liu, Dong-Mei; Zhao, Lin; Liu, Ting-Ting; Jiao, Pei-Lin; Zhao, Dian-Dian; Shih, Mei-Shu; Tao, Bei; Sun, Li-Hao; Zhao, Hong-Yan; Liu, Jian-Min

    2016-09-01

    Mammalian target of rapamycin (mTOR) is a Ser/Thr kinase conserved through evolution that coordinates extra cellular signals associated with cell growth. Main functions of mTOR present in the form of two complexes, namely mTORC1 and mTORC2, which are distinct in their unique components, raptor and rictor. In the current study, using a Cre/loxp system, we found an anabolic effect of mTORC2 signaling on skeleton. Osteoblast differentiation was reduced, with down-regulation of mTORC2 signaling activity in primary cultures of osteoblasts that did not contain rictor. Mice with a specific deletion of rictor in mature osteoblasts showed a significant reduction in lean mass and bone mineral density by dual energy x-ray absorptiometry analysis. Micro-computed tomography, histomorphometric, and molecular biological analyses revealed a marked impairment of the cortical bone mass and microarchitecture, as well as minor changes in trabecular bone, of the Rictorob(-/-) mice. Cortical bone mass and thickness of the femoral mid-shaft were dramatically reduced, with unusual increases in porosity and marrow area in Rictorob(-/-) mice. Thinner trabeculae were found in the L4 vertebrae with relatively normal structural indices of trabecular numbers and separation. A lower rate of bone turnover was observed, as the consequence of the decreased individual osteoblast activity and bone resorption. Furthermore, these changes were associated with significantly decreased bone biomechanical properties. In conclusion, expression of rictor in osteoblasts is essential for the maintenance of normal bone remodeling and microarchitecture, especially for the maintenance of the cortical bone. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Briolay, A.; Lencel, P.; Bessueille, L.

    2013-01-18

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

  10. Peroxidase Enzymes Regulate Collagen Biosynthesis and Matrix Mineralization by Cultured Human Osteoblasts.

    PubMed

    DeNichilo, Mark O; Shoubridge, Alexandra J; Panagopoulos, Vasilios; Liapis, Vasilios; Zysk, Aneta; Zinonos, Irene; Hay, Shelley; Atkins, Gerald J; Findlay, David M; Evdokiou, Andreas

    2016-03-01

    The early recruitment of inflammatory cells to sites of bone fracture and trauma is a critical determinant in successful fracture healing. Released by infiltrating inflammatory cells, myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are heme-containing enzymes, whose functional involvement in bone repair has mainly been studied in the context of providing a mechanism for oxidative defense against invading microorganisms. We report here novel findings that show peroxidase enzymes have the capacity to stimulate osteoblastic cells to secrete collagen I protein and generate a mineralized extracellular matrix in vitro. Mechanistic studies conducted using cultured osteoblasts show that peroxidase enzymes stimulate collagen biosynthesis at a post-translational level in a prolyl hydroxylase-dependent manner, which does not require ascorbic acid. Our studies demonstrate that osteoblasts rapidly bind and internalize both MPO and EPO, and the catalytic activity of these peroxidase enzymes is essential to support collagen I biosynthesis and subsequent release of collagen by osteoblasts. We show that EPO is capable of regulating osteogenic gene expression and matrix mineralization in culture, suggesting that peroxidase enzymes may play an important role not only in normal bone repair, but also in the progression of pathological states where infiltrating inflammatory cells are known to deposit peroxidases.

  11. Bone conditioned media (BCM) improves osteoblast adhesion and differentiation on collagen barrier membranes.

    PubMed

    Fujioka-Kobayashi, Masako; Caballé-Serrano, Jordi; Bosshardt, Dieter D; Gruber, Reinhard; Buser, Daniel; Miron, Richard J

    2016-07-04

    The use of autogenous bone chips during guided bone regeneration procedures has remained the gold standard for bone grafting due to its excellent combination of osteoconduction, osteoinduction and osteogenesis. Recent protocols established by our group have characterized specific growth factors and cytokines released from autogenous bone that have the potential to be harvested and isolated into bone conditioned media (BCM). Due to the advantageous osteo-promotive properties of BCM, the aims of the present study was to pre-coat collagen barrier membranes with BCM and investigate its effect on osteoblast adhesion, proliferation and differentiation for possible future clinical use. Scanning electron microscopy (SEM) was first used to qualitative assess BCM protein accumulation on the surface of collagen membranes. Thereafter, undifferentiated mouse ST2 stromal bone marrow cells were seeded onto BioGide porcine derived collagen barrier membranes (control) or barrier membranes pre-coated with BCM (test group). Control and BCM samples were compared for cell adhesion at 8 h, cell proliferation at 1, 3 and 5 days and real-time PCR at 5 days for osteoblast differentiation markers including Runx2, alkaline phosphatase (ALP), osteocalcin (OCN) and bone sialoprotein (BSP). Mineralization was further assessed with alizarin red staining at 14 days post seeding. SEM images demonstrated evidence of accumulated proteins found on the surface of collagen membranes following coating with BCM. Analysis of total cell numbers revealed that the additional pre-coating with BCM markedly increased cell attachment over 4 fold when compared to cells seeded on barrier membranes alone. No significant difference could be observed for cell proliferation at all time points. BCM significantly increased mRNA levels of osteoblast differentiation markers including ALP, OCN and BSP at 5 days post seeding. Furthermore, barrier membranes pre-coated with BCM demonstrated a 5-fold increase in alizarin

  12. Ubiquitous overexpression of Hey1 transcription factor leads to osteopenia and chondrocyte hypertrophy in bone.

    PubMed

    Salie, Rishard; Kneissel, Michaela; Vukevic, Mirko; Zamurovic, Natasa; Kramer, Ina; Evans, Glenda; Gerwin, Nicole; Mueller, Matthias; Kinzel, Bernd; Susa, Mira

    2010-03-01

    The transcription factor Hey1, a known Notch target gene of the HES family, has recently been described as a target gene of bone morphogenetic protein-2 (BMP-2) during osteoblastic differentiation in vitro. As the role of Hey1 in skeletal physiology is unknown, we analyzed bones of mice ubiquitously lacking or overexpressing Hey1. This strategy enabled us to evaluate whether Hey1 modulation in the whole organism could serve as a drug or antibody target for therapy of diseases associated with bone loss. Hey1 deficiency resulted in modest osteopenia in vivo and increased number and activity of osteoclasts generated ex vivo. Hey1 overexpression resulted in distinct progressive osteopenia and inhibition of osteoblasts ex vivo, an effect apparently dominant to a mild inhibition of osteoclasts. In both Hey1 deficient and overexpressing mice, males were less affected than females and skeleton was not affected during development. Bone histomorphometry did not reveal major changes in animals at 20 weeks, suggesting that modulation had occurred before. Adult Hey1 transgenics also displayed increased type X collagen expression and an enlarged hypertrophic zone in the growth plate. Taken together, our data suggest that ubiquitous in vivo Hey1 regulation affects osteoblasts, osteoclasts and chondrocytes. Due to the complex role of Hey1 in bone, inhibition of Hey1 does not appear to be a straightforward therapeutic strategy to increase the bone mass.

  13. The Influence of Primary Microenvironment on Prostate Cancer Osteoblastic Bone LesionDevelopment

    DTIC Science & Technology

    2016-11-01

    cancer- associated osteoblasts, but highly expressed in PCa cells. Thus we used the Tgfbr2Col1CreERT KO mice to study the mechanism with the goal...suggesting an indirect effect of bFGF, possibly through increases of angiogenesis and CAFs formation. Furthermore, to determine the mechanism by which...partially through increased PTHrP signaling. The significances of our study are the determination of the role and mechanism of osteoblast-specific

  14. Factors Affecting Wound Healing

    PubMed Central

    Guo, S.; DiPietro, L.A.

    2010-01-01

    Wound healing, as a normal biological process in the human body, is achieved through four precisely and highly programmed phases: hemostasis, inflammation, proliferation, and remodeling. For a wound to heal successfully, all four phases must occur in the proper sequence and time frame. Many factors can interfere with one or more phases of this process, thus causing improper or impaired wound healing. This article reviews the recent literature on the most significant factors that affect cutaneous wound healing and the potential cellular and/or molecular mechanisms involved. The factors discussed include oxygenation, infection, age and sex hormones, stress, diabetes, obesity, medications, alcoholism, smoking, and nutrition. A better understanding of the influence of these factors on repair may lead to therapeutics that improve wound healing and resolve impaired wounds. PMID:20139336

  15. Factors affecting wound healing.

    PubMed

    Guo, S; Dipietro, L A

    2010-03-01

    Wound healing, as a normal biological process in the human body, is achieved through four precisely and highly programmed phases: hemostasis, inflammation, proliferation, and remodeling. For a wound to heal successfully, all four phases must occur in the proper sequence and time frame. Many factors can interfere with one or more phases of this process, thus causing improper or impaired wound healing. This article reviews the recent literature on the most significant factors that affect cutaneous wound healing and the potential cellular and/or molecular mechanisms involved. The factors discussed include oxygenation, infection, age and sex hormones, stress, diabetes, obesity, medications, alcoholism, smoking, and nutrition. A better understanding of the influence of these factors on repair may lead to therapeutics that improve wound healing and resolve impaired wounds.

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

    PubMed

    Kanno, Sanae; Hirano, Seishiro; Kayama, Fujio

    2004-03-01

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

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yu, Haitao; Du, Yuxuan; Zhang, Xulong

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

  18. Osteocytes, not Osteoblasts or Lining Cells, are the Main Source of the RANKL Required for Osteoclast Formation in Remodeling Bone

    PubMed Central

    Xiong, Jinhu; Piemontese, Marilina; Onal, Melda; Campbell, Josh; Goellner, Joseph J.; Dusevich, Vladimir; Bonewald, Lynda; Manolagas, Stavros C.; O’Brien, Charles A.

    2015-01-01

    The cytokine receptor activator of nuclear factor kappa B ligand (RANKL), encoded by the Tnfsf11 gene, is essential for osteoclastogenesis and previous studies have shown that deletion of the Tnfsf11 gene using a Dmp1-Cre transgene reduces osteoclast formation in cancellous bone by more than 70%. However, the Dmp1-Cre transgene used in those studies leads to recombination in osteocytes, osteoblasts, and lining cells making it unclear whether one or more of these cell types produce the RANKL required for osteoclast formation in cancellous bone. Because osteoblasts, osteocytes, and lining cells have distinct locations and functions, distinguishing which of these cell types are sources of RANKL is essential for understanding the orchestration of bone remodeling. To distinguish between these possibilities, we have now created transgenic mice expressing the Cre recombinase under the control of regulatory elements of the Sost gene, which is expressed in osteocytes but not osteoblasts or lining cells in murine bone. Activity of the Sost-Cre transgene in osteocytes, but not osteoblast or lining cells, was confirmed by crossing Sost-Cre transgenic mice with tdTomato and R26R Cre-reporter mice, which express tdTomato fluorescent protein or LacZ, respectively, only in cells expressing the Cre recombinase or their descendants. Deletion of the Tnfsf11 gene in Sost-Cre mice led to a threefold decrease in osteoclast number in cancellous bone and increased cancellous bone mass, mimicking the skeletal phenotype of mice in which the Tnfsf11 gene was deleted using the Dmp1-Cre transgene. These results demonstrate that osteocytes, not osteoblasts or lining cells, are the main source of the RANKL required for osteoclast formation in remodeling cancellous bone. PMID:26393791

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

    PubMed

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

    2016-04-01

    Increased fragility has been described in humans with polycythemia vera (PV). Herein, we describe an osteoporotic phenotype associated with decreased osteoblast activity in a mouse model of PV and another mouse of polycythemia and elevated circulating erythropoietin (EPO). Our results are important for patients with PV or those treated with recombinant EPO (rEPO). PV and other myeloproliferative syndromes have been recently associated with an increased risk for fractures. However, the presence of osteoporosis in these patients has not been well documented. EPO, a hormone primarily known to stimulate erythropoiesis, has been shown recently to regulate bone homeostasis in mice. The aim of this study was to examine the bone phenotype of a mouse model of PV and compare it to that of animals with polycythemia caused by elevated circulating EPO. Bone mass and remodeling were evaluated by micro-computed tomography and histomorphometry. The JAK2(V617F) knock-in mouse, a model of human PV, manifests polycythemia and low circulating EPO levels. Results from this mouse were compared to wild type (wt) controls and the tg6 transgenic mouse that shows polycythemia caused by increased constitutive expression of EPO. Compared to wt, both JAK2(V617F) and tg6 mice had a decrease in trabecular bone mass. Tg6 mice showed an additional modest decrease in cortical thickness and cortical bone volume per tissue volume (P < 0.01) suggesting a more severe bone phenotype than JAK2(V617F). Decreased osteoblast numbers and bone formation along with normal osteoclast numbers and activity were found in both mice. This study indicates that PV is associated with low bone mass and decreased osteoblast activity in mice. Our results support future studies of osteoporosis in affected humans. Polycythemia caused by chronically elevated circulating EPO also results in bone loss, and implications on patients treated with rEPO should be evaluated.

  20. Receptors and effects of gut hormones in three osteoblastic cell lines.

    PubMed

    Pacheco-Pantoja, Elda L; Ranganath, Lakshminarayan R; Gallagher, James A; Wilson, Peter J M; Fraser, William D

    2011-07-29

    In recent years the interest on the relationship of gut hormones to bone processes has increased and represents one of the most interesting aspects in skeletal research. The proportion of bone mass to soft tissue is a relationship that seems to be controlled by delicate and subtle regulations that imply "cross-talks" between the nutrient intake and tissues like fat. Thus, recognition of the mechanisms that integrate a gastrointestinal-fat-bone axis and its application to several aspects of human health is vital for improving treatments related to bone diseases. This work analysed the effects of gut hormones in cell cultures of three osteoblastic cell lines which represent different stages in osteoblastic development. Also, this is the first time that there is a report on the direct effects of glucagon-like peptide 2, and obestatin on osteoblast-like cells. mRNA expression levels of five gut hormone receptors (glucose-dependent insulinotropic peptide [GIP], glucagon-like peptide 1 [GLP-1], glucagon-like peptide 2 [GLP-2], ghrelin [GHR] and obestatin [OB]) were analysed in three osteoblastic cell lines (Saos-2, TE-85 and MG-63) showing different stages of osteoblast development using reverse transcription and real time polymerase chain reaction. The responses to the gut peptides were studied using assays for cell viability, and biochemical bone markers: alkaline phosphatase (ALP), procollagen type 1 amino-terminal propeptides (P1NP), and osteocalcin production. The gut hormone receptor mRNA displayed the highest levels for GIP in Saos-2 and the lowest levels in MG-63, whereas GHR and GPR39 (the putative obestatin receptor) expression was higher in TE-85 and MG-63 and lower in Saos-2. GLP-1 and GLP-2 were expressed only in MG-63 and TE-85. Treatment of gut hormones to cell lines showed differential responses: higher levels in cell viability in Saos-2 after GIP, in TE-85 and MG-63 after GLP-1, GLP-2, ghrelin and obestatin. ALP showed higher levels in Saos-2 after GIP

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

    NASA Technical Reports Server (NTRS)

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

    2003-01-01

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

  2. Bacterium-Induced CXCL10 Secretion by Osteoblasts Can Be Mediated in Part through Toll-Like Receptor 4

    PubMed Central

    Gasper, Nancy A.; Petty, Cynthia C.; Schrum, Laura W.; Marriott, Ian; Bost, Kenneth L.

    2002-01-01

    Two common pathogens known to cause bone infection, Salmonella and Staphylococcus aureus, were investigated to determine their abilities to induce chemokine expression in cultured mouse and human osteoblasts. While these cells are responsible for bone formation, we were surprised to find that they could respond to bacterial infection by upregulating expression of the chemokine CXCL10 (IP-10). However, there were significant differences in the abilities of the gram-negative bacterium Salmonella and the gram-positive bacterium S. aureus to induce expression of CXCL10. Reverse transcription-PCR and enzyme-linked immunosorbent assay analyses showed high levels of Salmonella-induced CXCL10 mRNA and protein expression, respectively, whereas the osteoblast response to S. aureus was significantly less. Consistent with these findings, Salmonella-derived lipopolysaccharide (LPS), but not S. aureus-derived peptidoglycan, could induce expression of CXCL10. An antibody against toll-like receptor 4 (TLR4) could block the LPS-induced CXCL10 production, demonstrating the functional expression of TLR4 by osteoblasts. Despite the inducible nature of TLR2 mRNA expression by bacterium-infected osteoblasts, peptidoglycan failed to stimulate CXCL10 secretion. Immunofluorescent staining of bacterium-infected calvaria (i.e., skull bone) demonstrated the presence of CXCL10 in osteoblasts. The fact that osteoblasts did not express CXCR3 mRNA, whereas T lymphocytes can express high levels of this receptor, suggests that osteoblast-derived CXCL10 may recruit T lymphocytes to the sites of bone infections. PMID:12117914

  3. Optical diagnostics of osteoblast cells and osteogenic drug screening

    NASA Astrophysics Data System (ADS)

    Kolanti, Elayaraja; Veerla, Sarath C.; Khajuria, Deepak K.; Roy Mahapatra, D.

    2016-02-01

    Microfluidic device based diagnostics involving optical fibre path, in situ imaging and spectroscopy are gaining importance due to recent advances in diagnostics instrumentation and methods, besides other factors such as low amount of reagent required for analysis, short investigation times, and potential possibilities to replace animal model based study in near future. It is possible to grow and monitor tissues in vitro in microfluidic lab-on-chip. It may become a transformative way of studying how cells interact with drugs, pathogens and biomaterials in physiologically relevant microenvironments. To a large extent, progress in developing clinically viable solutions has been constrained because of (i) contradiction between in vitro and in vivo results and (ii) animal model based and clinical studies which is very expensive. Our study here aims to evaluate the usefulness of microfluidic device based 3D tissue growth and monitoring approach to better emulate physiologically and clinically relevant microenvironments in comparison to conventional in vitro 2D culture. Moreover, the microfluidic methodology permits precise high-throughput investigations through real-time imaging while using very small amounts of reagents and cells. In the present study, we report on the details of an osteoblast cell based 3D microfluidic platform which we employ for osteogenic drug screening. The drug formulation is functionalized with fluorescence and other biomarkers for imaging and spectroscopy, respectively. Optical fibre coupled paths are used to obtain insight regarding the role of stress/flow pressure fluctuation and nanoparticle-drug concentration on the osteoblast growth and osteogenic properties of bone.

  4. MicroRNA-29a ameliorates glucocorticoid-induced suppression of osteoblast differentiation by regulating β-catenin acetylation.

    PubMed

    Ko, Jih-Yang; Chuang, Pei-Chin; Chen, Ming-Wen; Ke, Huei-Ching; Wu, Shin-Long; Chang, Yu-Hsuan; Chen, Yu-Shan; Wang, Feng-Sheng

    2013-12-01

    Excess glucocorticoid treatment induces loss of osteoblast differentiation. Post-translational modification of β-catenin reportedly regulates osteogenic activities in bone cells. This study was undertaken to test whether miR-29a signaling regulates the acetylation status of β-catenin in the glucocorticoid-mediated osteoblast dysfunction. Murine osteoblast cultures were incubated under osteogenic conditions with or without supraphysiological glucocorticoid, miR-29a precursor, antisense oligonucleotides or histone deacetylase 4 (HDAC4) RNA interferences. Osteoblast differentiation was determined by alkaline phosphatase activity, calcium deposition, and von Kossa stain. β-Catenin acetylation and miR-29a transcription were detected by immunoblotting, chromatin immunoprecipitation and quantitative PCR. Protein interaction was detected by fluorescence protein ligation assay. Supraphysiological glucocorticoid treatment repressed osteoblast differentiation and induced loss of miR-29a expression and acetylated β-catenin levels in osteoblast cultures. Gain of miR-29a function attenuated the deleterious effects of glucocorticoid on osteogenic gene expression and mineralized nodule formation, whereas knockdown of miR-29a signaling accelerated loss of osteoblast differentiation capacity. miR-29a reduced HDAC4 signaling and attenuated the glucocorticoid-mediated β-catenin deacetylation and ubiquitination and restored nuclear β-catenin levels. Glucocorticoid-induced loss of miR-29a signaling occurred through transcriptional and translational regulation. Interruption of HDAC4 signaling attenuated the glucocorticoid-induced hypoacetylation of histone H3 at lysine 9 (H3K9Ac) and restored the enrichment of H3K9Ac in miR-29a proximal promoter region and miR-29a transcription in cell cultures. Taken together, excess glucocorticoid-induced loss of miR-29a signaling accelerates β-catenin deacetylation and ubiquitination that impairs osteogenic activities of osteoblast cultures. mi

  5. Intermittent PTH treatment can delay the transformation of mature osteoblasts into lining cells on the periosteal surfaces.

    PubMed

    Jang, Mi-Gyeong; Lee, Ji Yeon; Yang, Jae-Yeon; Park, Hyojung; Kim, Jung Hee; Kim, Jung-Eun; Shin, Chan Soo; Kim, Seong Yeon; Kim, Sang Wan

    2016-09-01

    Mature osteoblasts have three fates: as osteocytes, quiescent lining cells, or osteoblasts that undergo apoptosis. However, whether intermittent parathyroid hormone (PTH) can modulate the fate of mature osteoblasts in vivo is uncertain. We performed a lineage-tracing study using an inducible gene system. Dmp1-CreERt2 mice were crossed with Rosa26R reporter mice to obtain targeted mature osteoblasts and their descendants, lining cells or osteocytes, which were detected using X-gal staining. Rosa26R:Dmp1-CreERt2(+) mice were injected with 0.25 mg 4-OH-tamoxifen (4-OHTam) on postnatal days 5, 7, 9, 16, and 23. In a previous study, at 22 days after the last 4-OHTam, most LacZ+ cells on the periosteal surface were inactive lining cells. On day 25 (D25), the mice were challenged with an injection of human PTH (1-34, 80 μg/kg) or vehicle daily for 10 (D36) or 20 days (D46). We evaluated the number and thickness of LacZ+ osteoblast descendants in the calvaria and tibia. In the vehicle group, the number and thickness of LacZ+ osteoblast descendants at both D36 and D46 significantly decreased compared to D25, which was attenuated in the PTH group. In line with these results, PTH inhibited the decrease in the number of LacZ+/osteocalcin-positive cells compared to vehicle at both D36 and D46. As well, the serum levels of sclerostin decreased, as did the protein expression of sclerostin in the cortical bone. These results suggest that intermittent PTH treatment can increase the number of periosteal osteoblasts by preventing mature osteoblasts from transforming into lining cells in vivo.

  6. Factors Affecting Sign Retroreflectivity : final report.

    DOT National Transportation Integrated Search

    2001-01-01

    This study was undertaken to better understand the factors that may affect road sign retroreflectivity, specifically age and physical orientation. A better understanding of these factors could provide guidance to ODOT in managing its inventory of roa...

  7. Periostin inhibits mechanical stretch-induced apoptosis in osteoblast-like MG-63 cells.

    PubMed

    Yu, Kai-Wen; Yao, Chung-Chen; Jeng, Jiiang-Huei; Shieh, Hao-Ying; Chen, Yi-Jane

    2018-04-01

    Appropriate mechanical stress plays an important role in regulating the proliferation and differentiation of osteoblasts, whereas high-level mechanical stress may be harmful and compromise cell survival. Periostin, a matricellular protein, is essential in maintaining functional integrity of bone and collagen-rich connective tissue in response to mechanical stress. This study investigated whether or not high-level mechanical stretch induces cell apoptosis and the regulatory role of periostin in mechanical stretch-induced apoptosis in osteoblastic cells. Osteoblast-like MG-63 cells were seeded onto Bio-Flex I culture plates and subjected to cyclic mechanical stretching (15% elongation, 0.1 Hz) in a Flexercell tension plus system-5000. The same process was applied to cells pre-treated with exogenous human recombinant periostin before mechanical stretching. We used a chromatin condensation and membrane permeability dead cell apoptosis kit to evaluate the stretch-induced cell responses. Expression of caspase-3 and cPARP was examined by immunofluorescent stain and flow cytometry. The expression of periostin in MG-63 cells is involved in the TGF-β signaling pathway. High-level cyclic mechanical stretch induced apoptotic responses in MG-63 osteoblastic cells. The percentages of apoptotic cells and cells expressing cPARP protein increased in the groups of cells subjected to mechanical stretch, but these responses were absent in the presence of exogenous periostin. Our study revealed that high-level mechanical stretch induces apoptotic cell death, and that periostin plays a protective role against mechanical stretch-induced apoptosis in osteoblastic cells. Copyright © 2017. Published by Elsevier B.V.

  8. Cuscuta chinensis extract promotes osteoblast differentiation and mineralization in human osteoblast-like MG-63 cells.

    PubMed

    Yang, Hyun Mo; Shin, Hyun-Kyung; Kang, Young-Hee; Kim, Jin-Kyung

    2009-02-01

    The aim of the present study was to investigate whether the aqueous extract of To-Sa-Za (TSZ-AE), the seed of Cuscuta chinensis Lam., which is a traditional medicinal herb commonly used in Korea and other oriental countries, could induce osteogenic activity in human osteoblast-like MG-63 cells. TSZ-AE treatment mildly promoted the proliferation of MG-63 cells at doses of 500 and 1,000 microg/mL in the 24-hour culture period. Dose-dependent increases in alkaline phosphatase (ALP) activity and collagen synthesis were shown at 48 and 72 hours of incubation. The release of bone morphogenetic protein (BMP)-2 but not osteocalcin in the MG-63 cells was induced by TSZ-AE at 72 hours (100-1,000 microg/mL). In addition, TSZ-AE markedly increased mRNA expression of ALP, collagen, and BMP-2 in the MG-63 cells in a dose-dependent manner. Mineralization in the culture of MG-63 cells was significantly induced at 500 and 1,000 microg/mL TSZ-AE treatment. In conclusion, this study shows that TSZ-AE enhanced ALP activity, collagen synthesis, BMP-2 expression, and mineralization in MG-63 cells. These results strongly suggest that C. chinensis can play an important role in osteoblastic bone formation and may possibly lead to the development of bone-forming drugs.

  9. Retinoic acid receptor signalling directly regulates osteoblast and adipocyte differentiation from mesenchymal progenitor cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Green, A.C.; Department of Medicine at St. Vincent's Hospital, The University of Melbourne, Victoria 3065; Kocovski, P.

    Low and high serum retinol levels are associated with increased fracture risk and poor bone health. We recently showed retinoic acid receptors (RARs) are negative regulators of osteoclastogenesis. Here we show RARs are also negative regulators of osteoblast and adipocyte differentiation. The pan-RAR agonist, all-trans retinoic acid (ATRA), directly inhibited differentiation and mineralisation of early osteoprogenitors and impaired the differentiation of more mature osteoblast populations. In contrast, the pan-RAR antagonist, IRX4310, accelerated differentiation of early osteoprogenitors. These effects predominantly occurred via RARγ and were further enhanced by an RARα agonist or antagonist, respectively. RAR agonists similarly impaired adipogenesis in osteogenicmore » cultures. RAR agonist treatment resulted in significant upregulation of the Wnt antagonist, Sfrp4. This accompanied reduced nuclear and cytosolic β-catenin protein and reduced expression of the Wnt target gene Axin2, suggesting impaired Wnt/β-catenin signalling. To determine the effect of RAR inhibition in post-natal mice, IRX4310 was administered to male mice for 10 days and bones were assessed by µCT. No change to trabecular bone volume was observed, however, radial bone growth was impaired. These studies show RARs directly influence osteoblast and adipocyte formation from mesenchymal cells, and inhibition of RAR signalling in vivo impairs radial bone growth in post-natal mice. - Graphical abstract: Schematic shows RAR ligand regulation of osteoblast differentiation in vitro. RARγ antagonists±RARα antagonists promote osteoblast differentiation. RARγ and RARα agonists alone or in combination block osteoblast differentiation, which correlates with upregulation of Sfrp4, and downregulation of nuclear and cytosolic β-catenin and reduced expression of the Wnt target gene Axin2. Red arrows indicate effects of RAR agonists on mediators of Wnt signalling.« less

  10. TGF-β and BMP Signaling in Osteoblast Differentiation and Bone Formation

    PubMed Central

    Chen, Guiqian; Deng, Chuxia; Li, Yi-Ping

    2012-01-01

    Transforming growth factor-beta (TGF-β)/bone morphogenic protein (BMP) signaling is involved in a vast majority of cellular processes and is fundamentally important throughout life. TGF-β/BMPs have widely recognized roles in bone formation during mammalian development and exhibit versatile regulatory functions in the body. Signaling transduction by TGF-β/BMPs is specifically through both canonical Smad-dependent pathways (TGF-β/BMP ligands, receptors and Smads) and non-canonical Smad-independent signaling pathway (e.g. p38 mitogen-activated protein kinase pathway, MAPK). Following TGF-β/BMP induction, both the Smad and p38 MAPK pathways converge at the Runx2 gene to control mesenchymal precursor cell differentiation. The coordinated activity of Runx2 and TGF-β/BMP-activated Smads is critical for formation of the skeleton. Recent advances in molecular and genetic studies using gene targeting in mice enable a better understanding of TGF-β/BMP signaling in bone and in the signaling networks underlying osteoblast differentiation and bone formation. This review summarizes the recent advances in our understanding of TGF-β/BMP signaling in bone from studies of genetic mouse models and human diseases caused by the disruption of TGF-β/BMP signaling. This review also highlights the different modes of cross-talk between TGF-β/BMP signaling and the signaling pathways of MAPK, Wnt, Hedgehog, Notch, and FGF in osteoblast differentiation and bone formation. PMID:22298955

  11. Large gradient high magnetic field affects the association of MACF1 with actin and microtubule cytoskeleton.

    PubMed

    Qian, Ai-Rong; Hu, Li-Fang; Gao, Xiang; Zhang, Wei; Di, Sheng-Meng; Tian, Zong-Cheng; Yang, Peng-Fei; Yin, Da-Chuan; Weng, Yuan-Yuan; Shang, Peng

    2009-10-01

    The intense inhomogeneous magnetic fields acting on the diamagnetic materials naturally present in cells can generate strong magnetic forces. We have developed a superconducting magnet platform with large gradient high magnetic field (LG-HMF), which can produce three magnetic force fields of -1360, 0, and 1312 T(2)/m, and three corresponding apparent gravity levels, namely 0, 1, and 2-g for diamagnetic materials. In this study, the effects of different magnetic force fields on osteoblast-like cells (MG-63 and MC3T3-E1) viability, microtubule actin crosslinking factor 1 (MACF1) expression and its association with cytoskeleton were investigated. Results showed that cell viability increased to different degrees after exposure to 0 or 1-g conditions for 24 h, but it decreased by about 30% under 2-g conditions compared with control conditions. An increase in MACF1 expression at the RNA or protein level was observed in osteoblast-like cells under the magnetic force field of -1360 T(2)/m (0-g) relative to 1312 T(2)/m (2-g). Under control conditions, anti-MACF1 staining was scattered in the cytoplasm and partially colocalized with actin filaments (AFs) or microtubules (MTs) in the majority of osteoblast-like cells. Under 0-g conditions, MACF1 labeling was concentrated at perinuclear region and colocalization was not apparent. The patterns of anti-MACF1 labeling on MTs varied with MTs' changing under LG-HMF environment. In conclusion, LG-HMF affects osteoblast-like cell viability, MACF1 distribution, expression, and its association with cytoskeleton to some extent.

  12. Proliferation and osteoblastic differentiation of human bone marrow-derived stromal cells on akermanite-bioactive ceramics.

    PubMed

    Sun, Hongli; Wu, Chengtie; Dai, Kerong; Chang, Jiang; Tang, Tingting

    2006-11-01

    In the present study, the effects of a calcium magnesium silicate bioactive ceramic (akermanite) on proliferation and osteoblastic differentiation of human bone marrow stromal cells (hBMSC) have been investigated and compared with the classical ceramic (beta-tricalcium phosphate, beta-TCP). Akermanite and beta-TCP disks were seeded with hBMSC and kept in growth medium or osteogenic medium for 10 days. Proliferation and osteoblastic differentiation were evaluated on day 1, 4, 7 and 10. The data from the Alamar Blue assay and lactic acid production assay showed that hBMSC proliferated more significantly on akermanite than on beta-TCP. The analysis of osteoblast-related genes, including alkaline phosphatase (ALP), osteopontin (OPN), bone sialoprotein (BSP) and osteocalcin (OC), indicated that akermanite ceramics enhanced the expression of osteoblast-related genes, but type I collagen (COL I) showed no noticeable difference among akermanite and beta-TCP ceramics. Furthermore, this stimulatory effect was observed not only in osteogenic medium, but also in normal growth medium without osteogenic reagents such as l-ascorbic acid, glycerophosphate and dexamethasone. This result suggests that akermanite can promote osteoblastic differentiation of hBMSC in vitro even without osteogenic reagents, and may be used as a bioactive material for bone regeneration and tissue engineering applications.

  13. 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. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Differences in responses to X-ray exposure between osteoclast and osteoblast cells

    PubMed Central

    Zhang, Jian; Wang, Ziyang; Wu, Anqing; Nie, Jing; Pei, Hailong; Hu, Wentao; Wang, Bing; Shang, Peng; Li, Bingyan

    2017-01-01

    Abstract Radiation-induced bone loss is a potential health concern for cancer patients undergoing radiotherapy. Enhanced bone resorption by osteoclasts and decreased bone formation by osteoblasts were thought to be the main reasons. In this study, we showed that both pre-differentiating and differentiating osteoclasts were relatively sensitive to X-rays compared with osteoblasts. X-rays decreased cell viability to a greater degree in RAW264.7 cells and in differentiating cells than than in osteoblastic MC3T3-E1 cells. X-rays at up to 8 Gy had little effects on osteoblast mineralization. In contrast, X-rays at 1 Gy induced enhanced osteoclastogenesis by enhanced cell fusion, but had no effects on bone resorption. A higher dose of X-rays at 8 Gy, however, had an inhibitory effect on bone resorption. In addition, actin ring formation was disrupted by 8 Gy of X-rays and reorganized into clusters. An increased activity of Caspase 3 was found after X-ray exposure. Actin disorganization and increased apoptosis may be the potential effects of X-rays at high doses, by inhibiting osteoclast differentiation. Taken together, our data indicate high radiosensitivity of osteoclasts. X-ray irradiation at relatively low doses can activate osteoclastogenesis, but not osteogenic differentiation. The radiosensitive osteoclasts are the potentially responsive cells for X-ray-induced bone loss. PMID:28541506

  15. Parathyroid hormone modulates the response of osteoblast-like cells to mechanical stimulation

    NASA Technical Reports Server (NTRS)

    Ryder, K. D.; Duncan, R. L.

    2000-01-01

    Mechanical loading stimulates many responses in bone and osteoblasts associated with osteogenesis. Since loading and parathyroid hormone (PTH) activate similar signaling pathways in osteoblasts, we postulate that PTH can potentiate the effects of mechanical stimulation. Using an in vitro four-point bending device, we found that expression of COX-2, the inducible isoform of cyclooxygenase, was dependent on fluid forces generated across the culture plate, but not physiologic levels of strain in MC3T3-E1 osteoblast-like cells. Addition of 50 nM PTH during loading increased COX-2 expression at both subthreshold and threshold levels of fluid forces compared with either stimuli alone. We also demonstrated that application of fluid shear to MC3T3-E1 cells induced a rapid increase in [Ca(2+)](i). Although PTH did not significantly change [Ca(2+)](i) levels, flow and PTH did produce a significantly greater [Ca(2+)](i) response and increased the number of responding cells than is found in fluid shear alone. The [Ca(2+)](i) response to these stimuli was significantly decreased when the mechanosensitive channel inhibitor, gadolinium, was present. These studies indicate that PTH increases the cellular responses of osteoblasts to mechanical loading. Furthermore, this response may be mediated by alterations in [Ca(2+)](i) by modulating the mechanosensitive channel.

  16. Taurine suppresses osteoblastic differentiation of aortic valve interstitial cells induced by beta-glycerophosphate disodium, dexamethasone and ascorbic acid via the ERK pathway.

    PubMed

    Feng, Xiang; Li, Jian-ming; Liao, Xiao-bo; Hu, Ye-rong; Shang, Bao-peng; Zhang, Zhi-yuan; Yuan, Ling-qing; Xie, Hui; Sheng, Zhi-feng; Tang, Hao; Zhang, Wei; Gu, Lu; Zhou, Xin-min

    2012-10-01

    Aortic valve calcification (AVC) is an active process characterized by osteoblastic differentiation of the aortic valve interstitial cells (AVICs). Taurine is a free β-amino acid and plays important physiological roles including protective effect of cardiovascular events. To evaluate the possible role of taurine in AVC, we isolated human AVICs from patients with type A dissection without leaflet disease. We demonstrated that the cultured AVICs express SM α-actin, vimentin and taurine transporter (TAUT), but not CD31, SM-myosin or desmin. We also established the osteoblastic differentiation model of the AVICs induced by pro-calcific medium (PCM) containing β-glycerophosphate disodium, dexamethasone and ascorbic acid in vitro. The results showed that taurine attenuated the PCM-induced osteoblastic differentiation of AVICs by decreasing the alkaline phosphate (ALP) activity/expression and the expression of the core binding factor α1 (Cbfα1) in a dose-dependent manner (reaching the maximum protective effect at 10 mM), and taurine (10 mM) inhibited the mineralization level of AVICs in the form of calcium content significantly. Furthermore, taurine activated the extracellular signal-regulated protein kinase (ERK) pathway via TAUT, and the inhibitor of ERK (PD98059) abolished the effect of taurine on both ALP activity/expression and Cbfα1 expression. These results suggested that taurine could inhibit osteoblastic differentiation of AVIC via the ERK pathway.

  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. Copyright 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2010.

  18. Wnt-Lrp5 Signaling Regulates Fatty Acid Metabolism in the Osteoblast

    PubMed Central

    Frey, Julie L.; Li, Zhu; Ellis, Jessica M.; Zhang, Qian; Farber, Charles R.; Aja, Susan; Wolfgang, Michael J.; Clemens, Thomas L.

    2015-01-01

    The Wnt coreceptors Lrp5 and Lrp6 are essential for normal postnatal bone accrual and osteoblast function. In this study, we identify a previously unrecognized skeletal function unique to Lrp5 that enables osteoblasts to oxidize fatty acids. Mice lacking the Lrp5 coreceptor specifically in osteoblasts and osteocytes exhibit the expected reductions in postnatal bone mass but also exhibit an increase in body fat with corresponding reductions in energy expenditure. Conversely, mice expressing a high bone mass mutant Lrp5 allele are leaner with reduced plasma triglyceride and free fatty acid levels. In this context, Wnt-initiated signals downstream of Lrp5, but not the closely related Lrp6 coreceptor, regulate the activation of β-catenin and thereby induce the expression of key enzymes required for fatty acid β-oxidation. These results suggest that Wnt-Lrp5 signaling regulates basic cellular activities beyond those associated with fate specification and differentiation in bone and that the skeleton influences global energy homeostasis via mechanisms independent of osteocalcin and glucose metabolism. PMID:25802278

  19. In vitro osteoblastic differentiation of human bone marrow cells in the presence of metal ions.

    PubMed

    Morais, S; Dias, N; Sousa, J P; Fernandes, M H; Carvalho, G S

    1999-02-01

    For periods up to 21 days human bone marrow was cultured in control conditions that favor the proliferation and differentiation of osteoblastic cells. The effect of AISI 316L corrosion products and the corresponding major separate metal ions (Fe, Cr, and Ni) were studied in three different phases of the culture period in order to investigate the effects of metal ions in cell populations representative of osteoblastic cells in different stages of differentiation. Toxicity consequences of the presence of metal ions in bone marrow cultures were evaluated by biochemical parameters (enzymatic reduction of MTT, alkaline phosphatase activity, and total protein content), histochemical assays (identification of ALP-positive cells and Ca and phosphates deposits), and observation of the cultures by light and scanning electron microscopy. Culture media were analyzed for total and ionized Ca and P and also for metal ions (Fe, Cr, and Ni). The presence of AISI 316L corrosion products and Ni salt in bone marrow cultures during the first and second weeks of culture significantly disturbs the normal behavior of these cultures, interfering in the lag phase and exponential phase of cell growth and ALP expression. However, the presence of these species during the third week of culture, when expression of osteoblastic functions occurs (mineralization process), did not result in any detectable effect. Fe salt also disturbs the behavior of bone marrow cell cultures when present during the lag phase and proliferation phase, and a somewhat compromised response between the normal pattern (control cultures) and intense inhibition (AISI 316L corrosion products and Ni salt-added cultures) was observed. Fe did not affect the progression of the mineralization phase. Osteogenic cultures exposed to Cr salt (Cr3+) presented a pattern similar to the controls, indicating that this element does not interfere, in the concentration studied, in the osteoblastic differentiation of bone marrow cells

  20. Integrin expression by human osteoblasts cultured on degradable polymeric materials applicable for tissue engineered bone.

    PubMed

    El-Amin, Saadiq F; Attawia, Mohamed; Lu, Helen H; Shah, Asist K; Chang, Richard; Hickok, Noreen J; Tuan, Rocky S; Laurencin, Cato T

    2002-01-01

    The use of biodegradable polymers in the field of orthopaedic surgery has gained increased popularity, as surgical pins and screws, and as potential biological scaffolds for repairing cartilage and bone defects. One such group of polymers that has gained considerable attention are the polyesters, poly(lactide-co-glycolide) (PLAGA) and polylactic acid (PLA), because of their minimal tissue inflammatory response, favorable biocompatibility and degradation characteristics. The objective of this study was to evaluate human osteoblastic cell adherence and growth on PLAGA and PLA scaffolds by examining integrin receptor (alpha2, alpha3, alpha4, alpha5, alpha6 and beta1) expression. Primary human osteoblastic cells isolated from trabecular bone adhered efficiently to both PLAGA and PLA, with the rate of adherence on PLAGA comparable to that of control tissue culture polystyrene (TCPS), and significantly higher than on PLA polymers at 3, 6 and 12 h. Human osteoblastic phenotypic expression, alkaline phosphatase (ALP) activity was positive on both degradable matrices, whereas osteocalcin levels were significantly higher on cells grown on PLAGA than on PLA composites. Interestingly, the integrin subunits, alpha2, alpha3, alpha4, alpha5, alpha6 and beta1 were all expressed at higher levels by osteoblasts cultured on PLAGA than those on PLA as analyzed by westerns blots and by flow cytometry. Among the integrins, alpha2, beta5 and beta1 showed the greatest difference in levels between the two surfaces. Thus, both PLA and PLAGA support osteoblastic adhesion and its accompanying engagement of integrin receptor and expression of osteocalcin and ALP. However PLAGA consistently appeared to be a better substrate for osteoblastic cells based on these parameters. This study is one of the first to investigate the ability of primary human osteoblastic cells isolated from trabecular bone to adhere to the biodegradable polymers PLAGA and PLA, and to examine the expression of their key

  1. CCAAT/enhancer-binding protein delta is a critical regulator of insulin-like growth factor-I gene transcription in osteoblasts

    NASA Technical Reports Server (NTRS)

    Umayahara, Y.; Billiard, J.; Ji, C.; Centrella, M.; McCarthy, T. L.; Rotwein, P.

    1999-01-01

    Insulin-like growth factor-I (IGF-I) plays a major role in promoting skeletal growth by stimulating bone cell replication and differentiation. Prostaglandin E2 and other agents that induce cAMP production enhance IGF-I gene transcription in cultured rat osteoblasts through a DNA element termed HS3D, located in the proximal part of the major rat IGF-I promoter. We previously determined that CCAAT/enhancer-binding protein delta (C/EBPdelta) is the key cAMP-stimulated regulator of IGF-I transcription in these cells and showed that it transactivates the rat IGF-I promoter through the HS3D site. We now have defined the physical-chemical properties and functional consequences of the interactions between C/EBPdelta and HS3D. C/EBPdelta, expressed in COS-7 cells or purified as a recombinant protein from Escherichia coli, bound to HS3D with an affinity at least equivalent to that of the albumin D-site, a known high affinity C/EBP binding sequence, and both DNA elements competed equally for C/EBPdelta. C/EBPdelta bound to HS3D as a dimer, with protein-DNA contact points located on guanine residues on both DNA strands within and just adjacent to the core C/EBP half-site, GCAAT, as determined by methylation interference footprinting. C/EBPdelta also formed protein-protein dimers in the absence of interactions with its DNA binding site, as indicated by results of glutaraldehyde cross-linking studies. As established by competition gel-mobility shift experiments, the conserved HS3D sequence from rat, human, and chicken also bound C/EBPdelta with similar affinity. We also found that prostaglandin E2-induced expression of reporter genes containing human IGF-I promoter 1 or four tandem copies of the human HS3D element fused to a minimal promoter and show that these effects were enhanced by a co-transfected C/EBPdelta expression plasmid. Taken together, our results provide evidence that C/EBPdelta is a critical activator of IGF-I gene transcription in osteoblasts and potentially in

  2. Nano-scale surface morphology, wettability and osteoblast adhesion on nitrogen plasma-implanted NiTi shape memory alloy.

    PubMed

    Liu, X M; Wu, S L; Chu, Paul K; Chung, C Y; Chu, C L; Chan, Y L; Lam, K O; Yeung, K W K; Lu, W W; Cheung, K M C; Luk, K D K

    2009-06-01

    Plasma immersion ion implantation (PIII) is an effective method to increase the corrosion resistance and inhibit nickel release from orthopedic NiTi shape memory alloy. Nitrogen was plasma-implanted into NiTi using different pulsing frequencies to investigate the effects on the nano-scale surface morphology, structure, wettability, as well as biocompatibility. X-ray photoelectron spectroscopy (XPS) results show that the implantation depth of nitrogen increases with higher pulsing frequencies. Atomic force microscopy (AFM) discloses that the nano-scale surface roughness increases and surface features are changed from islands to spiky cones with higher pulsing frequencies. This variation in the nano surface structures leads to different surface free energy (SFE) monitored by contact angle measurements. The adhesion, spreading, and proliferation of osteoblasts on the implanted NiTi surface are assessed by cell culture tests. Our results indicate that the nano-scale surface morphology that is altered by the implantation frequencies impacts the surface free energy and wettability of the NiTi surfaces, and in turn affects the osteoblast adhesion behavior.

  3. Inhibition of osteoblastogenesis and promotion of apoptosis of osteoblasts and osteocytes by glucocorticoids. Potential mechanisms of their deleterious effects on bone.

    PubMed Central

    Weinstein, R S; Jilka, R L; Parfitt, A M; Manolagas, S C

    1998-01-01

    Glucocorticoid-induced bone disease is characterized by decreased bone formation and in situ death of isolated segments of bone (osteonecrosis) suggesting that glucocorticoid excess, the third most common cause of osteoporosis, may affect the birth or death rate of bone cells, thus reducing their numbers. To test this hypothesis, we administered prednisolone to 7-mo-old mice for 27 d and found decreased bone density, serum osteocalcin, and cancellous bone area along with trabecular narrowing. These changes were accompanied by diminished bone formation and turnover, as determined by histomorphometric analysis of tetracycline-labeled vertebrae, and impaired osteoblastogenesis and osteoclastogenesis, as determined by ex vivo bone marrow cell cultures. In addition, the mice exhibited a threefold increase in osteoblast apoptosis in vertebrae and showed apoptosis in 28% of the osteocytes in metaphyseal cortical bone. As in mice, an increase in osteoblast and osteocyte apoptosis was documented in patients with glucocorticoid-induced osteoporosis. Decreased production of osteoclasts explains the reduction in bone turnover, whereas decreased production and apoptosis of osteoblasts would account for the decline in bone formation and trabecular width. Furthermore, accumulation of apoptotic osteocytes may contribute to osteonecrosis. These findings provide evidence that glucocorticoid-induced bone disease arises from changes in the numbers of bone cells. PMID:9664068

  4. Effect of autogenous and fresh-frozen bone grafts on osteoblast differentiation.

    PubMed

    Ferraz, E P; Xavier, S P; Azevedo, F G; de Oliveira, F S; Beloti, M M; Rosa, A L

    2015-01-01

    Fresh-frozen bone allograft (FFBA) is an alternative to autogenous bone (AB) for reconstructing maxillary bone. Despite the promising clinical results, cell responses to FFBA and AB were not evaluated. Thus, our aim was to compare cells harvested from maxillary reconstructed sites with either AB or FFBA in terms of osteoblast differentiation and to evaluate the effect of culturing cells in contact with FFBA. Cells harvested from three patients submitted to bilateral maxillary reconstruction with AB and FFBA were cultured to evaluate: proliferation, alkaline phosphatase activity, extracellular matrix mineralization and gene expression of osteoblastic markers. The effect of FFBA on osteoblast differentiation was studied by culturing cells harvested from AB in contact with FFBA and evaluating the same parameters. Data were compared using either two-way ANOVA followed by Tukey-b test or Student's t test (p≤0.05). Cell proliferation was higher in cultures from AB grafted sites and extracellular matrix mineralization was higher in cultures derived from FFBA grafted sites. The gene expression of alkaline phosphatase, RUNX2, bone sialoprotein and osteocalcin was higher in cells derived from FFBA compared with cells from AB grafted sites. However, the exposure of cells derived from AB to FFBA particles did not have any remarkable effect on osteoblast differentiation. These results indicate the higher osteogenic activity of cells derived from FFBA compared with AB reconstructed sites, offering an explanation at cellular level of why FFBA could be a suitable alternative to AB for reconstructing maxillary bone defects. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Micro-/Nano- sized hydroxyapatite directs differentiation of rat bone marrow derived mesenchymal stem cells towards an osteoblast lineage

    NASA Astrophysics Data System (ADS)

    Huang, Yan; Zhou, Gang; Zheng, Lisha; Liu, Haifeng; Niu, Xufeng; Fan, Yubo

    2012-03-01

    Regenerative medicine consisting of cells and materials provides a new way for the repair and regeneration of tissues and organs. Nano-biomaterials are highlighted due to their advantageous features compared with conventional micro-materials. The aim of this study is to investigate the effects of micro-/nano- sized hydroxyapatite (μ/n-HA) on the osteogenic differentiation of rat bone marrow derived mesenchymal stem cells (rBMSCs). μ/n-HA were prepared by a microwave synthesizer and precipitation method, respectively. Different sizes of μ/n-HA were characterized by IR, XRD, SEM, TEM and co-cultured with rBMSCs. It was shown that rBMSCs expressed higher levels of osteoblast-related markers by n-HA than μ-HA stimulation. The size of HA is an important factor for affecting the osteogenic differentiation of rBMSCs. This provides a new avenue for mechanistic studies of stem cell differentiation and a new approach to obtain more committed differentiated cells.

  6. Human immunodeficiency virus type 1 enhancer-binding protein 3 is essential for the expression of asparagine-linked glycosylation 2 in the regulation of osteoblast and chondrocyte differentiation.

    PubMed

    Imamura, Katsuyuki; Maeda, Shingo; Kawamura, Ichiro; Matsuyama, Kanehiro; Shinohara, Naohiro; Yahiro, Yuhei; Nagano, Satoshi; Setoguchi, Takao; Yokouchi, Masahiro; Ishidou, Yasuhiro; Komiya, Setsuro

    2014-04-04

    Human immunodeficiency virus type 1 enhancer-binding protein 3 (Hivep3) suppresses osteoblast differentiation by inducing proteasomal degradation of the osteogenesis master regulator Runx2. In this study, we tested the possibility of cooperation of Hivep1, Hivep2, and Hivep3 in osteoblast and/or chondrocyte differentiation. Microarray analyses with ST-2 bone stroma cells demonstrated that expression of any known osteochondrogenesis-related genes was not commonly affected by the three Hivep siRNAs. Only Hivep3 siRNA promoted osteoblast differentiation in ST-2 cells, whereas all three siRNAs cooperatively suppressed differentiation in ATDC5 chondrocytes. We further used microarray analysis to identify genes commonly down-regulated in both MC3T3-E1 osteoblasts and ST-2 cells upon knockdown of Hivep3 and identified asparagine-linked glycosylation 2 (Alg2), which encodes a mannosyltransferase residing on the endoplasmic reticulum. The Hivep3 siRNA-mediated promotion of osteoblast differentiation was negated by forced Alg2 expression. Alg2 suppressed osteoblast differentiation and bone formation in cultured calvarial bone. Alg2 was immunoprecipitated with Runx2, whereas the combined transfection of Runx2 and Alg2 interfered with Runx2 nuclear localization, which resulted in suppression of Runx2 activity. Chondrocyte differentiation was promoted by Hivep3 overexpression, in concert with increased expression of Creb3l2, whose gene product is the endoplasmic reticulum stress transducer crucial for chondrogenesis. Alg2 silencing suppressed Creb3l2 expression and chondrogenesis of ATDC5 cells, whereas infection of Alg2-expressing virus promoted chondrocyte maturation in cultured cartilage rudiments. Thus, Alg2, as a downstream mediator of Hivep3, suppresses osteogenesis, whereas it promotes chondrogenesis. To our knowledge, this study is the first to link a mannosyltransferase gene to osteochondrogenesis.

  7. MiR-142-5p promotes bone repair by maintaining osteoblast activity.

    PubMed

    Tu, Manli; Tang, Juanjuan; He, Hongbo; Cheng, Peng; Chen, Chao

    2017-05-01

    MicroRNAs play important roles in regulating bone regeneration and remodeling. However, the pathophysiological roles of microRNAs in bone repair remain unclear. Here we identify a significant upregulation of miR-142-5p correlated with active osteoblastogenesis during the bone healing process. In vitro, miR-142-5p promoted osteoblast activity and matrix mineralization by targeting the gene encoding WW-domain-containing E3 ubiquitin protein ligase 1. We also found that the expression of miR-142-5p in the callus of aged mice was lower than that in the callus of young mice and directly correlated with the age-related delay in bone healing. Furthermore, treatment with agomir-142-5p in the fracture areas stimulated osteoblast activity which repaired the bone fractures in aged mice. Thus, our study revealed that miR-142-5p plays a crucial role in healing fractures by maintaining osteoblast activity, and provided a new molecular target therapeutic strategy for bone healing.

  8. Expression of extracellular calcium-sensing receptor in human osteoblastic MG-63 cell line

    NASA Technical Reports Server (NTRS)

    Yamaguchi, T.; Chattopadhyay, N.; Kifor, O.; Ye, C.; Vassilev, P. M.; Sanders, J. L.; Brown, E. M.

    2001-01-01

    We have previously shown the expression of the extracellular calcium (Ca2+o)-sensing receptor (CaR) in osteoblast-like cell lines, and others have documented its expression in sections of murine, bovine, and rat bone. The existence of the CaR in osteoblasts remains controversial, however, since some studies have failed to document its expression in the same osteoblast-like cell lines. The goals of the present study were twofold. 1) We sought to determine whether the CaR is expressed in the human osteoblast-like cell line, MG-63, which has recently been reported by others not to express this receptor. 2) We investigated whether the CaR, if present in MG-63 cells, is functionally active, since most previous studies have not proven the role of the CaR in mediating known actions of Ca2+o on osteoblast-like cells. We used immunocytochemistry and Western blotting with the specific, affinity-purified anti-CaR antiserum 4637 as well as Northern blot analysis and RT-PCR using a riboprobe and PCR primers specific for the human CaR, respectively, to show readily detectable CaR protein and mRNA expression in MG-63 cells. Finally, we employed the patch-clamp technique to show that an elevation in Ca2+o as well as the specific, allosteric CaR activator NPS R-467 (0.5 microM), but not its less active stereoisomer NPS S-467 (0.5 microM), activate an outward K+ channel in MG-63 cells, strongly suggesting that the CaR in MG-63 cells is not only expressed but is functionally active.

  9. Zirconium Ions Up-Regulate the BMP/SMAD Signaling Pathway and Promote the Proliferation and Differentiation of Human Osteoblasts

    PubMed Central

    Chen, Yongjuan; Roohani-Esfahani, Seyed-Iman; Lu, ZuFu; Zreiqat, Hala; Dunstan, Colin R.

    2015-01-01

    Zirconium (Zr) is an element commonly used in dental and orthopedic implants either as zirconia (ZrO2) or in metal alloys. It can also be incorporated into calcium silicate-based ceramics. However, the effects of in vitro culture of human osteoblasts (HOBs) with soluble ionic forms of Zr have not been determined. In this study, primary culture of human osteoblasts was conducted in the presence of medium containing either ZrCl4 or Zirconium (IV) oxynitrate (ZrO(NO3)2) at concentrations of 0, 5, 50 and 500 µM, and osteoblast proliferation, differentiation and calcium deposition were assessed. Incubation of human osteoblast cultures with Zr ions increased the proliferation of human osteoblasts and also gene expression of genetic markers of osteoblast differentiation. In 21 and 28 day cultures, Zr ions at concentrations of 50 and 500 µM increased the deposition of calcium phosphate. In addition, the gene expression of BMP2 and BMP receptors was increased in response to culture with Zr ions and this was associated with increased phosphorylation of SMAD1/5. Moreover, Noggin suppressed osteogenic gene expression in HOBs co-treated with Zr ions. In conclusion, Zr ions appear able to induce both the proliferation and the differentiation of primary human osteoblasts. This is associated with up-regulation of BMP2 expression and activation of BMP signaling suggesting this action is, at least in part, mediated by BMP signaling. PMID:25602473

  10. Osteoblast-secreted WISP-1 promotes adherence of prostate cancer cells to bone via the VCAM-1/integrin α4β1 system.

    PubMed

    Chang, An-Chen; Chen, Po-Chun; Lin, Yu-Feng; Su, Chen-Ming; Liu, Ju-Fang; Lin, Tien-Huang; Chuang, Show-Mei; Tang, Chih-Hsin

    2018-07-10

    Bone metastasis is a frequent occurrence in prostate cancer (PCa) that is associated with severe complications such as fracture, bone pain and hypercalcemia. The cross-talk between metastatic cancer cells and bone is critical to the development and progression of bone metastases. In our previous data, we have described how the involvement of the Wnt-induced secreted protein-1/vascular cell adhesion molecule-1 (WISP-1/VCAM-1) system in this tumor-bone interaction contributes to human PCa cell motility. In this study, we found that WISP-1 regulates bone mineralization by inducing bone morphogenetic protein-2 (BMP2), BMP4 and osteopontin (OPN) expression in osteoblasts. We also found that WISP-1 inhibited RANKL-dependent osteoclastogenesis. Moreover, osteoblast-derived WISP-1 enhanced VCAM-1 expression in PCa cells and subsequently promoted the adherence of cancer cells to osteoblasts. Furthermore, endothelin-1 (ET-1) expression in PCa cells was regulated by osteoblast-derived WISP-1, which promoted integrin α4β1 expression in osteoblasts via the MAPK pathway. Pretreatment of PCa cells with VCAM-1 antibody or osteoblasts with integrin α4β1 antibody attenuated the adherence of PCa cells to osteoblasts, suggesting that integrin α4β1 serves as a ligand that captures VCAM-1 + metastatic tumor cells adhering to osteoblasts. Our findings reveal that osteoblast-derived WISP-1 plays a key role in regulating the adhesion of PCa cells to osteoblasts via the VCAM-1/integrin α4β1 system. Osteoblast-derived WISP-1 is a promising target for the prevention and inhibition of PCa-bone interaction. Copyright © 2018 Elsevier B.V. All rights reserved.

  11. Bone morphogenetic protein Smads signaling in mesenchymal stem cells affected by osteoinductive calcium phosphate ceramics.

    PubMed

    Tang, Zhurong; Wang, Zhe; Qing, Fangzhu; Ni, Yilu; Fan, Yujiang; Tan, Yanfei; Zhang, Xingdong

    2015-03-01

    Porous calcium phosphate ceramics (CaP ceramics) could induce ectopic bone formation which was regulated by various signal molecules. In this work, bone marrow mesenchymal stem cells (MSCs) were cultured on the surface of osteoinductive hydroxyapatite (HA) and biphasic calcium phosphate (BCP) ceramics in comparison with control (culture plate) for up to 14 days to detect the signal molecules which might be affected by the CaP ceramics. Without adding osteogenic factors, MSCs cultured on HA and BCP both expressed higher Runx2, Osterix, collagen type I, osteopontin, bone sialoprotein, and osteocalcin at various stages compared with control, thus confirmed the osteoblastic differentiation of MSCs. Later study demonstrated the messenger RNA level of bone morphogenetic protein 2 (BMP2) and BMP4 were also significantly enhanced by HA and BCP. Furthermore, Smad1, 4, 5, and Dlx5, the main molecules in the BMP/Smads signaling pathway, were upregulated by HA and BCP. Moreover, the higher expression of Smads and BMP2, 4 in BCP over HA, corresponded to the better performance of BCP in stimulating in vitro osteoblastic differentiation of MSCs. This was in accordance with the better osteoinductivity of BCP over HA in vivo. Altogether, these results implied that the CaP ceramics may initiate the osteoblastic differentiation of MSCs by influencing the expression of molecules in BMP/Smads pathway. © 2014 Wiley Periodicals, Inc.

  12. Distinct requirements for cranial ectoderm and mesenchyme-derived wnts in specification and differentiation of osteoblast and dermal progenitors.

    PubMed

    Goodnough, L Henry; Dinuoscio, Gregg J; Ferguson, James W; Williams, Trevor; Lang, Richard A; Atit, Radhika P

    2014-02-01

    The cranial bones and dermis differentiate from mesenchyme beneath the surface ectoderm. Fate selection in cranial mesenchyme requires the canonical Wnt effector molecule β-catenin, but the relative contribution of Wnt ligand sources in this process remains unknown. Here we show Wnt ligands are expressed in cranial surface ectoderm and underlying supraorbital mesenchyme during dermal and osteoblast fate selection. Using conditional genetics, we eliminate secretion of all Wnt ligands from cranial surface ectoderm or undifferentiated mesenchyme, to uncover distinct roles for ectoderm- and mesenchyme-derived Wnts. Ectoderm Wnt ligands induce osteoblast and dermal fibroblast progenitor specification while initiating expression of a subset of mesenchymal Wnts. Mesenchyme Wnt ligands are subsequently essential during differentiation of dermal and osteoblast progenitors. Finally, ectoderm-derived Wnt ligands provide an inductive cue to the cranial mesenchyme for the fate selection of dermal fibroblast and osteoblast lineages. Thus two sources of Wnt ligands perform distinct functions during osteoblast and dermal fibroblast formation.

  13. Smart biomaterials: Surfaces functionalized with proteolytically stable osteoblast-adhesive peptides.

    PubMed

    Zamuner, Annj; Brun, Paola; Scorzeto, Michele; Sica, Giuseppe; Castagliuolo, Ignazio; Dettin, Monica

    2017-09-01

    Engineered scaffolds for bone tissue regeneration are designed to promote cell adhesion, growth, proliferation and differentiation. Recently, covalent and selective functionalization of glass and titanium surfaces with an adhesive peptide (HVP) mapped on [351-359] sequence of human Vitronectin allowed to selectively increase osteoblast attachment and adhesion strength in in vitro assays, and to promote osseointegration in in vivo studies. For the first time to our knowledge, in this study we investigated the resistance of adhesion sequences to proteolytic digestion: HVP was completely cleaved after 5 h. In order to overcome the enzymatic degradation of the native peptide under physiological conditions we synthetized three analogues of HVP sequence. A retro-inverted peptide D-2HVP, composed of D amino acids, was completely stable in serum-containing medium. In addition, glass surfaces functionalized with D-2HVP increased human osteoblast adhesion as compared to the native peptide and maintained deposition of calcium. Interestingly, D-2HVP increased expression of IBSP, VTN and SPP1 genes as compared to HVP functionalized surfaces. Total internal reflection fluorescence microscope analysis showed cells with numerous filopodia spread on D-2HVP-functionalized surfaces. Therefore, the D-2HVP sequence is proposed as new osteoblast adhesive peptide with increased bioactivity and high proteolytic resistance.

  14. Osteoblast Differentiation Decreases Hypergravity-Stimulated Release of PGE(sub 2)

    NASA Technical Reports Server (NTRS)

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

    2002-01-01

    We determined if progressive differentiation of osteoblasts influences their sensitivity to gravitational loading. Osteoblasts were cultured for 4 days (confluent monolayer), 6 days (prenodules), 9 days (nodules) and 19 days (mineralized nodules), then centrifuged at 10 times gravity (g) or 50-g for 3 hours using the NASA Ames 1-ft. Diameter Centrifuge. Stationary controls were placed in an adjacent incubator. Following centrifugation, conditioned media were collected and analyzed for PGE, by ELISA. Microtubules were fluorescently labeled and analyzed by confocal microscopy to determine microtubule network morphology and height. Centrifugation at 10-g reduced microtubule network height by 15% on d4 and 10% on d6, with variable changes in more mature cultures. No major changes in microtubule morphology were observed. PGE(sub 2) release by d4 cultures increased in a dose-dependent fashion (3-fold at 10-g and 6-fold at 50-g relative to controls). D6 cultures produced a 5-fold increase for both 10-g and 50-g. PGE(sub 2) increased only 1.5-fold by d9, and by d19, PGE(sub 2) was not delectable in either the control or hypergravity-stimulated cells. Thus, as osteoblasts differentiate in culture, responsiveness of the microtubule cytoskeleton and the PGE(sub 2) pathway to hypergravity declines.

  15. Osteoblasts extracellular matrix induces vessel like structures through glycosylated collagen I

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Palmieri, D.; Valli, M.; Viglio, S.

    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 ofmore » 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.« less

  16. Direct effects of casein phosphopeptides on growth and differentiation of in vitro cultured osteoblastic cells (MC3T3-E1).

    PubMed

    Tulipano, Giovanni; Bulgari, Omar; Chessa, Stefania; Nardone, Alessandro; Cocchi, Daniela; Caroli, Anna

    2010-02-25

    Casein phosphopeptides (CPPs) obtained by enzymatic hydrolysis in vitro of caseins, have been shown to enhance calcium solubility and to increase the calcification of embryonic rat bones in their diaphyseal area. Little is known about the direct effects of CPPs on cultured osteoblastic cells. Calcium in the microenvironment surrounding bone cells is not only important for the mineralization of the extracellular matrix, but it is believed to provide preosteblasts with a signal that modulates their proliferation and differentiation. The aim of the present study was to investigate the direct effects of four selected casein phosphopeptides on osteoblastic cell (MC3T3-E1 cells) viability and differentiation. The selected peptides have been obtained by chemical synthesis and differed in the number of phosphorylated sites and in the amino acid spacing out two phosphorylated sites, in order to further characterize the relationship between structure and function. The results obtained in this work demonstrated that CPPs may directly affect osteoblast-like cell growth, calcium uptake and ultimately calcium deposition in the extracellular matrix. The effects exerted by distinct CPPs on osteogenesis in vitro can be either stimulatory or inhibitory. Differential short amino acid sequences in their molecules, like the -SpEE- and the -SpTSpEE-motifs, are likely the molecular determinants for their biological activities on osteoblastic cells. Moreover, two genetic variants of CPPs showing one amino acid change in their sequence may profoundly differ in their biological activities. Finally, our data may also suggest important clues about the role of intrinsic phosphorylated peptides derived from endogenous phosphorylated proteins in bone metabolism, apart from extrinsic CPPs. Copyright 2009 Elsevier B.V. All rights reserved.

  17. miR-383 negatively regulates osteoblastic differentiation of bone marrow mesenchymal stem cells in rats by targeting Satb2.

    PubMed

    Tang, Jianfei; Zhang, Zeng; Jin, Xiangyun; Shi, Huipeng

    2018-06-14

    Emerging evidence indicates that microRNAs (miRNAs, miRs) play diverse roles in the regulation of biological processes, including osteoblastic differentiation. In this study, we found that miR-383 is a critical regulator of osteoblastic differentiation. We showed that miR-383 was downregulated during osteoblastic differentiation of rat bone marrow mesenchymal stem cells (BMSCs). Overexpression of miR-383 suppressed osteoblastic differentiation of BMSCs by downregulating alkaline phosphatase (ALP), matrix mineralization, and mRNA and protein levels of RUNX2 and OCN, whereas a knockdown of miR-383 promoted osteoblastic differentiation in vitro. The results of in vivo analysis indicated that inhibition of miR-383 expression enhanced the efficacy of new bone formation in a rat calvarial defect model. Mechanistic experiments revealed that special AT-rich-sequence-binding protein 2 (Satb2) was a direct and functional target of miR-383. Knockdown of Satb2 had inhibitory effects resembling those of miR-383 on the osteoblast differentiation of rat BMSCs. In addition, the positive effect of miR-383 suppression on osteoblastic differentiation was apparently abrogated by Satb2 silencing. Collectively, these results indicate that miR-383 plays an inhibitory role in osteogenic differentiation of rat BMSCs and may act by targeting Satb2. Copyright © 2017. Published by Elsevier Inc.

  18. MiR-9 is overexpressed in spontaneous canine osteosarcoma and promotes a metastatic phenotype including invasion and migration in osteoblasts and osteosarcoma cell lines.

    PubMed

    Fenger, Joelle M; Roberts, Ryan D; Iwenofu, O Hans; Bear, Misty D; Zhang, Xiaoli; Couto, Jason I; Modiano, Jaime F; Kisseberth, William C; London, Cheryl A

    2016-10-10

    MicroRNAs (miRNAs) regulate the expression of networks of genes and their dysregulation is well documented in human malignancies; however, limited information exists regarding the impact of miRNAs on the development and progression of osteosarcoma (OS). Canine OS exhibits clinical and molecular features that closely resemble the corresponding human disease and it is considered a well-established spontaneous animal model to study OS biology. The purpose of this study was to investigate miRNA dysregulation in canine OS. We evaluated miRNA expression in primary canine OS tumors and normal canine osteoblast cells using the nanoString nCounter system. Quantitative PCR was used to validate the nanoString findings and to assess miR-9 expression in canine OS tumors, OS cell lines, and normal osteoblasts. Canine osteoblasts and OS cell lines were stably transduced with pre-miR-9 or anti-miR-9 lentiviral constructs to determine the consequences of miR-9 on cell proliferation, apoptosis, invasion and migration. Proteomic and gene expression profiling of normal canine osteoblasts with enforced miR-9 expression was performed using 2D-DIGE/tandem mass spectrometry and RNA sequencing and changes in protein and mRNA expression were validated with Western blotting and quantitative PCR. OS cell lines were transduced with gelsolin (GSN) shRNAs to investigate the impact of GSN knockdown on OS cell invasion. We identified a unique miRNA signature associated with primary canine OS and identified miR-9 as being significantly overexpressed in canine OS tumors and cell lines compared to normal osteoblasts. Additionally, high miR-9 expression was demonstrated in tumor-specific tissue obtained from primary OS tumors. In normal osteoblasts and OS cell lines transduced with miR-9 lentivirus, enhanced invasion and migration were observed, but miR-9 did not affect cell proliferation or apoptosis. Proteomic and transcriptional profiling of normal canine osteoblasts overexpressing miR-9 identified

  19. Effect of Atmospheric Plasma Treatment to Titanium Surface on Initial Osteoblast-Like Cell Spreading. .

    PubMed

    Kim, In-Hye; Son, Jun-Sik; Kwon, Tae-Yub; Kim, Kyo-Han

    2015-01-01

    Plasma treatments are becoming a popular method for modifying the characteristics of a range of substrate surfaces. Atmospheric pressure plasma is cost-efficient, safe and simple compared to high-pressure plasma. This study examined the effects of atmospheric pressure plasma to a titanium (Ti) surface on osteoblast-like cell (osteoblast) spreading and cellular networks. The characteristics of the Ti surface before and after the atmospheric plasma treatment were analyzed by X-ray photoemission spectroscopy (XPS), scanning electron microscopy (SEM), contact angle measurements, and an optical 3D profiling system. The morphology of osteoblasts attached to the Ti surfaces was observed by SEM and confocal laser scanning microscopy. The atmospheric pressure plasma made the Ti surfaces more hydrophilic. The osteoblasts that adhered to the untreated surface were round and spherical, whereas the cells covered a larger surface area on the plasma-treated surface. The plasma-treated Ti surface showed enhanced cell spreading and migration with more developed cellular networks. In conclusion, an atmospheric plasma treatment is a potential surface modifying method that can enhance the initial the cell affinity at the early stages in vitro.

  20. Hydroxyapatite and Calcified Elastin Induce Osteoblast-like Differentiation in Rat Aortic Smooth Muscle Cells

    PubMed Central

    Lei, Yang; Sinha, Aditi; Nosoudi, Nasim; Grover, Ankit; Vyavahare, Naren

    2014-01-01

    Vascular calcification can be categorized into two different types. Intimal calcification related to atherosclerosis and elastin-specific medial arterial calcification (MAC). Osteoblast-like differentiation of vascular smooth muscle cells (VSMCs) has been shown in both types; however, how this relates to initiation of vascular calcification is unclear. We hypothesize that the initial deposition of hydroxyapatite-like mineral in MAC occurs on degraded elastin first and that causes osteogenic transformation of VSMCs. To test this, rat aortic smooth muscle cells (RASMCs) were cultured on hydroxyapatite crystals and calcified aortic elastin. Using RT-PCR and specific protein assays, we demonstrate that RASMCs lose their smooth muscle lineage markers like alpha smooth muscle actin (SMA) and myosin heavy chain (MHC) and undergo chondrogenic/osteogenic transformation. This is indicated by an increase in the expression of typical chondrogenic proteins such as aggrecan, collagen type II alpha 1(Col2a1) and bone proteins such as runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP) and osteocalcin (OCN). Furthermore, when calcified conditions are removed, cells return to their original phenotype. Our data supports the hypothesis that elastin degradation and calcification precedes VSMCs' osteoblast-like differentiation. PMID:24447384