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Primary Osteoblasts (primary + osteoblast)
Selected AbstractsAssociation of a single nucleotide polymorphism in the steroid and xenobiotic receptor (SXR) gene (IVS1-579A/G) with bone mineral densityGERIATRICS & GERONTOLOGY INTERNATIONAL, Issue 2 2007Tomohiko Urano Vitamin K2 plays an important role in the bone metabolism. The steroid and xenobiotic receptor (SXR) as a nuclear receptor activated by vitamin K2 as well as rifampicin could increase bone markers such as alkaline phosphatase in human osteoblastic cells. Thus, the SXR could mediate vitamin K2 signaling pathway in bone cells. Therefore, we analyzed expression of the SXR mRNA in human primary osteoblasts and chondrocytes. We also studied association of a single nucleotide polymorphism (SNP) in the SXR gene with bone mineral density (BMD). Expression levels of the SXR mRNA were analyzed during the culture course of human primary osteoblasts and chondrocytes. Association of a SNP in the SXR gene in intron 1 (IVS1-579A>G) with BMD was examined in 294 healthy postmenopausal Japanese women. The SXR mRNA increased at day 5 and then decreased at day 10 in human primary osteoblasts. Its mRNA gradually increased in human primary chondrocytes until day 10. As an association study of a SNP in the SXR gene (IVS1-579A/G), the subjects without the A allele (GG; n = 47) had significantly higher total BMD than the subjects bearing at least one A allele (AA + AG; n = 247) (Z score ± SD; 0.635 ± 1.031 versus 0.268 ± 1.061; P = 0.0298). The SXR mRNA was expressed and regulated in primary human osteoblasts and chondrocytes. A genetic variation at the SXR gene locus is associated with BMD, suggesting an involvement of the SXR gene in human bone metabolism. [source] Primary Cell Adhesion on RGD-Functionalized and Covalently Crosslinked Thin Polyelectrolyte Multilayer Films,ADVANCED FUNCTIONAL MATERIALS, Issue 1 2005C. Picart Abstract Polyelectrolyte multilayers (PEMs) are now widely used for biomedical applications. In this work, we investigated the primary osteoblast adhesion properties of PEMs of poly(L -lysine) (PLL), poly(L -glutamic acid) (PGA), poly(alginic acid) (Palg), and poly(galacturonic acid) (Pgal). In order to compensate for the poor adhesion of the as-synthesized films, two kinds of film modifications were achieved: a purely physical modification by film crosslinking, and a chemical modification by grafting a arginine,glycine,aspartic acid (RGD) peptide to PGA. Crosslinking was performed using a water-soluble carbodiimide in combination with N -hydroxysulfosuccinimide (sulfo-NHS) to induce amide formation. This reaction was followed by Fourier-transform IR spectroscopy. For film functionalization, a 15-amino-acid peptide was grafted to PGA and deposited as the top layer of the film. PLL/PGA, PLL/Palg, and PLL/Pgal films were crosslinked or functionalized. The films were tested for both short-term adhesion properties and long-term proliferation of primary osteoblasts. Whereas the effect of film crosslinking on short-term adhesion was moderate, it was much more important for the RGD-functionalized films. On the other hand, the long-term proliferation was the same or even higher for the crosslinked films as compared with the functionalized films. This effect was particularly enhanced for the PLL/Palg and PLL/Pgal films. Finally, we functionalized PLL/PGA that had been crosslinked prior to PGA-RGD deposition. These architectures exhibited even higher short-term adhesion and proliferation. These results clearly show the important role of the physical properties of the films, besides their chemical properties, for the modulation of primary cell-adhesion behavior. [source] Characterization of human fetal osteoblasts by microarray analysis following stimulation with 58S bioactive gel-glass ionic dissolution productsJOURNAL OF BIOMEDICAL MATERIALS RESEARCH, Issue 2 2006Ioannis Christodoulou Abstract Bioactive glasses dissolve upon immersion in culture medium, releasing their constitutive ions in solution. There is evidence suggesting that these ionic dissolution products influence osteoblast-specific processes. Here, we investigated the effect of 58S sol,gel-derived bioactive glass (60 mol % SiO2, 36 mol % CaO, 4 mol % P2O5) dissolution products on primary osteoblasts derived from human fetal long bone explant cultures (hFOBs). We used U133A human genome GeneChip® oligonucleotide arrays to examine 22,283 transcripts and variants, which represent over 18,000 well-substantiated human genes. Hybridization of samples (biotinylated cRNA) derived from monolayer cultures of hFOBs on the arrays revealed that 10,571 transcripts were expressed by these cells, with high confidence. These included transcripts representing osteoblast-related genes coding for growth factors and their associated molecules or receptors, protein components of the extracellular matrix (ECM), enzymes involved in degradation of the ECM, transcription factors, and other important osteoblast-associated markers. A 24-h treatment with a single dosage of ionic products of sol,gel 58S dissolution induced the differential expression of a number of genes, including IL-6 signal transducer/gp130, ISGF-3/STAT1, HIF-1 responsive RTP801, ERK1 p44 MAPK (MAPK3), MAPKAPK2, IGF-I and IGFBP-5. The over 2-fold up-regulation of gp130 and MAPK3 and down-regulation of IGF-I were confirmed by real-time RT-PCR analysis. These data suggest that 58S ionic dissolution products possibly mediate the bioactive effect of 58S through components of the IGF system and MAPK signaling pathways. © 2005 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2006 [source] Expression of Mouse Osteoclast K-Cl Co-Transporter-1 and Its Role During Bone Resorption,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2006Hiroshi Kajiya PhD Abstract To assess the role of Cl, transport during osteoclastic bone resorption, we studied the expression and function of K+/Cl, co-transporters (KCCs). KCC1 and chloride channel-7 were found to be expressed in mouse osteoclasts. The KCC inhibitor, R(+)-butylindazone (DIOA), KCC1 antisense oligo-nucleotides, and siRNA suppressed osteoclastic pit formation. DIOA also decreased Cl, extrusion and reduced H+ extrusion activity. These results show that KCC1 provides a Cl, extrusion mechanism accompanying the H+ extrusion during bone resorption. Introduction: Mice with deficient chloride (Cl,) channels, ClC7, show severe osteopetrosis, resulting from impairment of Cl, extrusion during osteoclastic bone resorption. However, the expression and functional role of Cl, transporters other than ClC7 in mammalian osteoclasts is unknown. The aim of this study was to determine expression of K+/Cl, co-transporters (KCCs) and their functional role for bone resorption in mouse osteoclasts. Materials and Methods: Mouse osteoclasts were derived from cultured bone marrow cells with macrophage-colony stimulating factor (M-CSF) and RANKL or from co-culture of bone marrow cells and primary osteoblasts. We examined the expression of Cl, transporters using RT-PCR, immunochemical, and Western blot methods. The effects of Cl, transport inhibitors on H+ and Cl, extrusion were assessed by measuring intracellular H+ ([H+]i) and Cl, ([Cl,]i). The effects of inhibitors, antisense oligo-nucleotides, and siRNA for Cl, transporters on bone resorption activities were evaluated using a pit formation assay. Results and Conclusions: Mouse osteoclasts express not only ClC7 but also K+/Cl, co-transporter mRNA. The existence of KCC1 in the cell membrane of mouse osteoclasts was confirmed by immunochemical staining and Western blot analysis. KCC inhibitors and Cl, channels blockers increased [Cl,]i and [H+]i in resorbing osteoclasts, suggesting that the suppression of Cl, extrusion through KCC and Cl, channels leads to reduced H+ extrusion activity. The combination of both inhibitors greatly suppressed these extrusion activities. KCC inhibitors and Cl, channel blockers also decreased osteoclastic bone resorption in our pit area essay. Furthermore, KCC1 antisense oligo-nucleotides and siRNA suppressed osteoclastic pit formation as well as treatment of ClC7 inhibitors. These results indicate that K+/Cl, co-transporter-1 expressed in mouse osteoclasts acts as a Cl, extruder and plays an important role for H+ extrusion during bone resorption. [source] Increased Bone Formation in Mice Lacking Plasminogen Activators,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2003E Daci Abstract Plasminogen activators tPA and uPA are involved in tissue remodeling, but their role in bone growth is undefined. Mice lacking tPA and uPA show increased bone formation and bone mass. The noncollagenous components of bone matrix are also increased, probably from defective degradation. This study underlines the importance of controlled bone matrix remodeling for normal endochondral ossification. Introduction: Proteolytic pathways are suggested to play a role in endochondral ossification. To elucidate the involvement of the plasminogen activators tPA and uPA in this process, we characterized the long bone phenotype in mice deficient in both tPA and uPA (tPA,/,:uPA,/,). Materials and Methods: Bones of 2- to 7-day-old tPA,/,:uPA,/, and wild-type (WT) mice were studied using bone histomorphometry, electron microscopy analysis, and biochemical assessment of bone matrix components. Cell-mediated degradation of metabolically labeled bone matrix, osteoblast proliferation, and osteoblast differentiation, both at the gene and protein level, were studied in vitro using cells derived from both genotypes. Results: Deficiency of the plasminogen activators led to elongation of the bones and to increased bone mass (25% more trabecular bone in the proximal tibial metaphysis), without altering the morphology of the growth plate. In addition, the composition of bone matrix was modified in plasminogen activator deficient mice, because an increased amount of proteoglycans (2×), osteocalcin (+45%), and fibronectin (+36%) was detected. Matrix degradation assays showed that plasminogen activators, by generating plasmin, participate in osteoblast-mediated degradation of the noncollagenous components of bone matrix. In addition, proliferation of primary osteoblasts derived from plasminogen activator-deficient mice was increased by 35%. Finally, osteoblast differentiation and formation of a mineralized bone matrix were enhanced in osteoblast cultures derived from tPA,/,:uPA,/, mice. Conclusions: The data presented indicate the importance of the plasminogen system in degradation of the noncollagenous components of bone matrix and suggest that the accumulation of these proteins in bone matrix,as occurs during plasminogen activator deficiency,may in turn stimulate osteoblast function, resulting in increased bone formation. [source] Fluid Flow Induction of Cyclo-Oxygenase 2 Gene Expression in Osteoblasts Is Dependent on an Extracellular Signal-Regulated Kinase Signaling Pathway,,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 2 2002Sunil Wadhwa Abstract Mechanical loading of bone may be transmitted to osteocytes and osteoblasts via shear stresses at cell surfaces generated by the flow of interstitial fluid. The stimulated production of prostaglandins, which mediates some effects of mechanical loading on bone, is dependent on inducible cyclo-oxygenase 2 (COX-2) in bone cells. We examined the fluid shear stress (FSS) induction of COX-2 gene expression in immortalized MC3T3-E1 osteoblastic cells stably transfected with ,371/+70 base pairs (bp) of the COX-2 5,-flanking DNA (Pluc371) and in primary osteoblasts (POBs) from calvaria of mice transgenic for Pluc371. Cells were plated on collagen-coated glass slides and subjected to steady laminar FSS in a parallel plate flow chamber. FSS, from 0.14 to10 dynes/cm2, induced COX-2 messenger RNA (mRNA) and protein. FSS (10 dynes/cm2) induced COX-2 mRNA within 30 minutes, with peak effects at 4 h in MC3T3-E1 cells and at ,8 h in POBs. An inhibitor of new protein synthesis puromycin blocked the peak induction of COX-2 mRNA by FSS. COX-2 promoter activity, measured as luciferase activity, correlated with COX-2 mRNA expression in both MC3T3-E1 and POB cells. FSS induced phosphorylation of extracellular signal-regulated kinase (ERK) in MC3T3-E1 cells, with peak effects at 5 minutes. Inhibiting ERK phosphorylation with the specific inhibitor PD98059 inhibited FSS induction of COX-2 mRNA by 55-70% and FSS stimulation of luciferase activity by ,80% in both MC3T3-E1 and POB cells. We conclude that FSS transcriptionally induces COX-2 gene expression in osteoblasts, that the maximum induction requires new protein synthesis, and that induction occurs largely via an ERK signaling pathway. [source] Role of the Latent Transforming Growth Factor ,,Binding Protein 1 in Fibrillin-Containing Microfibrils in Bone Cells In Vitro and In VivoJOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2000Sarah L. Dallas Abstract Latent transforming growth factor ,,binding proteins (LTBPs) are extracellular matrix (ECM) proteins that bind latent transforming growth factor , (TGF-,) and influence its availability in bone and other connective tissues. LTBPs have homology with fibrillins and may have related functions as microfibrillar proteins. However, at present little is known about their structural arrangement in the ECM. By using antibodies against purified LTBP1, against a short peptide in LTBP1, and against epitope-tagged LTBP1 constructs, we have shown colocalization of LTBP1 and fibrillin 1 in microfibrillar structures in the ECM of cultured primary osteoblasts. Immunoelectron microscopy confirmed localization of LTBP1 to 10- to 12-nm microfibrils and suggested an ordered aggregation of LTBP1 into these structures. Early colocalization of LTBP1 with fibronectin suggested a role for fibronectin in the initial assembly of LTBP1 into the matrix; however, in more differentiated osteoblast cultures, LTBP1 and fibronectin 1 were found in distinct fibrillar networks. Overexpression of LTBP1 deletion constructs in osteoblast-like cells showed that N-terminal amino acids 67,467 were sufficient for incorporation into fibrillin-containing microfibrils and suggested that LTBP1 can be produced by cells distant from the site of fibril formation. In embryonic long bones in vivo, LTBP1 and fibrillin 1 colocalized at the surface of newly forming osteoid and bone. However, LTBP1-positive fibrils, which did not contain fibrillin 1, were present in cartilage matrix. These studies show that in addition to regulating TGF,1, LTBP1 may function as a structural component of connective tissue microfibrils. LTBP1 may therefore be a candidate gene for Marfan-related connective tissue disorders in which linkage to fibrillins has been excluded. [source] Purification and characterization of heparan sulfate from human primary osteoblastsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2009Sadasivam Murali Abstract Heparan sulfate (HS) is a linear, highly variable, highly sulfated glycosaminoglycan sugar whose biological activity largely depends on internal sulfated domains that mediate specific binding to an extensive range of proteins. In this study we employed anion exchange chromatography, molecular sieving and enzymatic cleavage on HS fractions purified from three compartments of cultured osteoblasts,soluble conditioned media, cell surface, and extracellular matrix (ECM). We demonstrate that the composition of HS chains purified from the different compartments is structurally non-identical by a number of parameters, and that these differences have significant ramifications for their ligand-binding properties. The HS chains purified of conditioned medium had twice the binding affinity for FGF2 when compared with either cell surface or ECM HS. In contrast, similar binding of BMP2 to the three types of HS was observed. These results suggest that different biological compartments of cultured cells have structurally and functionally distinct HS species that help to modulate the flow of HS-dependent factors between the ECM and the cell surface. J. Cell. Biochem. 108: 1132,1142, 2009. © 2009 Wiley-Liss, Inc. [source] To go or not to go: Migration of human mesenchymal progenitor cells stimulated by isoforms of PDGFJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2004Jörg Fiedler Abstract The recruitment of mesenchymal progenitor cells (MPCs) and their subsequent differentiation to osteoblasts is mandatory for bone development, remodeling, and repair. To study the possible involvement of platelet-derived growth factor (PDGF) isoforms, primary human MPCs and osteogenic differentiated progenitor cells (dOB) were examined for chemotaxic response to homodimeric human platelet-derived growth factor AA, -BB, and heterodimeric PDGF-AB. The role of PDGF receptors was addressed by preincubation with PDGF receptor alpha and beta chain specific antibodies. Migration of MPCs, dOB, and primary osteoblasts (OB) was stimulated by the addition of rhPDGF-AA, rhPDGF-BB, and rhPDGF-AB. The effect was highest in MPCs and for rhPDGF-BB, and declining with osteogenic differentiation. Preincubation with the receptor alpha specific antibody decreased the CI to borderline values while pretreatment with the receptor beta specific antibody led to a complete loss of chemotactic response to PDGF isoforms. In control experiments, basal migration values and rhBMP-2 as well as rxBMP-4 induced chemotaxis of MPC were not influenced by the addition of receptor alpha or beta antibodies. Interestingly, without preincubation the parallel exposure of MPC to rhTGF-,1 instantaneously leads to a selective loss of migratory stimulation by rhPDGF-AA. The chemotactic effect of PDGF isoforms for primary human MPCs and the influence of osteogenic differentiation suggest a functional role for recruitment of MPCs during bone development and remodeling. Moreover, these observations may be useful for novel approaches towards guided tissue regeneration or tissue engineering of bone. © 2004 Wiley-Liss, Inc. [source] R-spondin 1 protects against inflammatory bone damage during murine arthritis by modulating the Wnt pathwayARTHRITIS & RHEUMATISM, Issue 8 2010Gerhard Krönke Objective During the course of different musculoskeletal diseases, joints are progressively damaged by inflammatory, infectious, or mechanical stressors, leading to joint destruction and disability. While effective strategies to inhibit joint inflammation, such as targeted cytokine-blocking therapy, have been developed during the last decade, the molecular mechanisms of joint damage are still poorly understood. This study was undertaken to investigate the role of the Wnt pathway modulator R-Spondin 1 (RSpo1) in protecting bone and cartilage in a mouse model of arthritis. Methods Tumor necrosis factor , (TNF,),transgenic mice were treated with vehicle or Rspo1. Mice were evaluated for signs of arthritis, and histologic analysis of the hind paws was performed. Moreover, we determined the effect of Rspo1 on Wnt signaling activity and osteoprotegerin (OPG) expression in murine primary osteoblasts. Results The secreted Wnt pathway modulator RSpo1 was highly effective in preserving the structural integrity of joints in a TNF,-transgenic mouse model of arthritis by protecting bone and cartilage from inflammation-related damage. RSpo1 antagonized the Wnt inhibitor Dkk-1 and modulated Wnt signaling in mouse mesenchymal cells. In osteoblasts, RSpo1 induced differentiation and expression of OPG, thereby inhibiting osteoclastogenesis in vitro. In vivo, RSpo1 promoted osteoblast differentiation and bone formation while blocking osteoclast development, thereby contributing to the integrity of joints during inflammatory arthritis. Conclusion Our results demonstrate the therapeutic potential of RSpo1 as an anabolic agent for the preservation of joint architecture. [source] A rotating bed system bioreactor enables cultivation of primary osteoblasts on well-characterized sponceram® regarding structural and flow propertiesBIOTECHNOLOGY PROGRESS, Issue 3 2010Kirstin Suck Abstract The development of bone tissue engineering depends on the availability of suitable biomaterials, a well-defined and controlled bioreactor system, and on the use of adequate cells. The biomaterial must fulfill chemical, biological, and mechanical requirements. Besides biocompatibility, the structural and flow characteristics of the biomaterial are of utmost importance for a successful dynamic cultivation of osteoblasts, since fluid percolation within the microstructure must be assured to supply to cells nutrients and waste removal. Therefore, the biomaterial must consist of a three-dimensional structure, exhibit high porosity and present an interconnected porous network. Sponceram®, a ZrO2 based porous ceramic, is characterized in the presented work with regard to its microstructural design. Intrinsic permeability is obtained through a standard Darcy's experiment, while Young's modulus is derived from a two plates stress,strain test in the linear range. Furthermore, the material is applied for the dynamic cultivation of primary osteoblasts in a newly developed rotating bed bioreactor. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] | ||||||||||||||||