WorldWideScience

Sample records for primary osteoblast culture

  1. Post-transcriptional regulation of osteoblastic platelet-derived growth factor receptor-alpha expression by co-cultured primary endothelial cells

    DEFF Research Database (Denmark)

    Finkenzeller, Günter; Mehlhorn, Alexander T; Schmal, Hagen

    2010-01-01

    -alpha downregulation is dependent on time and cell number. This effect was specific to endothelial cells and was not observed when hOBs were co-cultured with human primary chondrocytes or fibroblasts. Likewise, HUVEC-mediated suppression of PDGFR-alpha expression was only seen in hOBs and mesenchymal stem cells......Platelet-derived growth factor receptor (PDGFR) signaling plays an important role in osteoblast function. Inhibition of PDGFR activity leads to a suppression of osteoblast proliferation, whereas mineralized matrix production is enhanced. In previous experiments, we showed that co......-cultivation of human primary endothelial cells and human primary osteoblasts (hOBs) leads to a cell contact-dependent downregulation of PDGFR-alpha expression in the osteoblasts. In this study, we investigated this effect in more detail, revealing that human umbilical vein endothelial cell (HUVEC)-mediated PDGFR...

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

    International Nuclear Information System (INIS)

    Cao Xianying; Yin Meizhen; Zhang Lina; Li Shipu; Cao Yang

    2006-01-01

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

  3. Engraftment Outcomes after HPC Co-Culture with Mesenchymal Stromal Cells and Osteoblasts

    Directory of Open Access Journals (Sweden)

    Matthew M. Cook

    2013-09-01

    Full Text Available Haematopoietic stem cell (HSC transplantation is an established cell-based therapy for a number of haematological diseases. To enhance this therapy, there is considerable interest in expanding HSCs in artificial niches prior to transplantation. This study compared murine HSC expansion supported through co-culture on monolayers of either undifferentiated mesenchymal stromal cells (MSCs or osteoblasts. Sorted Lineage− Sca-1+ c-kit+ (LSK haematopoietic stem/progenitor cells (HPC demonstrated proliferative capacity on both stromal monolayers with the greatest expansion of LSK shown in cultures supported by osteoblast monolayers. After transplantation, both types of bulk-expanded cultures were capable of engrafting and repopulating lethally irradiated primary and secondary murine recipients. LSKs co-cultured on MSCs showed comparable, but not superior, reconstitution ability to that of freshly isolated LSKs. Surprisingly, however, osteoblast co-cultured LSKs showed significantly poorer haematopoietic reconstitution compared to LSKs co-cultured on MSCs, likely due to a delay in short-term reconstitution. We demonstrated that stromal monolayers can be used to maintain, but not expand, functional HSCs without a need for additional haematopoietic growth factors. We also demonstrated that despite apparently superior in vitro performance, co-injection of bulk cultures of osteoblasts and LSKs in vivo was detrimental to recipient survival and should be avoided in translation to clinical practice.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  6. Cellular lead toxicity and metabolism in primary and clonal osteoblastic bone cells

    International Nuclear Information System (INIS)

    Long, G.J.; Rosen, J.F.; Pounds, J.G.

    1990-01-01

    A knowledge of bone lead metabolism is critical for understanding the toxicological importance of bone lead, as a toxicant both to bone cells and to soft tissues of the body, as lead is mobilized from large reservoirs in hard tissues. To further understand the processes that mediate metabolism of lead in bone, it is necessary to determine lead metabolism at the cellular level. Experiments were conducted to determine the intracellular steady-state 210 Pb kinetics in cultures of primary and clonal osteoblastic bone cells. Osteoblastic bone cells obtained by sequential collagenase digestion of mouse calvaria or rat osteosarcoma (ROS 17/2.8) cells were labeled with 210 Pb as 5 microM lead acetate for 20 hr, and kinetic parameters were determined by measuring the efflux of 210 Pb from the cells over a 210 -min period. The intracellular metabolism of 210 Pb was characterized by three kinetic pools of 210 Pb in both cell types. Although the values of these parameters differed between the primary osteoblastic cells and ROS cells, the profile of 210 Pb was remarkably similar in both cell types. Both types exhibited one large, slowly exchanging pool (S3), indicative of mitochondrial lead. These data show that primary osteoblastic bone cells and ROS cells exhibit similar steady-state lead kinetics, and intracellular lead distribution. These data also establish a working model of lead kinetics in osteoblastic bone cells and now permit an integrated view of lead kinetics in bone

  7. Leptin promotes osteoblast differentiation and mineralization of primary cultures of vascular smooth muscle cells by inhibiting glycogen synthase kinase (GSK)-3{beta}

    Energy Technology Data Exchange (ETDEWEB)

    Zeadin, Melec G.; Butcher, Martin K.; Shaughnessy, Stephen G. [Department of Medicine, McMaster University, Hamilton, ON (Canada); Thrombosis and Atherosclerosis Research Institute, Hamilton, ON (Canada); Werstuck, Geoff H., E-mail: Geoff.Werstuck@taari.ca [Department of Medicine, McMaster University, Hamilton, ON (Canada); Thrombosis and Atherosclerosis Research Institute, Hamilton, ON (Canada)

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer Leptin promotes osteoblast differentiation of primary smooth muscle cells. Black-Right-Pointing-Pointer Leptin regulates the expression of genes involved in osteoblast differentiation. Black-Right-Pointing-Pointer Constitutively active GSK-3{beta} attenuates leptin-induced osteoblast differentiation. Black-Right-Pointing-Pointer This suggests that leptin signals through GSK-3{beta} to promote osteoblast differentiation. -- Abstract: In this study, we begin to investigate the underlying mechanism of leptin-induced vascular calcification. We found that treatment of cultured bovine aortic smooth muscle cells (BASMCs) with leptin (0.5-4 {mu}g/ml) induced osteoblast differentiation in a dose-dependent manner. Furthermore, we found that leptin significantly increased the mRNA expression of osteopontin and bone sialoprotein, while down-regulating matrix gla protein (MGP) expression in BASMCs. Key factors implicated in osteoblast differentiation, including members of the Wnt signaling pathway, were examined. Exposure to leptin enhanced phosphorylation of GSK-3{beta} on serine-9 thereby inhibiting activity and promoting the nuclear accumulation of {beta}-catenin. Transfection of BASMCs with an adenovirus that expressed constitutively active GSK-3{beta} (Ad-GSK-3{beta} S9A) resulted in a >2-fold increase in GSK-3{beta} activity and a significant decrease in leptin-induced alkaline phosphatase (ALP) activity. In addition, qRT-PCR analysis showed that GSK-3{beta} activation resulted in a significant decrease in the expression of osteopontin and bone sialoprotein, but a marked increase in MGP mRNA expression. When taken together, our results suggest a mechanism by which leptin promotes osteoblast differentiation and vascular calcification in vivo.

  8. Integrin expression by human osteoblasts cultured on degradable polymeric materials applicable for tissue engineered bone.

    Science.gov (United States)

    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

  9. Hypergravity Stimulates the Extracellular Matrix/Integrin-Signaling Axis and Proliferation in Primary Osteoblasts

    Science.gov (United States)

    Parra, M.; Vercoutere, W.; Roden, C.; Banerjee, I.; Krauser, W.; Holton, E.; Searby, N.; Globus, R.; Almeida, E.

    2003-01-01

    We set out to determine the molecular mechanisms involved in the proliferative response of primary rat osteoblasts to mechanical stimulation using cell culture centrifugation as a model for hypergravity. We hypothesized that this proliferative response is mediated by specific integrin/Extracellular Matrix (ECM) interactions. To investigate this question we developed a cell culture centrifuge and an automated system that performs cell fixation during hypergravity loading. We generated expression vectors for various focal adhesion and cytoskeletal proteins fused to GFP or dsRed and visualized these structures in transfected (or infected) osteoblasts. The actin cytoskeleton was also visualized using rhodamine-phalloidin staining and Focal Adhesion Kinase (FAK) levels were assessed biochemically. We observed that a 24 hour exposure to 50-g stimulated proliferation compared to the 1-g control when cells were plated on fibronectin, collagen Type I , and collagen Type IV, but not on uncoated tissue culture plastic surfaces. This proliferative response was greatest for osteoblasts grown on fibronectin (2-fold increase over 1-g control) and collagen Type I (1.4 fold increase over 1-g control), suggesting that specific matrices and integrins are involved in the signaling pathways required for proliferation. Exposing osteoblasts grown on different matrices to 10-g or 25-g showed that effects on proliferation depended on both matrix type and loading level. We found that osteoblasts exposed to a short pulse of hypergravity during adhesion spread further and had more GFP-FAK containing focal adhesions compared to their 1-g controls. While overall levels of FAK did not change, more FAK was in the active (phosphorylated) form under hypergravity than in the 1-g controls. Cytoskeletal F-actin organization into filaments was also more prominent after brief exposures to hypergravity during the first five minutes of adhesion. These results suggest that specific integrins sense

  10. Size-dependent cytotoxicity of yttrium oxide nanoparticles on primary osteoblasts in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Guoqiang, E-mail: zhougq1982@163.com; Li, Yunfei; Ma, Yanyan; Liu, Zhu; Cao, Lili; Wang, Da; Liu, Sudan; Xu, Wenshi; Wang, Wenying [Hebei University, Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of Ministry of Education, Key Laboratory of Chemical Biology of Hebei Province, College of Chemistry and Environmental Science (China)

    2016-05-15

    Yttrium oxide nanoparticles are an excellent host material for the rare earth metals and have high luminescence efficiency providing a potential application in photodynamic therapy and biological imaging. In this study, the effects of yttrium oxide nanoparticles with four different sizes were investigated using primary osteoblasts in vitro. The results demonstrated that the cytotoxicity generated by yttrium oxide nanoparticles depended on the particle size, and smaller particles possessed higher toxicological effects. For the purpose to elucidate the relationship between reactive oxygen species generation and cell damage, cytomembrane integrity, intracellular reactive oxygen species level, mitochondrial membrane potential, cell apoptosis rate, and activity of caspase-3 in cells were then measured. Increased reactive oxygen species level was also observed in a size-dependent way. Thus, our data demonstrated that exposure to yttrium oxide nanoparticles resulted in a size-dependent cytotoxicity in cultured primary osteoblasts, and reactive oxygen species generation should be one possible damage pathway for the toxicological effects produced by yttrium oxide particles. The results may provide useful information for more rational applications of yttrium oxide nanoparticles in the future.

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

    International Nuclear Information System (INIS)

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

    1989-01-01

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

  12. Osteocalcin, a marker of differentiated function during calcification of cultured chick osteoblasts

    International Nuclear Information System (INIS)

    Lian, J.; Chipman, S.; Glowacki, J.; Gerstenfeld, L.

    1986-01-01

    The expression of differentiated function was examined in cultured osteoblasts isolated from 17-day embryonic chicken calvarie. Cell cultures grown in the absence (control) or presence of 10 mM β-Glycerol Phosphate (βGP) (stimulus for calcification) were analyzed at 6-day intervals over a 30-day period for total mineral, alkaline phosphatase (AP) activity, osteocalcin levels and collagen. AP was first detected in both cultures between days 6 and 9 when cells became crowded. Control cultures maintained high levels of enzyme activity (30-50 fold) while β GPO 4 culture activity declined after day 18 when extensive mineralization occurred. Osteocalcin, the vitamin K-dependent, bone-specific, calcium-binding protein showed a similar pattern of induction as AP with at 50-100-fold increase in both cultures. Collagen accumulated through out the 30-day experimental period for both β GPO 4 and control cultures while collagen synthesis ( 3 H-proline pulse) peaked at day 15 in culture. These results suggest that with time in culture, osteoblast differentiation may be occurring. The increased mineralization of β GPO 4 cultures appeared to down regulate the enzyme activity of AP in comparison to control culture, while osteocalcin synthesis was enhanced. In conclusion, the chick osteoblast system offers a model to study bone cell differentiation, protein synthesis and matrix calcification

  13. Three-dimensional spheroid culture promotes odonto/osteoblastic differentiation of dental pulp cells.

    Science.gov (United States)

    Yamamoto, Mioko; Kawashima, Nobuyuki; Takashino, Nami; Koizumi, Yu; Takimoto, Koyo; Suzuki, Noriyuki; Saito, Masahiro; Suda, Hideaki

    2014-03-01

    Three-dimensional (3D) spheroid culture is a method for creating 3D aggregations of cells and their extracellular matrix without a scaffold mimicking the actual tissues. The aim of this study was to evaluate the effects of 3D spheroid culture on the phenotype of immortalized mouse dental papilla cells (MDPs) that have the ability to differentiate into odontoblasts. We cultured MDPs for 1, 3, 7, and 14 days in 96-well low-attachment culture plates for 3D spheroid culture or flat-bottomed plates for two-dimensional (2D) monolayer culture. Cell proliferation and apoptosis were detected by immunohistochemical staining of Ki67 and cleaved caspase-3, respectively. Hypoxia was measured by the hypoxia probe LOX-1. Odonto/osteoblastic differentiation marker gene expression was evaluated by quantitative PCR. We also determined mineralized nodule formation, alkaline phosphatase (ALP) activity, and dentine matrix protein-1 (DMP1) expression. Vinculin and integrin signalling-related proteins were detected immunohistochemically. Odonto/osteoblastic marker gene expression and mineralized nodule formation were significantly up-regulated in 3D spheroid-cultured MDPs compared with those in 2D monolayer-cultured MDPs (podonto/osteoblastic differentiation of MDPs, which may be mediated by integrin signalling. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

    International Nuclear Information System (INIS)

    Tang, C.-H.; Chiu, Y.-C.; Huang, C.-F.; Chen, Y.-W.; Chen, P.-C.

    2009-01-01

    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 stromal 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.

  15. Behaviour of moderately differentiated osteoblast-like cells cultured in contact with bioactive glasses

    Directory of Open Access Journals (Sweden)

    Hattar S.

    2002-12-01

    Full Text Available Bioactive glasses have been shown to stimulate osteogenesis both in vivo and in vitro. However, the molecular mechanisms underlying this process are still poorly understood. In this study, we have investigated the behaviour of osteoblast-like cells (MG63, cultured in the presence of bioglass particles. Three types of granules were used: 45S5registered bioactive glass, 45S5registered granules preincubated in tris buffer and 60S non-reactive glass, used as control. Phase contrast microscopy permitted step-by-step visualization of cell cultures in contact with the particles. Ultrastructural observations of undecalcified sections revealed direct contacts of the cells and an electron-dense layer located at the periphery of the material. Protein synthesis was evaluated biochemically and showed a gradual increase throughout the culture time in the three types of cultures. Alkaline phosphatase was detected in situ, in clusters of packed cells either in contact with the material or in the background cell layer. Semi-quantitative RT-PCR analysis of the main osteoblastic markers showed that gene expression was maintained in all three cultures. The fact that osteocalcin was not detected, supports the fact that the MG63 cell line is composed of less differentiated osteogenic cells rather than mature osteoblasts. We also demonstrated for the first time in this cell line, the expression of Msx-2, Dlx-3 and Dlx-7 homeogenes, known to regulate in vivo foetal skeletogenesis as well as adult skeletal regeneration. However, no significant differences could be recognised in the expression pattern of bone markers between the three types of cultures. Yet these preliminary results indicate that bioactive glasses provided a suitable environment for the growth and proliferation of osteoblasts in vitro, since no drastic changes in phenotype expression of pre-osteoblasts was noted.

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

    Science.gov (United States)

    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.

  17. Ex Vivo Maintenance of Primary Human Multiple Myeloma Cells through the Optimization of the Osteoblastic Niche.

    Science.gov (United States)

    Zhang, Wenting; Gu, Yexin; Sun, Qiaoling; Siegel, David S; Tolias, Peter; Yang, Zheng; Lee, Woo Y; Zilberberg, Jenny

    2015-01-01

    We previously reported a new approach for culturing difficult-to-preserve primary patient-derived multiple myeloma cells (MMC) using an osteoblast (OSB)-derived 3D tissue scaffold constructed in a perfused microfluidic environment and a culture medium supplemented with patient plasma. In the current study, we used this biomimetic model to show, for the first time, that the long-term survival of OSB is the most critical factor in maintaining the ex vivo viability and proliferative capacity of MMC. We found that the adhesion and retention of MMC to the tissue scaffold was meditated by osteoblastic N-cadherin, as one of potential mechanisms that regulate MMC-OSB interactions. However, in the presence of MMC and patient plasma, the viability and osteogenic activity of OSB became gradually compromised, and consequently MMC could not remain viable over 3 weeks. We demonstrated that the long-term survival of both OSB and MMC could be enhanced by: (1) optimizing perfusion flow rate and patient-derived plasma composition in the culture medium and (2) replenishing OSB during culture as a practical means of prolonging MMC's viability beyond several weeks. These findings were obtained using a high-throughput well plate-based perfusion device from the perspective of optimizing the ex vivo preservation of patient-derived MM biospecimens for downstream use in biological studies and chemosensitivity analyses.

  18. Ex Vivo Maintenance of Primary Human Multiple Myeloma Cells through the Optimization of the Osteoblastic Niche.

    Directory of Open Access Journals (Sweden)

    Wenting Zhang

    Full Text Available We previously reported a new approach for culturing difficult-to-preserve primary patient-derived multiple myeloma cells (MMC using an osteoblast (OSB-derived 3D tissue scaffold constructed in a perfused microfluidic environment and a culture medium supplemented with patient plasma. In the current study, we used this biomimetic model to show, for the first time, that the long-term survival of OSB is the most critical factor in maintaining the ex vivo viability and proliferative capacity of MMC. We found that the adhesion and retention of MMC to the tissue scaffold was meditated by osteoblastic N-cadherin, as one of potential mechanisms that regulate MMC-OSB interactions. However, in the presence of MMC and patient plasma, the viability and osteogenic activity of OSB became gradually compromised, and consequently MMC could not remain viable over 3 weeks. We demonstrated that the long-term survival of both OSB and MMC could be enhanced by: (1 optimizing perfusion flow rate and patient-derived plasma composition in the culture medium and (2 replenishing OSB during culture as a practical means of prolonging MMC's viability beyond several weeks. These findings were obtained using a high-throughput well plate-based perfusion device from the perspective of optimizing the ex vivo preservation of patient-derived MM biospecimens for downstream use in biological studies and chemosensitivity analyses.

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

    Science.gov (United States)

    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.

  20. Hypergravity Loading the Cultured Osteoblasts: Modeling and Experimental Analysis of Cellular Morphology and the Cytoskeleton

    Science.gov (United States)

    Searby, N. D.; Steele, C. R.; Globus, R. K.; Dalton, Bonnie P. (Technical Monitor)

    2001-01-01

    Bone forming cells, osteoblasts, respond to various mechanical forces, including mechanical strain and fluid-induced shear stress. This study examined whether osteoblasts detect changes in gravity as a mechanical force, as assessed by cellular morphology and dimensions of the cytoskeletal network. We used modeling to evaluate how gravity influences cell morphology given theoretical differences in densities between the surrounding medium, cytoplasm, and nucleus. A mechanical model was built based on analysis of axisymmetric shell structures (Fast4 software) to study the effects of 10 times gravity (10G) on cell height. The model indicated 0.02% decrease in overall cell height when the medium was 10% denser than the nucleus or cytoplasm, 5.9 x 10(exp-5)% decrease when the nucleus was 10% denser than the cytoplasm or medium, and 1.3 x 10(exp-5)% decrease when the cell cytoplasm was 10% denser than the nucleus or medium. To experimentally evaluate the influence of gravity, cultured primary fetal rat osteoblasts were grown to near confluence and centrifuged at 10G for 3 hours. Actin, microtubules, and nuclei were fluorescently labeled and analyzed by confocal microscopy to determine overall microtubule and actin network height. Centrifugation led to an apparent reduction in height of both the microtubule (-16%) and the actin (-20%) networks relative to stationary controls. Thus, both modeling and experiments indicate that hypergravity reduces the height of the osteoblast cell layer and their microtubule and actin networks. This combination of modeling and experimental analyses will help us to better understand the mechanical loading of osteoblasts.

  1. Immortalization and characterization of mouse floxed Bmp2/4 osteoblasts

    International Nuclear Information System (INIS)

    Wu, Li-An; Yuan, Guohua; Yang, Guobin; Ortiz-Gonzalez, Iris; Yang, Wuchen; Cui, Yong; MacDougall, Mary; Donly, Kevin J.; Harris, Stephen; Chen, Shuo

    2009-01-01

    Generation of a floxed Bmp2/4 osteoblast cell line is a valuable tool for studying the modulatory effects of Bmp2 and Bmp4 on osteoblast differentiation as well as relevant molecular events. In this study, primary floxed Bmp2/4 mouse osteoblasts were cultured and transfected with simian virus 40 large T-antigen. Transfection was verified by polymerase chain reaction (PCR) and immunohistochemistry. To examine the characteristics of the transfected cells, morphology, proliferation and mineralization were analyzed, expression of cell-specific genes including Runx2, ATF4, Dlx3, Osx, dentin matrix protein 1, bone sialoprotein, osteopontin, osteocalcin, osteonectin and collagen type I was detected. These results show that transfected floxed Bmp2/4 osteoblasts bypassed senescence with a higher proliferation rate, but retain the genotypic and phenotypic characteristics similar to the primary cells. Thus, we for the first time demonstrate the establishment of an immortalized mouse floxed Bmp2/4 osteoblast cell line.

  2. Immortalization and characterization of mouse floxed Bmp2/4 osteoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Li-An [Department of Pediatric Dentistry, The University of Texas Health Science Center at San Antonio, TX (United States); Department of Pediatric Dentistry, School of Stomatology, The Fourth Military Medical University, Xi-an (China); Yuan, Guohua; Yang, Guobin [Department of Pediatric Dentistry, The University of Texas Health Science Center at San Antonio, TX (United States); Key Laboratory of Oral Biomedical Engineering Ministry of Education, Wuhan (China); Ortiz-Gonzalez, Iris [Department of Pediatric Dentistry, The University of Texas Health Science Center at San Antonio, TX (United States); Yang, Wuchen; Cui, Yong [Department of Periodontics, Dental School, The University of Texas Health Science Center at San Antonio, TX (United States); MacDougall, Mary [Department of Oral/Maxillofacial Surgery, University of Alabama, Birmingham, AL (United States); Donly, Kevin J. [Department of Pediatric Dentistry, The University of Texas Health Science Center at San Antonio, TX (United States); Harris, Stephen [Department of Periodontics, Dental School, The University of Texas Health Science Center at San Antonio, TX (United States); Chen, Shuo, E-mail: chens0@uthscsa.edu [Department of Pediatric Dentistry, The University of Texas Health Science Center at San Antonio, TX (United States)

    2009-08-14

    Generation of a floxed Bmp2/4 osteoblast cell line is a valuable tool for studying the modulatory effects of Bmp2 and Bmp4 on osteoblast differentiation as well as relevant molecular events. In this study, primary floxed Bmp2/4 mouse osteoblasts were cultured and transfected with simian virus 40 large T-antigen. Transfection was verified by polymerase chain reaction (PCR) and immunohistochemistry. To examine the characteristics of the transfected cells, morphology, proliferation and mineralization were analyzed, expression of cell-specific genes including Runx2, ATF4, Dlx3, Osx, dentin matrix protein 1, bone sialoprotein, osteopontin, osteocalcin, osteonectin and collagen type I was detected. These results show that transfected floxed Bmp2/4 osteoblasts bypassed senescence with a higher proliferation rate, but retain the genotypic and phenotypic characteristics similar to the primary cells. Thus, we for the first time demonstrate the establishment of an immortalized mouse floxed Bmp2/4 osteoblast cell line.

  3. Expression and Dynamics of Podoplanin in Cultured Osteoblasts with Mechanostress and Mineralization Stimulus.

    Science.gov (United States)

    Takenawa, Tomohiro; Kanai, Takenori; Kitamura, Tetsuya; Yoshimura, Yoshitaka; Sawa, Yoshihiko; Iida, Junichiro

    2018-02-27

    This study investigates the significance of the expression and dynamics of podoplanin in mechanostress and mineralization in cultured murine osteoblasts. Podoplanin increased in osteoblasts subjected to straining in non-mineralization medium, suggesting that the mechanostress alone is a podoplanin induction factor. In osteoblasts subjected to vertical elongation straining in the mineralization medium, the mRNA amounts of podoplanin, osteopontin, and osteocalcin were significantly larger than those in cells not subjected to straining, suggesting that mechanostress is the cause of a synergistic effect in the expression of these proteins. In osteoblasts in the mineralization medium, significant increases in osteocalcin mRNA occurred earlier in cells subjected to straining than in the cells not subjected to straining, suggesting that the mechanostress is a critical factor to enhance the expression of osteocalcin. Western blot and ELISA analysis showed increased podoplanin production in osteoblasts with longer durations of straining. There was significantly less mineralization product in osteoblasts with antibodies for podoplanin, osteopontin, and osteocalcin. There was also less osteopontin and osteocalcin produced in osteoblasts with anti-podoplanin. These findings suggest that mechanostress induces the production of podoplanin in osteoblasts and that podoplanin may play a role in mineralization in cooperation with bone-associated proteins.

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

    Directory of Open Access Journals (Sweden)

    Zunich Samantha M

    2012-07-01

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

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

    Directory of Open Access Journals (Sweden)

    Yongjuan Chen

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

  6. Zirconium Ions Up-Regulate the BMP/SMAD Signaling Pathway and Promote the Proliferation and Differentiation of Human Osteoblasts

    Science.gov (United States)

    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

  7. Matrix metalloproteinases (MMPs) safeguard osteoblasts from apoptosis during transdifferentiation into osteocytes

    DEFF Research Database (Denmark)

    Karsdal, M A; Levin Andersen, Thomas; Bonewald, L

    2004-01-01

    of osteoblasts forced to transdifferentiate into osteocytes in 3D type I collagen gels were inhibited by more than 50% when exposed to 10 microM GM6001 and to Tissue Inhibitor of Metalloproteinase-2 (TIMP-2), a natural MT1-MMP inhibitor. This shows the importance of MMPs in safeguarding osteoblasts from......Osteoblasts undergo apoptosis or differentiate into either osteocytes or bone-lining cells after termination of bone matrix synthesis. In this study, we investigated the role of matrix metalloproteinases (MMPs) in differentiation of osteoblasts, bone formation, transdifferentiation into osteocytes......, and osteocyte apoptosis. This was accomplished by using calvarial sections from the MT1-MMP-deficient mouse and by culture of the mouse osteoblast cell line MC3T3-E1 and primary mouse calvarial osteoblasts. We found that a synthetic matrix metalloprotease inhibitor, GM6001, strongly inhibited bone formation...

  8. Extracellular matrix production by human osteoblasts cultured on biodegradable polymers applicable for tissue engineering.

    Science.gov (United States)

    El-Amin, S F; Lu, H H; Khan, Y; Burems, J; Mitchell, J; Tuan, R S; Laurencin, C T

    2003-03-01

    The nature of the extracellular matrix (ECM) is crucial in regulating cell functions via cell-matrix interactions, cytoskeletal organization, and integrin-mediated signaling. In bone, the ECM is composed of proteins such as collagen (CO), fibronectin (FN), laminin (LM), vitronectin (VN), osteopontin (OP) and osteonectin (ON). For bone tissue engineering, the ECM should also be considered in terms of its function in mediating cell adhesion to biomaterials. This study examined ECM production, cytoskeletal organization, and adhesion of primary human osteoblastic cells on biodegradable matrices applicable for tissue engineering, namely polylactic-co-glycolic acid 50:50 (PLAGA) and polylactic acid (PLA). We hypothesized that the osteocompatible, biodegradable polymer surfaces promote the production of bone-specific ECM proteins in a manner dependent on polymer composition. We first examined whether the PLAGA and PLA matrices could support human osteoblastic cell growth by measuring cell adhesion at 3, 6 and 12h post-plating. Adhesion on PLAGA was consistently higher than on PLA throughout the duration of the experiment, and comparable to tissue culture polystyrene (TCPS). ECM components, including CO, FN, LM, ON, OP and VN, produced on the surface of the polymers were quantified by ELISA and localized by immunofluorescence staining. All of these proteins were present at significantly higher levels on PLAGA compared to PLA or TCPS surfaces. On PLAGA, OP and ON were the most abundant ECM components, followed by CO, FN, VN and LN. Immunofluorescence revealed an extracellular distribution for CO and FN, whereas OP and ON were found both intracellularly as well as extracellularly on the polymer. In addition, the actin cytoskeletal network was more extensive in osteoblasts cultured on PLAGA than on PLA or TCPS. In summary, we found that osteoblasts plated on PLAGA adhered better to the substrate, produced higher levels of ECM molecules, and showed greater cytoskeletal

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

    International Nuclear Information System (INIS)

    Utting, J.C.; Robins, S.P.; Brandao-Burch, A.; Orriss, I.R.; Behar, J.; Arnett, T.R.

    2006-01-01

    We investigated the effect of hypoxia on rat osteoblast function in long-term primary cultures. Reduction of pO 2 from 20% to 5% and 2% decreased formation of mineralized bone nodules 1.7-fold and 11-fold, respectively. When pO 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 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 apoptosis of osteoblasts but induced a reversible state of quiescence. Transmission electron microscopy revealed that collagen fibrils deposited by osteoblasts cultured in 2% O 2 were less organized and much less abundant than in 20% O 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

  10. In vitro culture and characterization of alveolar bone osteoblasts isolated from type 2 diabetics

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Dao-Cai [Department of Implant Dentistry, School of Stomatology, Fourth Military Medical University, Xi' an (China); Department of Stomatology, The 291st Hospital of P.L.A, Baotou (China); Li, De-Hua [Department of Implant Dentistry, School of Stomatology, Fourth Military Medical University, Xi' an (China); Ji, Hui-Cang [Military Sanatorium of Retired Cadres, Baotou (China); Rao, Guo-Zhou [Center of Laboratory, School of Stomatology, Xi' an Jiaotong University, Xi' an (China); Liang, Li-Hua [Department of Implant Dentistry, School of Stomatology, Fourth Military Medical University, Xi' an (China); Ma, Ai-Jie [Xi' an Technology University, Xi' an (China); Xie, Chao; Zou, Gui-Ke; Song, Ying-Liang [Department of Implant Dentistry, School of Stomatology, Fourth Military Medical University, Xi' an (China)

    2012-04-05

    In order to understand the mechanisms of poor osseointegration following dental implants in type 2 diabetics, it is important to study the biological properties of alveolar bone osteoblasts isolated from these patients. We collected alveolar bone chips under aseptic conditions and cultured them in vitro using the tissue explants adherent method. The biological properties of these cells were characterized using the following methods: alkaline phosphatase (ALP) chemical staining for cell viability, Alizarin red staining for osteogenic characteristics, MTT test for cell proliferation, enzyme dynamics for ALP contents, radio-immunoassay for bone gla protein (BGP) concentration, and ELISA for the concentration of type I collagen (COL-I) in the supernatant. Furthermore, we detected the adhesion ability of two types of cells from titanium slices using non-specific immunofluorescence staining and cell count. The two cell forms showed no significant difference in morphology under the same culture conditions. However, the alveolar bone osteoblasts received from type 2 diabetic patients had slower growth, lower cell activity and calcium nodule formation than the normal ones. The concentration of ALP, BGP and COL-I was lower in the supernatant of alveolar bone osteoblasts received from type 2 diabetic patients than in that received from normal subjects (P < 0.05). The alveolar bone osteoblasts obtained from type 2 diabetic patients can be successfully cultured in vitro with the same morphology and biological characteristics as those from normal patients, but with slower growth and lower concentration of specific secretion and lower combining ability with titanium than normal ones.

  11. In vitro culture and characterization of alveolar bone osteoblasts isolated from type 2 diabetics

    International Nuclear Information System (INIS)

    Sun, Dao-Cai; Li, De-Hua; Ji, Hui-Cang; Rao, Guo-Zhou; Liang, Li-Hua; Ma, Ai-Jie; Xie, Chao; Zou, Gui-Ke; Song, Ying-Liang

    2012-01-01

    In order to understand the mechanisms of poor osseointegration following dental implants in type 2 diabetics, it is important to study the biological properties of alveolar bone osteoblasts isolated from these patients. We collected alveolar bone chips under aseptic conditions and cultured them in vitro using the tissue explants adherent method. The biological properties of these cells were characterized using the following methods: alkaline phosphatase (ALP) chemical staining for cell viability, Alizarin red staining for osteogenic characteristics, MTT test for cell proliferation, enzyme dynamics for ALP contents, radio-immunoassay for bone gla protein (BGP) concentration, and ELISA for the concentration of type I collagen (COL-I) in the supernatant. Furthermore, we detected the adhesion ability of two types of cells from titanium slices using non-specific immunofluorescence staining and cell count. The two cell forms showed no significant difference in morphology under the same culture conditions. However, the alveolar bone osteoblasts received from type 2 diabetic patients had slower growth, lower cell activity and calcium nodule formation than the normal ones. The concentration of ALP, BGP and COL-I was lower in the supernatant of alveolar bone osteoblasts received from type 2 diabetic patients than in that received from normal subjects (P < 0.05). The alveolar bone osteoblasts obtained from type 2 diabetic patients can be successfully cultured in vitro with the same morphology and biological characteristics as those from normal patients, but with slower growth and lower concentration of specific secretion and lower combining ability with titanium than normal ones

  12. Surface modification of parylene-N films for the culture of osteoblast-like cells (MG-63)

    Energy Technology Data Exchange (ETDEWEB)

    Liaqat, Usman [Graduate Program of Nano Science and Technology, Yonsei University, 50-Yonsei Ro, Seodaemun-gu, Seoul 120-749 (Korea, Republic of); Ko, Hyuk [Department of Materials Science and Engineering, Yonsei University, 50-Yonsei Ro, Seodaemun-gu, Seoul 120-749 (Korea, Republic of); Suh, Hwal [Graduate Program of Nano Science and Technology, Yonsei University, 50-Yonsei Ro, Seodaemun-gu, Seoul 120-749 (Korea, Republic of); Department of Medical Engineering, College of Medicine, Yonsei University, 50-Yonsei Ro, Seodaemun-gu, Seoul, 120-749 (Korea, Republic of); Lee, Misu [Division of Life Sciences, College of Life Science and Bioengineering, Incheon National University, Incheon 406-772 (Korea, Republic of); Pyun, Jae-Chul, E-mail: jcpyun@yonsei.ac.kr [Department of Materials Science and Engineering, Yonsei University, 50-Yonsei Ro, Seodaemun-gu, Seoul 120-749 (Korea, Republic of)

    2016-08-15

    Highlights: • Osteoblast-like cells (MG-63) was cultured on differently modified surfaces of parylene films. • Proliferation of MG-63 was observed to be far increased on UV-treated parylene-N film. • The influences of UV-treatment were found out on cell viability, proliferation rate and cell cycle. • The influence was estimated to be negligible on the protein synthesis, cell differentiation. • The UV-treated parylene-N was demonstrated to be effectively used for the culture of MG-63. - Abstract: The influence of microenvironments on the culture of osteoblast-like cells (MG-63) has been investigated using parylene films with different surfaces, such as parylene-N film, UV-modified parylene-N film, functional parylene film with amine groups (parylene-A), and UV-modified parylene-A film. In this work, parylene-N film was found to induce dramatic changes in cell adhesion and cell viability before and after UV-treatment with respect to the culture of osteoblast-like cells (MG-63). The influences of such a chemical environment on cell culture were investigated in relation to the cell proliferation (viability and proliferation rate) and the cell physiology (cell cycle, protein synthesis, and differentiation) of cells grown on parylene-N film, UV-modified parylene-N film, parylene-A film, and UV-modified parylene-A film in comparison with cells grown on a polystyrene surface.

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

    DEFF Research Database (Denmark)

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

    2006-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-11-01

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

  15. Monosodium urate monohydrate crystals inhibit osteoblast viability and function: implications for development of bone erosion in gout.

    Science.gov (United States)

    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.

  16. Influence of sterilization methods on cell behavior and functionality of osteoblasts cultured on TiO2 nanotubes

    International Nuclear Information System (INIS)

    Oh, Seunghan; Brammer, Karla S.; Moon, Kyung-Suk; Bae, Ji-Myung; Jin, Sungho

    2011-01-01

    We investigated the adhesion, proliferation and osteogenic functionality of osteoblasts cultured on titanium dioxide (TiO 2 ) nanotubes in response to different sterilization methods (dry autoclaving vs. wet autoclaving). We prepared various sizes (30-100 nm diameter) of TiO 2 nanotubes on titanium substrates by anodization, sterilized nanotubes by different conditions, and seeded osteoblast cells onto the nanotube surfaces with two different cell seeding densities (10,000 vs. 50,000 cells/well in 12-culture well). The result of this study indicates that the adhesion, proliferation and alkaline phosphatase activity of osteoblasts cultured on only the larger 70 and 100 nm TiO 2 nanotube arrays were dramatically changed by the different sterilization conditions at a low cell seeding density. However, with a higher cell seeding density (50,000 cells/well in 12-cell culture well), the results revealed no significant difference among altered nanotube geometry, 30-100 nm diameters, nor sterilization methods. Next, it was revealed that the nanofeatures of proteins adhered on nanotubular TiO 2 morphology are altered by the sterilization method. It was determined that this protein adhesion effect, in combination with the cell density of osteoblasts seeded onto such TiO 2 nanotube surfaces, has profound effects on cell behavior. This study clearly shows that these are some of the important in vitro culture factors that need to be taken into consideration, as well as TiO 2 nanotube diameters which play an important role in the improvement of cell behavior and functionality.

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

    Science.gov (United States)

    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.

  18. Primary Osteoblastic Osteosarcoma of the Rib in an Adult: A Case Report

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Jung Ah; Kang, Heung Sik [Dept. of Radiology, Seoul National University Bundang Hospital, Seongnam (Korea, Republic of); Ryoo, In Seon [Dept. of Radiology and Institute of Radiation Medicine, Seoul National University College of Medicine, Seoul (Korea, Republic of); Park, Hyo Ah; Chung, Jin Haeng [Dept. of Patholgy, Seoul National University Bundang Hospital, Seongnam (Korea, Republic of); Oh, Joo Han [Dept. of Orthopedic Surgery, Seoul National University Bundang Hospital, Seongnam (Korea, Republic of)

    2011-12-15

    We report the CT and magnetic resonance (MR) imaging appearances in an adult case of primary osteoblastic osteosarcoma of the rib. Osteosarcoma of the rib presents a diagnostic challenge because of the rarity of the lesion, especially with plain radiographs. The tumor should be suspected if CT and MR images demonstrate mineralization, suggestive of an osteoid matrix.

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

    International Nuclear Information System (INIS)

    Zhang, Dawei; Liu, Dandan; Zhang, Jinchao; Fong, Chichun; Yang, Mengsu

    2014-01-01

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

  20. Lecithin blended polyamide-6 high aspect ratio nanofiber scaffolds via electrospinning for human osteoblast cell culture

    Energy Technology Data Exchange (ETDEWEB)

    Nirmala, R. [Bio-nano System Engineering, College of Engineering, Chonbuk National University, Jeonju, 561 756 (Korea, Republic of); Park, Hye-Min [Department of Pharmacology and Toxicology, College of Veterinary Medicine, Chonbuk National University, Jeonju 561 756 (Korea, Republic of); Navamathavan, R. [School of Advanced Materials Engineering, Chonbuk National University, Jeonju 561 756 (Korea, Republic of); Kang, Hyung-Sub [Department of Pharmacology and Toxicology, College of Veterinary Medicine, Chonbuk National University, Jeonju 561 756 (Korea, Republic of); El-Newehy, Mohamed H. [Petrochemical Research Chair, Department of Chemistry, College of Science, King Saud University, Riyadh 11451 (Saudi Arabia); Kim, Hak Yong, E-mail: khy@jbnu.ac.kr [Petrochemical Research Chair, Department of Chemistry, College of Science, King Saud University, Riyadh 11451 (Saudi Arabia); Center for Healthcare Technology and Development, Chonbuk National University, Jeonju, 561 756 (Korea, Republic of)

    2011-03-12

    In this study, we focused on the preparation and characterization of lecithin blended polyamide-6 nanofibers via an electrospinning process for human osteoblastic (HOB) cell culture applications. The morphological, structural characterizations and thermal properties of polyamide-6/lecithin nanofibers were determined by using scanning electron microscopy (SEM), field-emission scanning electron microscopy (FE-SEM), X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, differential scanning calorimetry (DSC) and thermogravimetry (TGA). SEM images revealed that the nanofibers were well-oriented with good incorporation of lecithin. FT-IR results indicated the presence of amino groups of lecithin in the blended nanofibers. TGA analysis revealed that the onset degradation temperature decreased with increasing lecithin content in the blended nanofibers. The morphological features of cells attached on polyamide-6/lecithin nanofibers were confirmed by SEM. The adhesion, viability and proliferation properties of osteoblast cells on the polyamide-6/lecithin blended nanofibers were analyzed by in vitro cell compatibility test. This study demonstrated the non-cytotoxic behavior of electrospun polyamide-6/lecithin nanofibers for the osteoblast cell culture.

  1. Lecithin blended polyamide-6 high aspect ratio nanofiber scaffolds via electrospinning for human osteoblast cell culture

    International Nuclear Information System (INIS)

    Nirmala, R.; Park, Hye-Min; Navamathavan, R.; Kang, Hyung-Sub; El-Newehy, Mohamed H.; Kim, Hak Yong

    2011-01-01

    In this study, we focused on the preparation and characterization of lecithin blended polyamide-6 nanofibers via an electrospinning process for human osteoblastic (HOB) cell culture applications. The morphological, structural characterizations and thermal properties of polyamide-6/lecithin nanofibers were determined by using scanning electron microscopy (SEM), field-emission scanning electron microscopy (FE-SEM), X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, differential scanning calorimetry (DSC) and thermogravimetry (TGA). SEM images revealed that the nanofibers were well-oriented with good incorporation of lecithin. FT-IR results indicated the presence of amino groups of lecithin in the blended nanofibers. TGA analysis revealed that the onset degradation temperature decreased with increasing lecithin content in the blended nanofibers. The morphological features of cells attached on polyamide-6/lecithin nanofibers were confirmed by SEM. The adhesion, viability and proliferation properties of osteoblast cells on the polyamide-6/lecithin blended nanofibers were analyzed by in vitro cell compatibility test. This study demonstrated the non-cytotoxic behavior of electrospun polyamide-6/lecithin nanofibers for the osteoblast cell culture.

  2. Production and action of transforming growth factor-beta in human osteoblast cultures: dependence on cell differentiation and modulation by calcitriol

    DEFF Research Database (Denmark)

    Kassem, M; Kveiborg, Marie; Eriksen, E F

    2000-01-01

    Transforming growth factor beta (TGF-beta) plays an important role in skeletal remodelling. However, few studies have examined its effects on cultured human osteoblasts. Our aim is to characterise the biological effects of TGF-beta1 on human osteoblasts and to examine the interaction between TGF-...

  3. Establishment of 3D culture and induction of osteogenic differentiation of pre-osteoblasts using wet-collected aligned scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Ding, Huifen [Hubei-MOSTKLOS & KLOBM, School and Hospital of Stomatology, Wuhan University, Wuhan 430079 (China); Chongqing Affiliated Hospital of Stomatology, Chongqing University of Medical Sciences, Chongqing 400015 (China); Zhong, Junwen [Wuhan National Laboratory for Optoelectronics, School of Optical and Electronic Information, Huazhong University of Science and Technology, Wuhan 430074 (China); Xu, Fei; Song, Fangfang; Yin, Miao; Wu, Yanru; Hu, Qiyi [Hubei-MOSTKLOS & KLOBM, School and Hospital of Stomatology, Wuhan University, Wuhan 430079 (China); Wang, Jiawei, E-mail: wangjwei@hotmail.com [Hubei-MOSTKLOS & KLOBM, School and Hospital of Stomatology, Wuhan University, Wuhan 430079 (China)

    2017-02-01

    Aligned fibrous scaffolds have attracted much interest in bone tissue engineering, because they are supposed to induce osteogenic differentiation. For the first time, aligned silk fibroin nanofibres were loosely packed using a novel wet-collection electrospinning method. Moreover, three-dimensional (3D) culture of MC3T3-E1 pre-osteoblasts was established on these fibrous scaffolds. Physicochemical properties of the scaffolds and the behaviour of MC3T3-E1 pre-osteoblasts on the scaffolds were analysed and compared with scaffolds obtained using traditional method. Ethanol bath improved the uniformity and alignment of the fibres and increased the thickness and porosity of the scaffolds. Structures of the fibres were well maintained after immediate crosslinking in ethanol. Cells on the wet-collected scaffolds exhibited more ordered arrangement and elongated morphology as well as faster and deeper infiltration. The ordered infiltration resulted in the establishment of the 3D culture of cells, promoting proliferation and osteogenic differentiation of the pre-osteoblasts. Thus, the wet-collected aligned scaffolds with improved topographical and physicochemical properties presents significant potential application in bone regeneration. - Highlights: • Aligned silk fibroin nanofibres were loosely packed using a novel wet-collection electrospinning method. • Structural properties of the aligned nanofibres were improved. • Three-dimensional culture of MC3T3-E1 pre-osteoblasts was established. • The arrangement, morphology, infiltration, proliferation and osteogenic differentiation of cells were enhanced.

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

    Science.gov (United States)

    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.

  5. Vitamin D activation of functionally distinct regulatory miRNAs in primary human osteoblasts.

    Science.gov (United States)

    Lisse, Thomas S; Chun, Rene F; Rieger, Sandra; Adams, John S; Hewison, Martin

    2013-06-01

    When bound to the vitamin D receptor (VDR), the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25D) is a potent regulator of osteoblast transcription. Less clear is the impact of 1,25D on posttranscriptional events in osteoblasts, such as the generation and action of microRNAs (miRNAs). Microarray analysis using replicate (n = 3) primary cultures of human osteoblasts (HOBs) identified human miRNAs that were differentially regulated by >1.5-fold following treatment with 1,25D (10 nM, 6 hours), which included miRNAs 637 and 1228. Quantitative reverse transcription PCR analyses showed that the host gene for miR-1228, low-density lipoprotein receptor-related protein 1 (LRP1), was coinduced with miR-1228 in a dose-dependent fashion following treatment with 1,25D (0.1-10 nM, 6 hours). By contrast, the endogenous host gene for miR-637, death-associated protein kinase 3 (DAPK3), was transcriptionally repressed by following treatment with 1,25D. Analysis of two potential targets for miR-637 and miR-1228 in HOB, type IV collagen (COL4A1) and bone morphogenic protein 2 kinase (BMP2K), respectively, showed that 1,25D-mediates suppression of these targets via distinct mechanisms. In the case of miR-637, suppression of COL4A1 appears to occur via decreased levels of COL4A1 mRNA. By contrast, suppression of BMP2K by miR-1228 appears to occur by inhibition of protein translation. In mature HOBs, small interfering RNA (siRNA) inactivation of miR-1228 alone was sufficient to abrogate 1,25D-mediated downregulation of BMP2K protein expression. This was associated with suppression of prodifferentiation responses to 1,25D in HOB, as represented by parallel decrease in osteocalcin and alkaline phosphatase expression. These data show for the first time that the effects of 1,25D on human bone cells are not restricted to classical VDR-mediated transcriptional responses but also involve miRNA-directed posttranscriptional mechanisms. Copyright © 2013 American Society for Bone and

  6. Four-point bending protocols to study the effects of dynamic strain in osteoblastic cells in vitro.

    Science.gov (United States)

    Galea, Gabriel L; Price, Joanna S

    2015-01-01

    Strain engendered within bone tissue by mechanical loading of the skeleton is a major influence on the processes of bone modeling and remodeling and so a critical determinant of bone mass and architecture. The cells best placed to respond to strain in bone tissue are the resident osteocytes and osteoblasts. To address the mechanisms of strain-related responses in osteoblast-like cells, our group uses both in vivo and in vitro approaches, including a system of four-point bending of the substrate on which cells are cultured. A range of cell lines can be studied using this system but we routinely compare their responses to those in primary cultures of osteoblast-like cells derived from explants of mouse long bones. These cells show a range of well-characterized responses to physiological levels of strain, including increased proliferation, which in vivo is a feature of the osteogenic response.

  7. Tridax procumbens flavonoids promote osteoblast differentiation and bone formation

    Directory of Open Access Journals (Sweden)

    Md. Abdullah Al Mamun

    Full Text Available BACKGROUND: Tridaxprocumbens 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 RESULTS: 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 CONCLUSION: 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

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

    Science.gov (United States)

    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

  9. Activating AMP-activated protein kinase by an α1 selective activator compound 13 attenuates dexamethasone-induced osteoblast cell death

    International Nuclear Information System (INIS)

    Guo, Shiguang; Mao, Li; Ji, Feng; Wang, Shouguo; Xie, Yue; Fei, Haodong; Wang, Xiao-dong

    2016-01-01

    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 other 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.

  10. Activating AMP-activated protein kinase by an α1 selective activator compound 13 attenuates dexamethasone-induced osteoblast cell death

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Shiguang [Department of Intensive Care Unit, Huai' an First People' s Hospital, Nanjing Medical University, Huai' an (China); Mao, Li [Department of Endocrinology, Huai' an First People' s Hospital, Nanjing Medical University, Huai' an (China); Ji, Feng, E-mail: huaiaifengjidr@163.com [Department of Orthopedics, Huai' an First People' s Hospital, Nanjing Medical University, Huai' an (China); Wang, Shouguo; Xie, Yue; Fei, Haodong [Department of Orthopedics, Huai' an First People' s Hospital, Nanjing Medical University, Huai' an (China); Wang, Xiao-dong, E-mail: xiaodongwangsz@163.com [The Center of Diagnosis and Treatment for Children' s Bone Diseases, The Children' s Hospital Affiliated to Soochow University, Suzhou (China)

    2016-03-18

    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 other 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.

  11. Ihh signaling is directly required for the osteoblast lineage in the endochondral skeleton.

    Science.gov (United States)

    Long, Fanxin; Chung, Ung-il; Ohba, Shinsuke; McMahon, Jill; Kronenberg, Henry M; McMahon, Andrew P

    2004-03-01

    Indian hedgehog (Ihh) is indispensable for development of the osteoblast lineage in the endochondral skeleton. In order to determine whether Ihh is directly required for osteoblast differentiation, we have genetically manipulated smoothened (Smo), which encodes a transmembrane protein that is essential for transducing all Hedgehog (Hh) signals. Removal of Smo from perichondrial cells by the Cre-LoxP approach prevents formation of a normal bone collar and also abolishes development of the primary spongiosa. Analysis of chimeric embryos composed of wild-type and Smo(n/n) cells indicates that Smo(n/n) cells fail to contribute to osteoblasts in either the bone collar or the primary spongiosa but generate ectopic chondrocytes. In order to assess whether Ihh is sufficient to induce bone formation in vivo, we have analyzed the bone collar in the long bones of embryos in which Ihh was artificially expressed in all chondrocytes by the UAS-GAL4 bigenic system. Although ectopic Ihh does not induce overt ossification along the entire cartilage anlage, it promotes progression of the bone collar toward the epiphysis, suggesting a synergistic effect between ectopic Ihh and endogenous factors such as the bone morphogenetic proteins (BMPs). In keeping with this model, Hh signaling is further found to be required in BMP-induced osteogenesis in cultures of a limb-bud cell line. Taken together, these results demonstrate that Ihh signaling is directly required for the osteoblast lineage in the developing long bones and that Ihh functions in conjunction with other factors such as BMPs to induce osteoblast differentiation. We suggest that Ihh acts in vivo on a potential progenitor cell to promote osteoblast and prevent chondrocyte differentiation.

  12. Differentiation and cytokine synthesis of human alveolar osteoblasts compared to osteoblast-like cells (MG63) in response to titanium surfaces.

    Science.gov (United States)

    Rausch-fan, Xiaohui; Qu, Zhe; Wieland, Marco; Matejka, Michael; Schedle, Andreas

    2008-01-01

    The aim of this study was to investigate the influence of different implant surface topographies and chemistries on the expression of differentiation/proliferation markers on MG63 cells and primary human alveolar osteoblasts. Hydrophobic acid-etched (A) and hydrophobic coarse-grit-blasted, acid-etched (SLA) surfaces and hydrophilic acid-etched (modA) and hydrophilic coarse-grit-blasted (modSLA) surfaces were produced. Thereby, modA and modSLA surfaces were rinsed under nitrogen protection and stored in a sealed glass tube containing isotonic NaCl solution at pH 4-6. Tissue culture plates without specimens served as controls. The behavior of MG63 cells and primary human alveolar osteoblasts (AOB) grown on all surfaces was compared through determination of alkaline phosphatase (ALP) activity, cell proliferation ((3)H-thymidin incorporation, MTT colorimetric assay) and expression of osteocalcin (OC), osteoprotegerin (OPG), transforming growth factor-beta1 (TGF-beta(1)) and vascular endothelial growth factor (VEGF), detected with commercial available test kits. Proliferation of MG63 and primary cells was highest on controls, followed by A surfaces, modA and SLA surfaces being almost on the same level and lowest on modSLA surfaces. modSLA surfaces exhibited highest ALP and OC production, followed by SLA, modA and A surfaces. Proliferation and OC production were comparable for MG63 cells and AOB. OPG, TGF-beta(1) and VEGF produced on primary cells showed a slightly different rank order on different surfaces compared to MG63 cells. modSLA still showed the highest production of OPG, TGF-beta(1) and VEGF, but was followed by modA, SLA and A. Statistical significance was checked by ANOVA (pmodA surfaces showed enhanced expression of OPG, TGF-beta(1) and VEGF on MG63 cells compared to primary human alveolar osteoblasts. Overall, the lowest proliferation rates and the highest expressions of differentiation markers and growth factor productions were observed on modSLA.

  13. Effects of imatinib and nilotinib on the whole transcriptome of cultured murine osteoblasts.

    Science.gov (United States)

    Kirschner, Gyöngyi; Balla, Bernadett; Horváth, Péter; Kövesdi, Andrea; Lakatos, Gergely; Takács, István; Nagy, Zsolt; Tóbiás, Bálint; Árvai, Kristóf; Kósa, János Pál; Lakatos, Péter

    2016-09-01

    Numerous clinical observations have confirmed that breakpoint cluster region-abelson fusion oncoprotein tyrosine kinase inhibitors used in leukemia treatment alter bone physiology in a complex manner. The aim of the present study was to analyze the whole transcriptome of cultured murine osteoblasts and determine the changes following treatment with imatinib and nilotinib using Sequencing by Oligonucleotide Ligation and Detection next generation RNA sequencing. This study also aimed to identify candidate signaling pathways and network regulators by multivariate Ingenuity Pathway Analysis. Based on the right-tailed Fisher's exact test, significantly altered pathways including upstream regulators were defined for each drug. The correlation between these pathways and bone metabolism was also examined. The preliminary results suggest the two drugs have different mechanisms of action on osteoblasts, and imatinib was shown to have a greater effect on gene expression. Data also indicated the potential role of a number of genes and signaling cascades that may contribute to identifying novel targets for the treatment of metabolic bone diseases.

  14. The Role of MKP-1 in the Anti-Proliferative Effects of Glucocorticoids in Primary Rat Pre-Osteoblasts.

    Directory of Open Access Journals (Sweden)

    Micheline Sanderson

    Full Text Available Glucocorticoid (GC-induced osteoporosis has been attributed to a GC-induced suppression of pre-osteoblast proliferation. Our previous work identified a critical role for mitogen-activated protein kinase (MAPK phosphatase-1 (MKP-1 in mediating the anti-proliferative effects of GCs in immortalized pre-osteoblasts, but we subsequently found that MKP-1 null mice were not protected against the pathological effects of GCs on bone. In order to reconcile this discrepancy, we have assessed the effects of GCs on proliferation, activation of the MAPK ERK1/2 and MKP-1 expression in primary adipose-derived stromal cells (ADSCs and ADSC-derived pre-osteoblasts (ADSC-OBs. ADSCs were isolated by means of collagenase digestion from adipose tissue biopsies harvested from adult male Wistar rats. ADSC-OBs were prepared by treating ADSCs with osteoblast differentiation media for 7 days. The effects of increasing concentrations of the GC dexamethasone on basal and mitogen-stimulated cell proliferation were quantified by tritiated thymidine incorporation. ERK1/2 activity was measured by Western blotting, while MKP-1 expression was quantified on both RNA and protein levels, using semi-quantitative real-time PCR and Western blotting, respectively. GCs were strongly anti-proliferative in both naïve ADSCs and ADSC-OBs, but had very little effect on mitogen-induced ERK1/2 activation and did not upregulate MKP-1 protein expression. These findings suggest that the anti-proliferative effects of GCs in primary ADSCs and ADSC-OBs in vitro do not require the inhibition of ERK1/2 activation by MKP-1, which is consistent with our in vivo findings in MKP-1 null mice.

  15. Osteoblast cell response to surface-modified carbon nanotubes

    International Nuclear Information System (INIS)

    Zhang Faming; Weidmann, Arne; Nebe, J. Barbara; Burkel, Eberhard

    2012-01-01

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

  16. Plant-derived pectin nanocoatings to prevent inflammatory cellular response of osteoblasts following Porphyromonas gingivalis infection

    Directory of Open Access Journals (Sweden)

    Meresta A

    2017-01-01

    Full Text Available Anna Meresta,1 Justyna Folkert,1 Timo Gaber,2 Korneliusz Miksch,1 Frank Buttgereit,2 Jacqueline Detert,2 Nicole Pischon,3,* Katarzyna Gurzawska3,4,* 1Environmental Biotechnology Department, Faculty of Power and Environmental, Silesian University of Technology, Gliwice, Poland; 2Department of Rheumatology and Clinical Immunology, 3Department of Periodontology, Charité University Medicine, Berlin, Germany; 4Oral Surgery Department, The School of Dentistry, University of Birmingham, Birmingham, UK *These authors contributed equally to this work Background: Bioengineered plant-derived Rhamnogalacturonan-Is (RG-Is from pectins are potential candidates for surface nanocoating of medical devices. It has recently been reported that RG-I nanocoatings may prevent bacterial infection and improve the biocompatibility of implants. The aim of the study was to evaluate in vitro impact of bioengineered RG-I nanocoatings on osteogenic capacity and proinflammatory cytokine response of murine osteoblasts following Porphyromonas gingivalis infection.Methods: Murine MC3T3-E1 osteoblasts and isolated primary calvarial osteoblasts from C57BL/6J (B6J osteoblasts mice were infected with P. gingivalis and incubated on tissue culture polystyrene plates with or without nanocoatings of unmodified RG-Is isolated from potato pulps (PU or dearabinanated RG-Is (PA. To investigate a behavior of infected osteoblasts cultured on RG-Is cell morphology, proliferation, metabolic activity, mineralization and osteogenic and pro-inflammatory gene expression were examined.Results: Following P. gingivalis infection, PA, but not PU, significantly promoted MC3T3-E1 and BJ6 osteoblasts proliferation, metabolic activity, and calcium deposition. Moreover, Il-1b, Il-6, TNF-α, and Rankl gene expressions were downregulated in cells cultured on PU and to a higher extent on PA as compared to the corresponding control, whereas Runx, Alpl, Col1a1, and Bglap gene expressions were upregulated vice

  17. 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.

    Science.gov (United States)

    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

  18. Toxicity of iron oxide nanoparticles against osteoblasts

    International Nuclear Information System (INIS)

    Shi Sifeng; Jia Jingfu; Guo Xiaokui; Zhao Yaping; Liu Boyu; Chen Desheng; Guo Yongyuan; Zhang Xianlong

    2012-01-01

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

  19. Toxicity of iron oxide nanoparticles against osteoblasts

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-09-15

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

  20. Retroviral-mediated gene therapy for the differentiation of primary cells into a mineralizing osteoblastic phenotype.

    Science.gov (United States)

    Phillips, Jennifer E; García, Andrés J

    2008-01-01

    Bone tissue engineering has emerged as a promising strategy for the repair of critical-sized skeletal fractures. However, the clinical application of this approach has been limited by the availability of a robust mineralizing cell source. Non-osteogenic cells, such as skin fibroblasts, are an attractive cell-source alternative because they are easy to harvest from autologous donor skin biopsies and display a high capacity for in vitro expansion. We have recently demonstrated that retroviral gene delivery of the osteoblastic transcription factor Runx2/Cbfa1 promotes osteogenic differentiation in primary dermal fibroblasts cultured in monolayer. Notably, sustained expression of Runx2 was not sufficient to promote functional osteogenesis in these cells, and co-treatment with the steroid hormone dexamethasone was required to induce deposition of biologically-equivalent matrix mineralization. On the basis of these results, we then investigated the osteogenic capacity of these genetically engineered fibroblasts when seeded on polymeric scaffolds in vitro and in vivo. These experiments demonstrated that Runx2-expressing fibroblasts seeded on collagen scaffolds produce significant levels of matrix mineralization after 28 days in vivo implantation in a subcutaneous, heterotopic site. Overall, these results offer evidence that transcription factor-based gene therapy may be a powerful strategy for the conversion of a non-osteogenic cellular phenotype into a mineralizing cell source for bone repair applications. This concept may also be applied to control functional differentiation in a broad range of cell types and tissue engineering applications. The chapter below outlines detailed methods for the isolation and ex vivo genetic modification of primary dermal fibroblasts using retroviral-mediated delivery of the Runx2 transgene in both monolayer culture and three-dimensional scaffolds.

  1. Strontium-Substituted Bioceramics Particles: A New Way to Modulate MCP-1 and Gro-α Production by Human Primary Osteoblastic Cells

    Directory of Open Access Journals (Sweden)

    Julien Braux

    2016-12-01

    Full Text Available Background: To avoid morbidity and limited availability associated with autografts, synthetic calcium phosphate (CaP ceramics were extensively developed and used as bone filling materials. Controlling their induced-inflammatory response nevertheless remained a major concern. Strontium-containing CaP ceramics were recently demonstrated for impacting cytokines’ secretion pattern of human primary monocytes. The present study focuses on the ability of strontium-containing CaP to control the human primary bone cell production of two major inflammatory and pro-osteoclastogenic mediators, namely MCP-1 and Gro-α, in response to ceramics particles. Methods: This in vitro study was performed using human primary osteoblasts in which their response to ceramics was evaluated by PCR arrays, antibody arrays were used for screening and real-time PCR and ELISA for more focused analyses. Results: Study of mRNA and protein expression highlights that human primary bone cells are able to produce these inflammatory mediators and reveal that the adjunction of CaP in the culture medium leads to their enhanced production. Importantly, the current work determines the down-regulating effect of strontium-substituted CaP on MCP-1 and Gro-α production. Conclusion: Our findings point out a new capability of strontium to modulate human primary bone cells’ communication with the immune system.

  2. Optimization of the Static Human Osteoblast/Osteoclast Co-culture System

    Directory of Open Access Journals (Sweden)

    James Jam Jolly

    2018-03-01

    Full Text Available Osteoblasts (OBs and osteoclasts (OCs are 2 major groups of bone cells. Their cell-to-cell interactions are important to ensure the continuity of the bone-remodeling process. Therefore, the present study was carried out to optimize an OB/OC co-culture system utilizing the human OB cell line hFOB 1.19 and OCs extracted from peripheral blood mononuclear cells (PBMNCs. It was a 2-step procedure, involving the optimization of the OB culture and the co-culture of the OBs with PBMNCs at an optimum ratio. Firstly, pre-OBs were cultured to 90% confluency and the time required for differentiation was determined. OB differentiation was determined using the van Gieson staining to detect the presence of collagen and Alizarin Red for calcium. Secondly, OBs and OCs were co-cultured at the ratios of 1 OC: 1 OB, 1 OC: 4 OBs, 2 OCs: 1 OB, and 1 OC: 2 OBs. Tartrate-resistant acid phosphatase (TRAP staining was used to detect the differentiation of the OCs. The results showed that collagen was present on day 1, whereas calcium was detected as early as day 3. Based on the result of TRAP staining, 1 OC: 2 OBs was taken as the most appropriate ratio. No macrophage colony-stimulating factor and receptor activator of the nuclear factor-κB ligand were added because they were provided by the OBs. In conclusion, these optimization processes are vital as they ensure the exact time point and ratio of the OB/OC co-culture in order to produce a reliable and reproducible co-culture system.

  3. Osteoblast-specific transcription factor Osterix increases vitamin D receptor gene expression in osteoblasts.

    Directory of Open Access Journals (Sweden)

    Chi Zhang

    Full Text Available Osterix (Osx is an osteoblast-specific transcription factor required for osteoblast differentiation from mesenchymal stem cells. In Osx knock-out mice, no bone formation occurs. The vitamin D receptor (VDR is a member of the nuclear hormone receptor superfamily that regulates target gene transcription to ensure appropriate control of calcium homeostasis and bone development. Here, we provide several lines of evidence that show that the VDR gene is a target for transcriptional regulation by Osx in osteoblasts. For example, calvaria obtained from Osx-null embryos displayed dramatic reductions in VDR expression compared to wild-type calvaria. Stable overexpression of Osx stimulated VDR expression in C2C12 mesenchymal cells. Inhibition of Osx expression by siRNA led to downregulation of VDR. In contrast, Osx levels remained unchanged in osteoblasts in VDR-null mice. Mechanistic approaches using transient transfection assays showed that Osx directly activated a 1 kb fragment of the VDR promoter in a dose-dependent manner. To define the region of the VDR promoter that was responsive to Osx, a series of VDR promoter deletion mutants were examined and the minimal Osx-responsive region was refined to the proximal 120 bp of the VDR promoter. Additional point mutants were used to identify two GC-rich regions that were responsible for VDR promoter activation by Osx. Chromatin immunoprecipitation assays demonstrated that endogenous Osx was associated with the native VDR promoter in primary osteoblasts in vivo. Cumulatively, these data strongly support a direct regulatory role for Osx in VDR gene expression. They further provide new insight into potential mechanisms and pathways that Osx controls in osteoblasts and during the process of osteoblastic cell differentiation.

  4. Quantification of carbon nanotube induced adhesion of osteoblast on hydroxyapatite using nano-scratch technique

    International Nuclear Information System (INIS)

    Lahiri, Debrupa; Agarwal, Arvind; Benaduce, Ana Paula; Kos, Lidia

    2011-01-01

    This paper explores the nano-scratch technique for measuring the adhesion strength of a single osteoblast cell on a hydroxyapatite (HA) surface reinforced with carbon nanotubes (CNTs). This technique efficiently separates out the contribution of the environment (culture medium and substrate) from the measured adhesion force of the cell, which is a major limitation of the existing techniques. Nano-scratches were performed on plasma sprayed hydroxyapatite (HA) and HA-CNT coatings to quantify the adhesion of the osteoblast. The presence of CNTs in HA coating promotes an increase in the adhesion of osteoblasts. The adhesion force and energy of an osteoblast on a HA-CNT surface are 17 ± 2 μN/cell and 78 ± 14 pJ/cell respectively, as compared to 11 ± 2 μN/cell and 45 ± 10 pJ/cell on a HA surface after 1 day of incubation. The adhesion force and energy of the osteoblasts increase on both the surfaces with culture periods of up to 5 days. This increase is more pronounced for osteoblasts cultured on HA-CNT. Staining of actin filaments revealed a higher spreading and attachment of osteoblasts on a surface containing CNTs. The affinity of CNTs to conjugate with integrin and other proteins is responsible for the enhanced attachment of osteoblasts. Our results suggest that the addition of CNTs to surfaces used in medical applications may be beneficial when stronger adhesion of osteoblasts is desired.

  5. Serotonin regulates osteoblast proliferation and function in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Dai, S.Q.; Yu, L.P. [Department of Orthopedic Surgery, The First Affiliated Hospital, Nanjing Medical University, Nanjing, Jiangsu (China); Shi, X. [Department of Obstetrics and Gynecology, The First Affiliated Hospital, Nanjing Medical University, Nanjing, Jiangsu (China); Wu, H. [Emergency Department, The First Affiliated Hospital, Soochow University, Suzhou (China); Shao, P.; Yin, G.Y.; Wei, Y.Z. [Department of Orthopedic Surgery, The First Affiliated Hospital, Nanjing Medical University, Nanjing, Jiangsu (China)

    2014-08-01

    The monoamine serotonin (5-hydroxytryptamine, 5-HT), a well-known neurotransmitter, also has important functions outside the central nervous system. The objective of this study was to investigate the role of 5-HT in the proliferation, differentiation, and function of osteoblasts in vitro. We treated rat primary calvarial osteoblasts with various concentrations of 5-HT (1 nM to 10 µM) and assessed the rate of osteoblast proliferation, expression levels of osteoblast-specific proteins and genes, and the ability to form mineralized nodules. Next, we detected which 5-HT receptor subtypes were expressed in rat osteoblasts at different stages of osteoblast differentiation. We found that 5-HT could inhibit osteoblast proliferation, differentiation, and mineralization at low concentrations, but this inhibitory effect was mitigated at relatively high concentrations. Six of the 5-HT receptor subtypes (5-HT{sub 1A}, 5-HT{sub 1B}, 5-HT{sub 1D}, 5-HT{sub 2A}, 5-HT{sub 2B}, and 5-HT{sub 2C}) were found to exist in rat osteoblasts. Of these, 5-HT{sub 2A} and 5-HT{sub 1B} receptors had the highest expression levels, at both early and late stages of differentiation. Our results indicated that 5-HT can regulate osteoblast proliferation and function in vitro.

  6. Culture conditions for equine bone marrow mesenchymal stem cells and expression of key transcription factors during their differentiation into osteoblasts

    Science.gov (United States)

    2013-01-01

    Background The use of equine bone marrow mesenchymal stem cells (BMSC) is a novel method to improve fracture healing in horses. However, additional research is needed to identify optimal culture conditions and to determine the mechanisms involved in regulating BMSC differentiation into osteoblasts. The objectives of the experiments were to determine: 1) if autologous or commercial serum is better for proliferation and differentiation of equine BMSC into osteoblasts, and 2) the expression of key transcription factors during the differentiation of equine BMSC into osteoblasts. Equine BMSC were isolated from the sterna of 3 horses, treated with purchased fetal bovine serum (FBS) or autologous horse serum (HS), and cell proliferation determined. To induce osteoblast differentiation, cells were incubated with L-ascorbic acid-2-phosphate and glycerol-2-phosphate in the presence or absence of human bone morphogenetic protein2 (BMP2), dexamethasone (DEX), or combination of the two. Alkaline phosphatase (ALP) activity, a marker of osteoblast differentiation, was determined by ELISA. Total RNA was isolated from differentiating BMSC between d 0 to 18 to determine expression of runt-related transcription factor2 (Runx2), osterix (Osx), and T-box3 (Tbx3). Data were analyzed by ANOVA. Results Relative to control, FBS and HS increased cell number (133 ± 5 and 116 ± 5%, respectively; P  0.8). Runt-related transcription factor2 expression increased 3-fold (P equine BMSC into osteoblasts. In addition, expression of Runx2 and osterix increased and expression of Tbx3 is reduced during differentiation. PMID:24169030

  7. Platelet-rich plasma stimulates osteoblastic differentiation in the presence of BMPs

    International Nuclear Information System (INIS)

    Tomoyasu, Akihiro; Higashio, Kanji; Kanomata, Kazuhiro; Goto, Masaaki; Kodaira, Kunihiko; Serizawa, Hiroko; Suda, Tatsuo; Nakamura, Atsushi; Nojima, Junya; Fukuda, Toru; Katagiri, Takenobu

    2007-01-01

    Platelet-rich plasma (PRP) is clinically used as an autologous blood product to stimulate bone formation in vivo. In the present study, we examined the effects of PRP on proliferation and osteoblast differentiation in vitro in the presence of bone morphogenetic proteins (BMPs). PRP and its soluble fraction stimulated osteoblastic differentiation of myoblasts and osteoblastic cells in the presence of BMP-2, BMP-4, BMP-6 or BMP-7. The soluble PRP fraction stimulated osteoblastic differentiation in 3D cultures using scaffolds made of collagen or hydroxyapatite. Moreover, heparin-binding fractions obtained from serum also stimulated osteoblastic differentiation in the presence of BMP-4. These results suggested that platelets contain not only growth factors for proliferation but also novel potentiator(s) for BMP-dependent osteoblastic differentiation

  8. Resveratrol inhibits myeloma cell growth, prevents osteoclast formation, and promotes osteoblast differentiation

    DEFF Research Database (Denmark)

    Boissy, Patrice; Andersen, Thomas L; Abdallah, Basem M

    2005-01-01

    , a challenge for treating multiple myeloma is discovering drugs targeting not only myeloma cells but also osteoclasts and osteoblasts. Because resveratrol (trans-3,4',5-trihydroxystilbene) is reported to display antitumor activities on a variety of human cancer cells, we investigated the effects...... of this natural compound on myeloma and bone cells. We found that resveratrol reduces dose-dependently the growth of myeloma cell lines (RPMI 8226 and OPM-2) by a mechanism involving cell apoptosis. In cultures of human primary monocytes, resveratrol inhibits dose-dependently receptor activator of nuclear factor......RNA and cell surface protein levels and a decrease of NFATc1 stimulation and NF-kappaB nuclear translocation, whereas the gene expression of c-fms, CD14, and CD11a is up-regulated. Finally, resveratrol promotes dose-dependently the expression of osteoblast markers like osteocalcin and osteopontin in human bone...

  9. Induction of a program gene expression during osteoblast differentiation with strontium ranelate

    International Nuclear Information System (INIS)

    Zhu Lingling; Zaidi, Samir; Peng Yuanzhen; Zhou Hang; Moonga, Baljit S.; Blesius, Alexia; Dupin-Roger, Isabelle; Zaidi, Mone; Sun Li

    2007-01-01

    Strontium ranelate, a new agent for the treatment of osteoporosis, has been shown stimulate bone formation in various experimental models. This study examines the effect of strontium ranelate on gene expression in osteoblasts, as well as the formation of mineralized (von Kossa-positive) colony-forming unit-osteoblasts (CFU-obs). Bone marrow-derived stromal cells cultured for 21 days under differentiating conditions, when exposed to strontium ranelate, displayed a significant time- and concentration-dependent increase in the expression of the master gene, Runx2, as well as bone sialoprotein (BSP), but interestingly without effects on osteocalcin. This was associated with a significant increase in the formation of CFU-obs at day 21 of culture. In U-33 pre-osteoblastic cells, strontium ranelate significantly enhanced the expression of Runx2 and osteocalcin, but not BSP. Late, more mature osteoblastic OB-6 cells showed significant elevations in BSP and osteocalcin, but with only minimal effects on Runx2. In conclusion, strontium ranelate stimulates osteoblast differentiation, but the induction of the program of gene expression appears to be cell type-specific. The increased osteoblastic differentiation is the likely basis underlying the therapeutic bone-forming actions of strontium ranelate

  10. Evaluation of low doses of gamma irradiation in the formation of mineralization nodules in osteoblasts culture

    Energy Technology Data Exchange (ETDEWEB)

    Targino, Bárbara; Pinto, Thais Lazarine; Silva, Evily Fernandes; Somessari, E.S.R.; Bellini, Maria Helena; Affonso, Regina [Instituto De Pesquisas Energéticas e Nucleares (IPEN/CNEN-SP), São Paulo, SP (Brazil)

    2017-07-01

    Full text: Introduction: Osteoblasts are specialized fibroblasts that secrete and mineralize the bone matrix. The mineralized extracellular matrix is mainly composed of type I collagen, osteocalcin, and the inorganic mineral hydroxylapatite1. The use of radiation as therapy in some cancers causes great bone loss. However, low dose radiation may have the opposite effect. Low dose X-irradiation on osteoblastic culture had effects on proliferation and differentiation with increase of mineralization nodules2. However, there is little information on the potential therapeutic efficacy of low-dose gamma-irradiation in the formation of mineralization nodules. Objective: To evaluate the effects of irradiation with 60Co γ-rays in low doses in the formation of mineralization nodules in culture of osteoblasts. Methods: MC3T3-E1 cells were bought by the Banco de Células do Rio de Janeiro, Brazil (MC3T3-E1 Subclone 14). The cells were cultured in α-MEM medium consisting of 10% FBS and without β-glycerophosphate and L-ascorbic acid (GIBCO, Custom Product, Catalog No. A1049001) (Zhao Y, Guan H, Liu S et al. Biol. Pharm. Bull. 2005, 28(8):1371-1376). Plating efficiency assays: cells were plated at a density of 100 cell/plate into 60 mm Petri dishes. After 14 days the places were stained with violet crystal and the colonies were counted. -glycerophosphate and 50 mg/ml ascorbic acid, and analyzed on days 7, 14 and 21. Osteoblast culture irradiation assay: cells were plated at a density of 1 x 105 cells/plate on 60 mm dishes and the next day were irradiated by 60Co source with 0 (as the control), 0.5, 1.0, 1.5 and 2.0 Gy using the GammaCell 220 - Irradiation Unit of Canadian-Atomic Energy Commission Ltd. (CTR-IPEN). On day 21 of culture, undifferentiated (without ascorbic acid and β-glycerophosphate), differentiating cells (0 Gy) and irradiated cells at different doses, the medium was removed, cells were washed with phosphate buffer saline, fixed with 70% ethyl alcohol and

  11. Evaluation of low doses of gamma irradiation in the formation of mineralization nodules in osteoblasts culture

    International Nuclear Information System (INIS)

    Targino, Bárbara; Pinto, Thais Lazarine; Silva, Evily Fernandes; Somessari, E.S.R.; Bellini, Maria Helena; Affonso, Regina

    2017-01-01

    Full text: Introduction: Osteoblasts are specialized fibroblasts that secrete and mineralize the bone matrix. The mineralized extracellular matrix is mainly composed of type I collagen, osteocalcin, and the inorganic mineral hydroxylapatite1. The use of radiation as therapy in some cancers causes great bone loss. However, low dose radiation may have the opposite effect. Low dose X-irradiation on osteoblastic culture had effects on proliferation and differentiation with increase of mineralization nodules2. However, there is little information on the potential therapeutic efficacy of low-dose gamma-irradiation in the formation of mineralization nodules. Objective: To evaluate the effects of irradiation with 60Co γ-rays in low doses in the formation of mineralization nodules in culture of osteoblasts. Methods: MC3T3-E1 cells were bought by the Banco de Células do Rio de Janeiro, Brazil (MC3T3-E1 Subclone 14). The cells were cultured in α-MEM medium consisting of 10% FBS and without β-glycerophosphate and L-ascorbic acid (GIBCO, Custom Product, Catalog No. A1049001) (Zhao Y, Guan H, Liu S et al. Biol. Pharm. Bull. 2005, 28(8):1371-1376). Plating efficiency assays: cells were plated at a density of 100 cell/plate into 60 mm Petri dishes. After 14 days the places were stained with violet crystal and the colonies were counted. -glycerophosphate and 50 mg/ml ascorbic acid, and analyzed on days 7, 14 and 21. Osteoblast culture irradiation assay: cells were plated at a density of 1 x 105 cells/plate on 60 mm dishes and the next day were irradiated by 60Co source with 0 (as the control), 0.5, 1.0, 1.5 and 2.0 Gy using the GammaCell 220 - Irradiation Unit of Canadian-Atomic Energy Commission Ltd. (CTR-IPEN). On day 21 of culture, undifferentiated (without ascorbic acid and β-glycerophosphate), differentiating cells (0 Gy) and irradiated cells at different doses, the medium was removed, cells were washed with phosphate buffer saline, fixed with 70% ethyl alcohol and

  12. Detachment strength of human osteoblasts cultured on hydroxyapatite with various surface roughness. Contribution of integrin subunits.

    Science.gov (United States)

    Kokkinos, Petros A; Koutsoukos, Petros G; Deligianni, Despina D

    2012-06-01

    Hydroxyapatite (HA) has been widely used as a bone substitute in dental, maxillofacial and orthopaedic surgery and as osteoconductive bone substitute or precoating of pedicle screws and cages in spine surgery. The aim of the present study was to investigate the osteoblastic adhesion strength on HA substrata with different surface topography and biochemistry (pre-adsorption of fibronectin) after blocking of specific integrin subunits with monoclonal antibodies. Stoichiometric HA was prepared by precipitation followed by ageing and characterized by SEM, EDX, powder XRD, Raman spectroscopy, TGA, and specific surface area analysis. Human bone marrow derived osteoblasts were cultured on HA disc-shaped substrata which were sintered and polished resulting in two surface roughness grades. For attachment evaluation, cells were incubated with monoclonal antibodies and seeded for 2 h on the substrata. Cell detachment strength was determined using a rotating disc device. Cell detachment strength was surface roughness, fibronectin preadsorption and intergin subunit sensitive.

  13. Lactate induces osteoblast differentiation by stabilization of HIF1α.

    Science.gov (United States)

    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.

  14. Osteoblast Differentiation Decreases Hypergravity-Stimulated Release of PGE(sub 2)

    Science.gov (United States)

    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. Maturation of osteoblast-like SaoS2 induced by carbon nanotubes

    International Nuclear Information System (INIS)

    Li Xiaoming; Uo, Motohiro; Akasaka, Tsukasa; Abe, Shigeaki; Watari, Fumio; Gao Hong; Sato, Yoshinori; Feng Qingling; Cui Fuzhai

    2009-01-01

    Osteogenic maturation of the osteoblast is crucial for bone formation. In this study, multi-walled carbon nanotubes (MWCNTs) and graphite (GP) were pressed as compacts. The greater ability of carbon nanotubes to adsorb proteins, compared with graphite, was shown. Human osteoblast-like SaoS2 cells were cultured and the cell response to the two kinds of compacts was compared in vitro. Meanwhile, we used cell culture on the culture plate as a control. Assays for osteonectin, osteopontin and osteocalcin gene expression, total protein (TP) amount, alkaline phosphatase activity (ALP) and DNA of cells cultured on the samples were done. During the conventional culture, significantly higher osteonectin, osteopontin and osteocalcin gene expression level, ALP/DNA and TP/DNA on carbon nanotubes were found. To confirm the hypothesis that the larger amount of specific proteins adsorbed on the carbon nanotubes was crucial for this, the compacts were pre-soaked in culture medium having additional recombinant human bone morphogenetic protein-2 (rhBMP-2) before cell culture. Compared with GP, osteonectin, osteopontin and osteocalcin gene expression level, ALP/DNA and TP/DNA of the cells tested increased more on the MWCNTs after the compacts were pre-soaked in the culture medium with rhBMP-2. The results indicated that the carbon nanotubes might induce osteogenic maturation of the osteoblast by adsorbing more specific proteins.

  16. Primary cultures of astrocytes

    DEFF Research Database (Denmark)

    Lange, Sofie C; Bak, Lasse Kristoffer; Waagepetersen, Helle S

    2012-01-01

    During the past few decades of astrocyte research it has become increasingly clear that astrocytes have taken a central position in all central nervous system activities. Much of our new understanding of astrocytes has been derived from studies conducted with primary cultures of astrocytes...... subsequently found in vivo. Nevertheless, primary cultures of astrocytes are an in vitro model that does not fully mimic the complex events occurring in vivo. Here we present an overview of the numerous contributions generated by the use of primary astrocyte cultures to uncover the diverse functions...... of astrocytes. Many of these discoveries would not have been possible to achieve without the use of astrocyte cultures. Additionally, we address and discuss the concerns that have been raised regarding the use of primary cultures of astrocytes as an experimental model system....

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  19. Design of a bioresorbable polymeric scaffold for osteoblast culture

    Science.gov (United States)

    Ditaranto, Vincent M., Jr.

    Bioresorbable polymeric scaffolds were designed for the purpose of growing rat osteosarcoma cells (ROS 17/2.8) using the compression molding method. The material used in the construction of the scaffolds was a mixture of polycaprolactone (PCL), Hydroxyapatite (HA), Glycerin (GL) and salt (NaCl) for porosity. The concentration of the several materials utilized, was determined by volume. Past research at the University of Massachusetts Lowell (UML) has successfully utilized the compression molding method for the construction of scaffolds, but was unable to accomplish the goal of long term cell survival and complete cellular proliferation throughout a three dimensional scaffold. This research investigated various concentrations of the materials and molding temperatures used for the manufacture of scaffolds in order to improve the scaffold design and address those issues. The design of the scaffold using the compression molding process is detailed in the Method and Materials section of this thesis. The porogen (salt) used for porosity was suspected as a possible source of contamination causing cell apoptosis in past studies. This research addressed the issues for cell survival and proliferation throughout a three dimensional scaffold. The leaching of the salt was one major design modification. This research successfully used ultrasonic leaching in addition to the passive method. Prior to cell culture, the scaffolds were irradiated to 2.75 Mrad, with cobalt-60 gamma radionuclide. The tissue culture consisted of two trials: (1) cell culture in scaffolds cleaned with passive leaching; (2) cell culture with scaffolds cleaned with ultrasonic leaching. Cell survival and proliferation was accomplished only with the addition of ultrasonic leaching of the scaffolds. Analysis of the scaffolds included Scanning Electron Microscopy (SEM), Nikon light microscopy and x-ray mapping of the calcium, sodium and chloride ion distribution. The cells were analyzed by Environmental Scanning

  20. Mechanotransductive Regulation of Gap-Junction Activity Between MLO-Y4 Osteocyte-Like and MC3T3-E1 Osteoblast-Like Cells in Three-Dimensional Co-Culture.

    Science.gov (United States)

    Juran, C. M.; Blaber, E. A.; Almeida, E. A. C.

    2016-01-01

    Cell and animal studies conducted onboard the International Space Station and formerly on Shuttle flights have provided groundbreaking data illuminating the deleterious biological response of bone to mechanical unloading. However the intercellular communicative mechanisms associated with the regulation of bone synthesis and bone resorption cells are still largely unknown. Connexin-43 (CX43), a gap junction protein, is hypothesized to play a significant role in osteoblast and osteocyte signaling. The purpose of this investigation was to evaluate within a novel three-dimensional microenvironment how the osteocyte-osteoblast gap-junction expression changes when cultures are exposed to exaggerated mechanical load. MLO-Y4 osteocyte-like cells were cultured on a 3D-Biotek polystyrene insert and placed in direct contact with an MC3T3-E1 pre-osteoblast co-cultured monolayer and exposed to 48 h of mechanical stimulation (pulsatile fluid flow (PFF) or monolayer cyclic stretch (MCS)) then evaluated for viability, proliferation, metabolism, and CX43 expression. Mono-cultured MLO-Y4 and MC3T3-E1 control experiments were conducted under PFF and MCS stimulation to observe how strain application stimuli (PFF cell membrane shear or MCS cell focal adhesion/attachment loading) initiates different signaling pathways or downstream regulatory controls. TotalLive cell count, viability and metabolic reduction (Trypan Blue, LIVEDead and Alamar Blue analysis respectively) indicate that mechanical activation of MC3T3-E1 cells inhibits proliferation while maintaining an average 1.04E4 reductioncell metabolic rate, *p0.05 n4. MLO-Y4s in monolayer culture increase in number when exposed to MCS loading but the percent of live cells within the population is low (46.3 total count, *p0.05 n4), these results may indicate an apoptotic signaling cascade. PFF stimulation of the three-dimensional co-cultures elicits a universal increase in CX43 in MLO-Y4 and MC3T3-E1 cells, illustrated by

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  3. Osteogenic differentiation of mesenchymal stem cells is regulated by osteocyte and osteoblast cells in a simplified bone niche

    Directory of Open Access Journals (Sweden)

    LM McNamara

    2012-01-01

    Full Text Available Mesenchymal stem cells (MSCs within their native environment of the stem cell niche in bone receive biochemical stimuli from surrounding cells. These stimuli likely influence how MSCs differentiate to become bone precursors. The ability of MSCs to undergo osteogenic differentiation is well established in vitro;however, the role of the natural cues from bone’s regulatory cells, osteocytes and osteoblasts in regulating the osteogenic differentiation of MSCs in vivo are unclear. In this study we delineate the role of biochemical signalling from osteocytes and osteoblasts, using conditioned media and co-culture experiments, to understand how they direct osteogenic differentiation of MSCs. Furthermore, the synergistic relationship between osteocytes and osteoblasts is examined by transwell co-culturing of MSCs with both simultaneously. Osteogenic differentiation of MSCs was quantified by monitoring alkaline phosphatase (ALP activity, calcium deposition and cell number. Intracellular ALP was found to peak earlier and there was greater calcium deposition when MSCs were co-cultured with osteocytes rather than osteoblasts, suggesting that osteocytes are more influential than osteoblasts in stimulating osteogenesis in MSCs. Osteoblasts initially stimulated an increase in the number of MSCs, but ultimately regulated MSC differentiation down the same pathway. Our novel co-culture system confirmed a synergistic relationship between osteocytes and osteoblasts in producing biochemical signals to stimulate the osteogenic differentiation of MSCs. This study provides important insights into the mechanisms at work within the native stem cell niche to stimulate osteogenic differentiation and outlines a possible role for the use of co-culture or conditioned media methodologies for tissue engineering applications.

  4. Effects of platelet rich plasma (PRP) on human gingival fibroblast, osteoblast and periodontal ligament cell behaviour.

    Science.gov (United States)

    Kobayashi, Eizaburo; Fujioka-Kobayashi, Masako; Sculean, Anton; Chappuis, Vivianne; Buser, Daniel; Schaller, Benoit; Dőri, Ferenc; Miron, Richard J

    2017-06-02

    The use of platelet rich plasma (PRP, GLO) has been used as an adjunct to various regenerative dental procedures. The aim of the present study was to characterize the influence of PRP on human gingival fibroblasts, periodontal ligament (PDL) cells and osteoblast cell behavior in vitro. Human gingival fibroblasts, PDL cells and osteoblasts were cultured with conditioned media from PRP and investigated for cell migration, proliferation and collagen1 (COL1) immunostaining. Furthermore, gingival fibroblasts were tested for genes encoding TGF-β, PDGF and COL1a whereas PDL cells and osteoblasts were additionally tested for alkaline phosphatase (ALP) activity, alizarin red staining and mRNA levels of osteoblast differentiation markers including Runx2, COL1a2, ALP and osteocalcin (OCN). It was first found that PRP significantly increased cell migration of all cells up to 4 fold. Furthermore, PRP increased cell proliferation at 3 and 5 days of gingival fibroblasts, and at 3 days for PDL cells, whereas no effect was observed on osteoblasts. Gingival fibroblasts cultured with PRP increased TGF-β, PDGF-B and COL1 mRNA levels at 7 days and further increased over 3-fold COL1 staining at 14 days. PDL cells cultured with PRP increased Runx2 mRNA levels but significantly down-regulated OCN mRNA levels at 3 days. No differences in COL1 staining or ALP staining were observed in PDL cells. Furthermore, PRP decreased mineralization of PDL cells at 14 days post seeding as assessed by alizarin red staining. In osteoblasts, PRP increased COL1 staining at 14 days, increased COL1 and ALP at 3 days, as well as increased ALP staining at 14 days. No significant differences were observed for alizarin red staining of osteoblasts following culture with PRP. The results demonstrate that PRP promoted gingival fibroblast migration, proliferation and mRNA expression of pro-wound healing molecules. While PRP induced PDL cells and osteoblast migration and proliferation, it tended to have

  5. Enhanced osteoblast adhesion to drug-coated anodized nanotubular titanium surfaces

    Directory of Open Access Journals (Sweden)

    George E Aninwene II

    2008-06-01

    Full Text Available George E Aninwene II1, Chang Yao2, Thomas J Webster21Department of Biochemical Engineering, University of Maryland, Baltimore, MD; 2Division of Engineering, Brown University, Providence, RI, USAAbstract: Current orthopedic implants have functional lifetimes of only 10–15 years due to a variety of reasons including infection, extensive inflammation, and overall poor osseointegration (or a lack of prolonged bonding of the implant to juxtaposed bone. To improve properties of titanium for orthopedic applications, this study anodized and subsequently coated titanium with drugs known to reduce infection (penicillin/streptomycin and inflammation (dexamethasone using simple physical adsorption and the deposition of such drugs from simulated body fluid (SBF. Results showed improved drug elution from anodized nanotubular titanium when drugs were coated in the presence of SBF for up to 3 days. For the first time, results also showed that the simple physical adsorption of both penicillin/streptomycin and dexamethasone on anodized nanotubular titanium improved osteoblast numbers after 2 days of culture compared to uncoated unanodized titanium. In addition, results showed that depositing such drugs in SBF on anodized titanium was a more efficient method to promote osteoblast numbers compared to physical adsorption for up to 2 days of culture. In addition, osteoblast numbers increased on anodized titanium coated with drugs in SBF for up to 2 days of culture compared to unanodized titanium. In summary, compared to unanodized titanium, this preliminary study provided unexpected evidence of greater osteoblast numbers on anodized titanium coated with either penicillin/streptomycin or dexamethasone using simple physical adsorption or when coated with SBF; results which suggest the need for further research on anodized titanium orthopedic implants possessing drug-eluting nanotubes.Keywords: anodization, titanium, adhesion, simulated body fluid, nanotubes

  6. Competitive time- and density-dependent adhesion of staphylococci and osteoblasts on crosslinked poly(ethylene glycol)-based polymer coatings in co-culture flow chambers.

    Science.gov (United States)

    Saldarriaga Fernández, Isabel C; Busscher, Henk J; Metzger, Steve W; Grainger, David W; van der Mei, Henny C

    2011-02-01

    Biomaterial-associated infections (BAI) remain a serious clinical complication, often arising from an inability of host tissue-implant integration to out-compete bacterial adhesion and growth. A commercial polymer coating based on polyethylene glycol (PEG), available in both chemically inert and NHS-activated forms (OptiChem(®)), was compared for simultaneous growth of staphylococci and osteoblasts. In the absence of staphylococci, osteoblasts adhered and proliferated well on glass controls and on the NHS-reactive PEG-based coating over 48 h, but not on the inert PEG coating. Staphylococcal growth was low on both PEG-based coatings. When staphylococci were pre-adhered on surfaces for 1.5 h to mimic peri-operative contamination, osteoblast growth and spreading was reduced on glass but virtually absent on both reactive and inert PEG-based coatings. Thus although NHS-reactive, PEG-based coatings stimulated tissue-cell interactions in the absence of contaminating staphylococci, the presence of adhering staphylococci eliminated osteoblast adhesion advantages on the PEG surface. This study demonstrates the importance of using bacterial and cellular co-cultures compared to monocultures when assessing functionalized biomaterials coatings for infectious potential. Copyright © 2010 Elsevier Ltd. All rights reserved.

  7. Edaravone protects osteoblastic cells from dexamethasone through inhibiting oxidative stress and mPTP opening.

    Science.gov (United States)

    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.

  8. Direct conversion of human fibroblasts into functional osteoblasts by defined factors.

    Science.gov (United States)

    Yamamoto, Kenta; Kishida, Tsunao; Sato, Yoshiki; Nishioka, Keisuke; Ejima, Akika; Fujiwara, Hiroyoshi; Kubo, Toshikazu; Yamamoto, Toshiro; Kanamura, Narisato; Mazda, Osam

    2015-05-12

    Osteoblasts produce calcified bone matrix and contribute to bone formation and remodeling. In this study, we established a procedure to directly convert human fibroblasts into osteoblasts by transducing some defined factors and culturing in osteogenic medium. Osteoblast-specific transcription factors, Runt-related transcription factor 2 (Runx2), and Osterix, in combination with Octamer-binding transcription factor 3/4 (Oct4) and L-Myc (RXOL) transduction, converted ∼ 80% of the fibroblasts into osteocalcin-producing cells. The directly converted osteoblasts (dOBs) induced by RXOL displayed a similar gene expression profile as normal human osteoblasts and contributed to bone repair after transplantation into immunodeficient mice at artificial bone defect lesions. The dOBs expressed endogenous Runx2 and Osterix, and did not require continuous expression of the exogenous genes to maintain their phenotype. Another combination, Oct4 plus L-Myc (OL), also induced fibroblasts to produce bone matrix, but the OL-transduced cells did not express Osterix and exhibited a more distant gene expression profile to osteoblasts compared with RXOL-transduced cells. These findings strongly suggest successful direct reprogramming of fibroblasts into functional osteoblasts by RXOL, a technology that may provide bone regeneration therapy against bone disorders.

  9. Osteoblasts generate an osteogenic microenvironment when grown on surfaces with rough microtopographies

    Directory of Open Access Journals (Sweden)

    Boyan B. D.

    2003-10-01

    Full Text Available Osteoblasts respond to microarchitectural features of their substrate. On smooth surfaces (tissue culture plastic, tissue culture glass, and titanium, the cells attach and proliferate but they exhibit relatively low expression of differentiation markers in monolayer cultures, even when confluent. When grown on microrough Ti surfaces with an average roughness (Ra of 4-7 µm, proliferation is reduced but differentiation is enhanced and in some cases, is synergistic with the effects of surface microtopography. In addition, cells on microrough Ti substrates form hydroxyapatite in a manner that is more typical of bone than do cells cultured on smooth surfaces. Osteoblasts also respond to growth factors and cytokines in a surface-dependent manner. On rougher surfaces, the effects of regulatory factors like 1alpha,25(OH2D3 or 17beta-estradiol are enhanced. The response to the surface is mediated by integrins, which signal to the cell through many of the same mechanisms used by growth factors and hormones. Studies using PEG-modified surfaces indicate that increased differentiation may be related to altered attachment to the surface. When osteoblasts are grown on surfaces with chemistries or microarchitectures that reduce cell attachment and proliferation, and enhance differentiation, the cells tend to increase production of factors like TGF-beta1 that promote osteogenesis while decreasing osteoclastic activity. Thus, on microrough Ti surface, osteoblasts create a microenvironment conducive to new bone formation.

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

    International Nuclear Information System (INIS)

    Tai Guangping; Christodoulou, Ioannis; Bishop, Anne E.; Polak, Julia M.

    2005-01-01

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

  11. Treatment with 1,25-dihydroxyvitamin D3 reduces impairment of human osteoblast functions during cellular aging in culture

    DEFF Research Database (Denmark)

    Kveiborg, M.; Rattan, Suresh; Eriksen, E.F.

    2001-01-01

    is due to impaired responsiveness to calcitriol known to be important for the regulation of biological activities of the osteoblasts. Thus, we examined changes in vitamin D receptor (VDR) system and the osteoblastic responses to calcitriol treatment during in vitro osteoblast aging. We found no change...... in the amount of VDR at either steady state mRNA level or protein level with increasing in vitro osteoblast age and examination of VDR localization, nuclear translocation and DNA binding activity revealed no in vitro age-related changes. Furthermore, calcitriol (10(-8)M) treatment of early-passage osteoblastic......Adequate responses to various hormones, such as 1,25-dihydroxyvitamin D(3) (calcitriol) are a prerequisite for optimal osteoblast functions. We have previously characterized several human diploid osteoblastic cell lines that exhibit typical in vitro aging characteristics during long...

  12. Thin films of single-walled carbon nanotubes promote human osteoblastic cells (Saos-2) proliferation in low serum concentrations

    International Nuclear Information System (INIS)

    Akasaka, Tsukasa; Yokoyama, Atsuro; Matsuoka, Makoto; Hashimoto, Takeshi; Watari, Fumio

    2010-01-01

    One strategy used for the regeneration of bone is the development of cell culture substrates and scaffolds that can control osteoblast proliferation and differentiation. In recent investigations, carbon nanotubes (CNTs) have been utilized as scaffolds for osteoblastic cell cultures; however, there are only a few reports describing the proliferation of osteoblastic cells on thin CNT films; in particular, the effects of serum concentration on cell proliferation have not been studied. In the present study, we prepared culture dishes with homogeneous thin or thick films of non-modified CNTs and examined the effect of serum concentrations on human osteoblastic cells (Saos-2) proliferation in these culture dishes. We demonstrated that the ratio of cell proliferation was strongly affected by the concentration of serum. Interestingly, single-walled carbon nanotube (SWNT) thin films were found to be the most effective substrate for the proliferation of Saos-2 cells in low concentrations of serum. Thus, thin SWNT films may be used as an effective biomaterial for the culture of Saos-2 cells in low serum concentrations.

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

    DEFF Research Database (Denmark)

    Gurzawska, Katarzyna Aleksandra; Svava, Rikke; Syberg, Susanne

    2012-01-01

    -I) on surface properties and osteoblasts response. Three different RG-Is from apple and lupin pectins were modified and coated on amino-functionalized tissue culture polystyrene plates (aminated TCPS). Surface properties were evaluated by scanning electron microscopy, contact angle measurement, atomic force...... microscopy, and X-ray photoelectron spectroscopy. The effects of nanocoating on proliferation, matrix formation and mineralization, and expression of genes (real-time PCR) related to osteoblast differentiation and activity were tested using human osteoblast-like SaOS-2 cells. It was shown that RG-I coatings...

  14. The Impact of Metal Ion Exposure on the Cellular Behavior of Human Osteoblasts and PBMCs: In Vitro Analyses of Osteolytic Processes

    Directory of Open Access Journals (Sweden)

    Anika Jonitz-Heincke

    2017-07-01

    Full Text Available Osteolysis in the periprosthetic tissue can be caused by metallic wear particles and ions that can originate from implant surface corrosion. These products influence cellular behavior and stimulate the expression of proinflammatory cytokines. The purpose of this study was to evaluate the impact of CoCr29Mo6 ions on cell survival, differentiation, and cytokine expression in human osteoblasts and peripheral blood mononuclear cells (PBMCs. Thus, we exposed cells with a mixture of 200 µg/L ion solution and determined cell viability and apoptosis/necrosis. Gene expression analyses of osteoblastic and osteoclastic differentiation markers as well as pro-osteolytic mediators (IL-6, IL-8, TNF-α, MCP-1, MMP1, TIMP1 were performed. These markers were also investigated in mixed cultures of adherent and non-adherent PBMCs as well as in co-cultures of human osteoblasts and PBMCs. The ion solution induced necrosis in osteoblasts and PBMCs in single cultures. All examined mediators were highly expressed in the co-culture of osteoblasts and PBMCs whereas in the single cell cultures only IL-6, IL-8, and MMP1 were found to be stimulated. While the applied concentration of the CoCr29Mo6 ion solutions had only marginal effects on human osteoblasts and PBMCs alone, the co-culture may provide a comprehensive model to study osteolytic processes in response to Co and Cr ions.

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

    Science.gov (United States)

    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

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

    International Nuclear Information System (INIS)

    Vezeridis, Peter S.; Semeins, Cornelis M.; Chen Qian; Klein-Nulend, Jenneke

    2006-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Michael S Stalvey

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Bao, Quanwei; Chen, Sixu; Qin, Hao [State Key Laboratory of Trauma, Burn and Combined injury, Department of Trauma Surgery, Daping Hospital, Third Military Medical University, ChongQing 400042 (China); Feng, Jianquan [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A& M Health Science Center, Dallas, TX 75246 (United States); Liu, Huayu; Liu, Daocheng; Li, Ang; Shen, Yue; Zhong, Xiaozheng; Li, Junfeng [State Key Laboratory of Trauma, Burn and Combined injury, Department of Trauma Surgery, Daping Hospital, Third Military Medical University, ChongQing 400042 (China); Zong, Zhaowen, E-mail: zongzhaowen@sina.cn [State Key Laboratory of Trauma, Burn and Combined injury, Department of Trauma Surgery, Daping Hospital, Third Military Medical University, ChongQing 400042 (China)

    2017-01-01

    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. 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 the bone

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

    Directory of Open Access Journals (Sweden)

    Guofeng Qian

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

  20. Effect of Irradiation on Apoptosis, Cell Cycle Arrest and Calcified Nodule Formation of Rat Calvarial Osteoblast

    International Nuclear Information System (INIS)

    Lee, Young Mi; Choi, Hang Moon; Heo, Min Suk; Lee, Sam Sun; Choi, Soon Chul; Park, Tae Won

    2000-01-01

    The study was aimed to detect the induction of apoptosis, cell cycle arrest and calcified nodule formation after irradiation on primarily cultured osteoblasts. Using rat calvarial osteoblasts, the effects of irradiation on apoptosis, cell cycle arrest, and calcified nodule formation were studied. The single irradiation of 10, 20 Gy was done with 5.38 Gy/min dose rate using the 137 Cs cell irradiator at 4th and 14th day of culture. Apoptosis induction and cell cycle arrest were assayed by the flow cytometry at 1, 2, 3, and 4 days after irradiation. The formation of calcified nodules was observed by alizarin red staining at 1, 3, 10, 14 days after irradiation at 4th day of culture, and at 1, 4, 5 days after irradiation at 14th day of culture. Apoptosis was not induced by 10 or 20 Gy independent of irradiation and culture period. Irradiation did not induced G1 arrest in post-irradiated osteoblasts. After irradiation at 4th-day of culture, G2 arrest was induced but it was not statistically significant after irradiation at 14th-day of culture. In the case of irradiated cells at 4th day of culture, calcified nodules were not formed and at 14th-day of culture after irradiation, calcified nodule formation did not affected. Taken together, these results suggest that irradiation at the dose of 10-20 Gy would not affect apoptosis induction of osteoblasts. Cell cycle and calcified nodule formation were influenced by the level of differentiation of osteblasts.

  1. Effect of porous titanium coating thickness on in vitro osteoblast phenotype expression

    Directory of Open Access Journals (Sweden)

    Antonio Canabarro

    2011-03-01

    Full Text Available Aim: This study aimed at determining the effect of different thickness of porous titanium (Ti coating, 0.5, 1.0 and 1.5 mm thick (PC-0.5, PC-1.0 and PC-1.5, on osteoblast phenotype expression. Materials and methods: Dense Ti discs coated with 0.5, 1.0 and 1.5 mm of porous Ti (PC-0.5, PC-1.0 and PC-1.5, respectively were fabricated by powder metallurgy process with pore size typically between 50 and 400 μm and porosity of 60%. Osteoblastic cells obtained from human alveolar bone were cultured on dense Ti (D-Ti and PC-Ti discs for periods of up to 17 days. Results: Cultures grown on PC-Ti exhibited higher cell proliferation rate than on D-Ti. By comparing PC-Ti groups, it was observed statistical differences on culture grown only at day 10 (PC-0.5cultured on PC-1.5 presented the highest gene expression of type I collagen (COL, alkaline phosphatase (ALP, and osteocalcin (OC. The calcium content was significantly greater on PC-1.5 compared to all other groups. Conclusion: These results indicate that PC-Ti favored osteoblastic cell proliferation. In addition, they increased gene expression of osteoblastic markers and higher content of mineralized matrix was observed on the thicker PC-Ti coating (PC-1.5. Therefore, further in vivo evaluations should be done in order to investigate whether this structure should be considered for clinical implant applications.

  2. Dysregulated gene expression in the primary osteoblasts and osteocytes isolated from hypophosphatemic Hyp mice.

    Directory of Open Access Journals (Sweden)

    Kazuaki Miyagawa

    Full Text Available Osteocytes express multiple genes involved in mineral metabolism including PHEX, FGF23, DMP1 and FAM20C. In Hyp mice, a murine model for X-linked hypophosphatemia (XLH, Phex deficiency results in the overproduction of FGF23 in osteocytes, which leads to hypophosphatemia and impaired vitamin D metabolism. In this study, to further clarify the abnormality in osteocytes of Hyp mice, we obtained detailed gene expression profiles in osteoblasts and osteocytes isolated from the long bones of 20-week-old Hyp mice and wild-type (WT control mice. The expression of Fgf23, Dmp1, and Fam20c was higher in osteocytic cells than in osteoblastic cells in both genotypes, and was up-regulated in Hyp cells. Interestingly, the up-regulation of these genes in Hyp bones began before birth. On the other hand, the expression of Slc20a1 encoding the sodium/phosphate (Na+/Pi co-transporter Pit1 was increased in osteoblasts and osteocytes from adult Hyp mice, but not in Hyp fetal bones. The direct effects of extracellular Pi and 1,25-dihydroxyvitamin D3 [1,25(OH2D3] on isolated osteoblastic and osteocytic cells were also investigated. Twenty-four-hour treatment with 10-8 M 1,25(OH2D3 increased the expression of Fgf23 in WT osteoblastic cells but not in osteocytic cells. Dmp1 expression in osteocytic cells was increased due to the 24-hour treatment with 10 mM Pi and was suppressed by 10-8 M 1,25(OH2D3 in WT osteocytic cells. We also found the up-regulation of the genes for FGF1, FGF2, their receptors, and Egr-1 which is a target of FGF signaling, in Hyp osteocytic cells, suggesting the activation of FGF/FGFR signaling. These results implicate the complex gene dysregulation in osteoblasts and osteocytes of Hyp mice, which might contribute to the pathogenesis.

  3. SIRT3/SOD2 maintains osteoblast differentiation and bone formation by regulating mitochondrial stress

    OpenAIRE

    Gao, Jing; Feng, Zhihui; Wang, Xueqiang; Zeng, Mengqi; Liu, Jing; Han, Shujun; Xu, Jie; Chen, Lei; Cao, Ke; Long, Jiangang; Li, Zongfang; Shen, Weili; Liu, Jiankang

    2017-01-01

    Recent studies have revealed robust metabolic changes during cell differentiation. Mitochondria, the organelles where many vital metabolic reactions occur, may play an important role. Here, we report the involvement of SIRT3-regulated mitochondrial stress in osteoblast differentiation and bone formation. In both the osteoblast cell line MC3T3-E1 and primary calvarial osteoblasts, robust mitochondrial biogenesis and supercomplex formation were observed during differentiation, accompanied by in...

  4. Stimulatory effect of undecylenic acid on mouse osteoblast differentiation.

    Science.gov (United States)

    Kim, Myung Hee; Shim, Ki Shuk; Lee, Su-Ui; Kim, Young Sup; Min, Yong Ki; Kim, Seong Hwan

    2010-04-01

    Natural compounds with bone-forming (or anabolic) activity have been recently focused on in bone research. The present study investigated the effect of undecylenic acid (UA) on osteoblast differentiation in mouse osteoblastic MC3T3-E1 subclone 4 cells and primary mouse calvarial cells. Low concentrations of UA (up to 5 microM) exhibited no cytotoxicity and significantly increased the expression and activity of alkaline phosphatase (early differentiation marker of osteoblast) and calcium deposition with the induction of expression of the osteocalcin gene in both cells. Interestingly, at low concentration of UA, the induction of NF-kappaB p65 translocation into nucleus and the up-regulation of AP-1 and NFATc1 transcript levels were also observed, suggesting that the stimulatory effect of UA on osteoblast differentiation could be mediated through the activation of transcription factors. Additionally, although the patterns of UA-induced activation of MAP kinases (JNK and p38) were not completely consistent with the increase of both ALP activity and calcium deposition by UA, MAP kinases might be partially involved in the biological function of UA during the early and late stages of osteoblast differentiation. Copyright (c) 2009 John Wiley & Sons, Ltd.

  5. Ca uptake and its influence by growth hormone in osteoblasts of fetal rat calvaria

    International Nuclear Information System (INIS)

    Wang Hongfu; Jin Weifang; Sekimoto Haku

    1994-01-01

    Uptake and release of Ca 2+ are important functions of osteoblasts. In the paper we studied the uptake of calcium and influence by Growth Hormone in osteoblasts of fetal rat calvaria by liquid scintillation spectrometry of 45 Ca 2+ . In short-term cultures of the bone derived cells, the uptake of 45 Ca 2+ increased steadily. The activity of 45 Ca 2+ in the cells of 15 minute cultures was 2∼3 times of that in the 0 minute cultures. It continued to increase in the cells of 30 minute cultures. Exposure of the bone cells to GH at 55.3 ng/ml increased the uptake of 45 Ca 2+ by 2.3 times in the 30 minute cultures and 1.5 times in the 60 minute cultures than those of the control

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

    Directory of Open Access Journals (Sweden)

    Ho MH

    2014-09-01

    Full Text Available Ming-Hua Ho,1,2 Mei-Hsiu Liao,3 Yi-Ling Lin,2 Chien-Hao Lai,3 Pei-I Lin,3 Ruei-Ming Chen2–4 1Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei, Taiwan; 2Cell Physiology and Molecular Image Research Center and Department of Anesthesiology, Wan Fang Hospital, 3Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan; 4Anesthetics and Toxicology Research Center, Taipei Medical University Hospital, Taipei, Taiwan Abstract: 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 (mRNA 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

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

    Science.gov (United States)

    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.

  8. Strontium ranelate changes the composition and crystal structure of the biological bone-like apatite produced in osteoblast cell cultures.

    Science.gov (United States)

    Querido, William; Campos, Andrea P C; Martins Ferreira, Erlon H; San Gil, Rosane A S; Rossi, Alexandre M; Farina, Marcos

    2014-09-01

    We evaluate the effects of strontium ranelate on the composition and crystal structure of the biological bone-like apatite produced in osteoblast cell cultures, a system that gave us the advantage of obtaining mineral samples produced exclusively during treatment. Cells were treated with strontium ranelate at concentrations of 0.05 and 0.5 mM Sr(2+). Mineral substances were isolated and analyzed by using a combination of methods: Fourier transform infrared spectroscopy, solid-state (1)H nuclear magnetic resonance, X-ray diffraction, micro-Raman spectroscopy and energy dispersive X-ray spectroscopy. The minerals produced in all cell cultures were typical bone-like apatites. No changes occurred in the local structural order or crystal size of the minerals. However, we noticed several relevant changes in the mineral produced under 0.5 mM Sr(2+): (1) increase in type-B CO3 (2-) substitutions, which often lead to the creation of vacancies in Ca(2+) and OH(-) sites; (2) incorporation of Sr(2+) by substituting slightly less than 10 % of Ca(2+) in the apatite crystal lattice, resulting in an increase in both lattice parameters a and c; (3) change in the PO4 (3-) environments, possibly because of the expansion of the lattice; (4) the Ca/P ratio of this mineral was reduced, but its (Ca+Sr)/P ratio was the same as that of the control, indicating that its overall cation/P ratio was preserved. Thus, strontium ranelate changes the composition and crystal structure of the biological bone-like apatite produced in osteoblast cell cultures.

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

    LENUS (Irish Health Repository)

    Solayar, Gandhi N

    2011-10-28

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

  10. Effects of bioglass powders with and without mesoporous structures on fibroblast and osteoblast responses

    Energy Technology Data Exchange (ETDEWEB)

    Shih, Chi-Jen, E-mail: cjshih@kmu.edu.tw [Department of Fragrance and Cosmetic Science, College of Pharmacy, Kaohsiung Medical University, 100 Shi-Chuan 1st Road, Kaohsiung 80708, Taiwan (China); Lu, Pei-Shan [Department of Fragrance and Cosmetic Science, College of Pharmacy, Kaohsiung Medical University, 100 Shi-Chuan 1st Road, Kaohsiung 80708, Taiwan (China); Hsieh, Chih-Hsin [Department of Orthopaedics, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China); Chen, Wen-Cheng [Advanced Medical Devices and Composites Laboratory, Department of Fiber and Composite Materials, College of Engineering, Feng Chia University, Taichung 40724, Taiwan (China); Chen, Jian-Chih [Department of Orthopaedics, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China); Department of Orthopaedics, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China)

    2014-09-30

    Highlights: • Fluorescent microscopy images show that BG-M has excellent cellular affinity. • Both the BG and BG-M substrates had positive effects on the proliferation of the osteoblastic cells. • Cells cultured on BG-M had approximately 1.4 times higher proliferation activity. - Abstract: The main objective of this study was to compare the responses of fibroblasts and osteoblasts to bioglass (BG) and bioglass-containing mesoporous structure (BG-M) powders. The BG-M powders exhibited specific surface areas approximately three times larger than those of the BG powders. The formation of a hysteresis loop also signified the presence of mesoporous structures in the BG-M samples; however, a hysteresis loop was not observed for the BG samples, resulting in 1/5 the pore volume of the BG-M samples. The viabilities of the fibroblasts and osteoblasts cultured in media containing the BG-M powders for 1, 2, and 3 days were greater than 90%. Importantly, the results of fluorescent microscopy images show that BG-M has excellent cellular affinity. Both the BG and BG-M substrates had positive effects on the proliferation of the osteoblastic cells. However, cells cultured on BG-M had approximately 1.4 times higher proliferation activity.

  11. Effects of bioglass powders with and without mesoporous structures on fibroblast and osteoblast responses

    International Nuclear Information System (INIS)

    Shih, Chi-Jen; Lu, Pei-Shan; Hsieh, Chih-Hsin; Chen, Wen-Cheng; Chen, Jian-Chih

    2014-01-01

    Highlights: • Fluorescent microscopy images show that BG-M has excellent cellular affinity. • Both the BG and BG-M substrates had positive effects on the proliferation of the osteoblastic cells. • Cells cultured on BG-M had approximately 1.4 times higher proliferation activity. - Abstract: The main objective of this study was to compare the responses of fibroblasts and osteoblasts to bioglass (BG) and bioglass-containing mesoporous structure (BG-M) powders. The BG-M powders exhibited specific surface areas approximately three times larger than those of the BG powders. The formation of a hysteresis loop also signified the presence of mesoporous structures in the BG-M samples; however, a hysteresis loop was not observed for the BG samples, resulting in 1/5 the pore volume of the BG-M samples. The viabilities of the fibroblasts and osteoblasts cultured in media containing the BG-M powders for 1, 2, and 3 days were greater than 90%. Importantly, the results of fluorescent microscopy images show that BG-M has excellent cellular affinity. Both the BG and BG-M substrates had positive effects on the proliferation of the osteoblastic cells. However, cells cultured on BG-M had approximately 1.4 times higher proliferation activity

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-07-06

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

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

    International Nuclear Information System (INIS)

    Muthukumaran, Padmalosini; Lim, Chwee Teck; Lee, Taeyong

    2012-01-01

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

  16. Effects of an injectable platelet-rich fibrin on osteoblast behavior and bone tissue formation in comparison to platelet-rich plasma.

    Science.gov (United States)

    Wang, Xuzhu; Zhang, Yufeng; Choukroun, Joseph; Ghanaati, Shahram; Miron, Richard J

    2018-01-01

    Platelet-rich plasma (PRP) has been utilized for many years as a regenerative agent capable of inducing vascularization of various tissues using blood-derived growth factors. Despite this, drawbacks mostly related to the additional use of anti-coagulants found in PRP have been shown to inhibit the wound healing process. For these reasons, a novel platelet concentrate has recently been developed with no additives by utilizing lower centrifugation speeds. The purpose of this study was therefore to investigate osteoblast behavior of this novel therapy (injectable-platelet-rich fibrin; i-PRF, 100% natural with no additives) when compared to traditional PRP. Human primary osteoblasts were cultured with either i-PRF or PRP and compared to control tissue culture plastic. A live/dead assay, migration assay as well as a cell adhesion/proliferation assay were investigated. Furthermore, osteoblast differentiation was assessed by alkaline phosphatase (ALP), alizarin red and osteocalcin staining, as well as real-time PCR for genes encoding Runx2, ALP, collagen1 and osteocalcin. The results showed that all cells had high survival rates throughout the entire study period irrespective of culture-conditions. While PRP induced a significant 2-fold increase in osteoblast migration, i-PRF demonstrated a 3-fold increase in migration when compared to control tissue-culture plastic and PRP. While no differences were observed for cell attachment, i-PRF induced a significantly higher proliferation rate at three and five days when compared to PRP. Furthermore, i-PRF induced significantly greater ALP staining at 7 days and alizarin red staining at 14 days. A significant increase in mRNA levels of ALP, Runx2 and osteocalcin, as well as immunofluorescent staining of osteocalcin was also observed in the i-PRF group when compared to PRP. In conclusion, the results from the present study favored the use of the naturally-formulated i-PRF when compared to traditional PRP with anti

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

    International Nuclear Information System (INIS)

    Schindeler, Aaron; Little, David G.

    2005-01-01

    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 [ 14 C]-labeled ZA confirmed that the bisphosphonate bound in a concentration-dependent manner over the 1-50 μM dose range. When grown on ZA-treated discs, the viability of bone-marrow derived osteoclasts was greatly reduced, while the viability and mineralization of the osteoblastic MC3T3-E1 cell line were largely unaffected. This suggests that only bone resorbing cells are affected by bound bisphosphonate. However, this system does not account for transient exposure to unbound bisphosphonate in the hours following a clinical dosing. To model this event, we transiently treated osteoblasts with ZA in the absence of a calcified surface. Osteoblasts proved highly resistant to all transitory treatment regimes, even when utilizing ZA concentrations that prevented mineralization and/or induced cell death when dosed chronically. This study represents a pharmacologically more relevant approach to modeling bisphosphonate treatment on cultured bone cells and implies that bisphosphonate therapies may not directly affect osteoblasts at bone surfaces

  18. Palmitate attenuates osteoblast differentiation of fetal rat calvarial cells

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-07-18

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

  19. Palmitate attenuates osteoblast differentiation of fetal rat calvarial cells

    International Nuclear Information System (INIS)

    Yeh, Lee-Chuan C.; Ford, Jeffery J.; Lee, John C.; Adamo, Martin L.

    2014-01-01

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

  20. Factorial Study of Compressive Mechanical Properties and Primary In Vitro Osteoblast Response of PHBV/PLLA Scaffolds

    Directory of Open Access Journals (Sweden)

    Naznin Sultana

    2012-01-01

    Full Text Available For bone tissue regeneration, composite scaffolds containing biodegradable polymers and nanosized osteoconductive bioceramics have been regarded as promising biomimetic systems. Polymer blends of poly(hydroxybutyrate-co-hydroxyvalerate (PHBV and poly(L-lactic acid (PLLA can be used as the polymer matrix to control the degradation rate. In order to render the scaffolds osteoconductive, nano-sized hydroxyapatite (nHA particles can be incorporated into the polymer matrix. In the first part of this study, a factorial design approach to investigate the influence of materials on the initial compressive mechanical properties of the scaffolds was studied. In the second part, the protein adsorption behavior and the attachment and morphology of osteoblast-like cells (Saos-2 of the scaffolds in vitro were also studied. It was observed that nHA incorporated PHBV/PLLA composite scaffolds adsorbed more bovine serum albumin (BSA protein than PHBV or PHBV/PLLA scaffolds. In vitro studies also revealed that the attachment of human osteoblastic cells (SaOS-2 was significantly higher in nHA incorporated PHBV/PLLA composite scaffolds. From the SEM micrographs of nHA incorporated PHBV/PLLA composite scaffolds seeded with SaOS-2 cells after a 7-day cell culture period, it was observed that the cells were well expanded and spread in all directions on the scaffolds.

  1. Interaction of osteoblast-like cells with serum and fibronectin: effects on cell motility and proliferation in vitro

    International Nuclear Information System (INIS)

    Zuk, A.

    1986-01-01

    Osteoblast migration and proliferation are believed to occur during bone remodelling, in particular after osteoclastic bone resorption and prior to osteoblastic bone formation. In order to study migration and proliferation in vitro, the model of Alessandri et al. (1983) was modified. The model entailed seeding osteoblast-like cells into wells cut in agar and quantifying migration and proliferation peripheral to the well. Cell morphology also was described. The data indicated that on growth surfaces enriched with varying concentrations of fetal calf serum (FSC), the quantification of migration and proliferation was related both to percent cell attachment and to FCS-concentration. Because few osteoblast-like cells incorporated ( 3 H-TdR), it was concluded that the appearance of cells peripheral to the well was due to migration, and not to proliferation. Cell morphology and myosin distribution and organization indicated that osteoblast-like cells at the periphery of the cell culture (i.e. leading edge) may have been directionally migrating whereas cells behind the leading edge may have been engaged in non-directional migration. The migration, proliferation, and morphology of osteoblast-like cells cultured on fibronectin (FN) enriched growth surfaces also was examined. The quantification of migration and proliferation was related to the FN-concentration applied to the growth surface. Because few osteoblast-like cells incorporated 3 H-TdR and cell morphology indicated migration, it was concluded that osteoblast-like cells on FN-enriched growth surfaces are specialized, in part, for migration

  2. Specific proliferation rates of human osteoblasts on calcium phosphate surfaces with variable concentrations of α-TCP

    International Nuclear Information System (INIS)

    Santos, Euler A. dos; Farina, Marcos; Soares, Gloria A.

    2007-01-01

    Ideally, ceramics used in the repair of bone defects need to be resorbed and replaced by newly formed bone in vivo. Tricalcium phosphate (TCP) has been widely used in association with hydroxyapatite (HA) due to its higher resorption kinetics when compared with HA alone. The aim of our study was to quantitatively investigate the effect of α-tricalcium phosphate (α-TCP) on human osteoblasts' adhesion and proliferation. Ceramic samples with variable concentrations of α-TCP and HA were produced by the calcination of calcium-deficient and stoichiometric HA. Human osteoblasts were cultured on the materials in three distinct experiments with different concentrations of cells. Numerical evaluation of cellular growth along time in culture was performed for each condition. The quantity of cells seeded onto the ceramics seems to influence the osteoblast behavior once proliferation was lower when more cells were seeded onto the samples. However, a smaller content of α-TCP in relation to that of HA did not significantly modify the specific proliferation rates of the osteoblasts. Only after a long time in culture, the increasing of the α-TCP content seems to change the cells' behavior

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-05-02

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

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

    International Nuclear Information System (INIS)

    Kaneko, Keiko; Ito, Masako; Naoe, Yoshinori; Lacy-Hulbert, Adam; Ikeda, Kyoji

    2014-01-01

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

  5. Non-enzymatic glycosylation of a type I collagen matrix: effects on osteoblastic development and oxidative stress

    Directory of Open Access Journals (Sweden)

    Barrio Daniel A

    2001-08-01

    Full Text Available Abstract Background The tissue accumulation of protein-bound advanced glycation endproducts (AGE may be involved in the etiology of diabetic chronic complications, including osteopenia. The aim of this study was to investigate the effect of an AGE-modified type I collagen substratum on the adhesion, spreading, proliferation and differentiation of rat osteosarcoma UMR106 and mouse non-transformed MC3T3E1 osteoblastic cells. We also studied the role of reactive oxygen species (ROS and nitric oxide synthase (NOS expression on these AGE-collagen mediated effects. Results AGE-collagen decreased the adhesion of UMR106 cells, but had no effect on the attachment of MC3T3E1 cells. In the UMR106 cell line, AGE-collagen also inhibited cellular proliferation, spreading and alkaline phosphatase (ALP activity. In preosteoblastic MC3T3E1 cells (24-hour culture, proliferation and spreading were significantly increased by AGE-collagen. After one week of culture (differentiated MC3T3E1 osteoblasts AGE-collagen inhibited ALP activity, but had no effect on cell number. In mineralizing MC3T3E1 cells (3-week culture AGE-collagen induced a decrease in the number of surviving cells and of extracellular nodules of mineralization, without modifying their ALP activity. Intracellular ROS production, measured after a 48-hour culture, was decreased by AGE-collagen in MC3T3E1 cells, but was increased by AGE-collagen in UMR106 cells. After a 24-hour culture, AGE-collagen increased the expression of endothelial and inducible NOS, in both osteoblastic cell lines. Conclusions These results suggest that the accumulation of AGE on bone extracellular matrix could regulate the proliferation and differentiation of osteoblastic cells. These effects appear to depend on the stage of osteoblastic development, and possibly involve the modulation of NOS expression and intracellular ROS pathways.

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

    Science.gov (United States)

    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

  7. Effect of nanometer scale surface roughness of titanium for osteoblast function

    Directory of Open Access Journals (Sweden)

    Satoshi Migita

    2017-02-01

    Full Text Available Surface roughness is an important property for metallic materials used in medical implants or other devices. The present study investigated the effects of surface roughness on cellular function, namely cell attachment, proliferation, and differentiation potential. Titanium (Ti discs, with a hundred nanometer- or nanometer-scale surface roughness (rough and smooth Ti surface, respectively were prepared by polishing with silicon carbide paper. MC3T3-E1 mouse osteoblast-like cells were cultured on the discs, and their attachment, spreading area, proliferation, and calcification were analyzed. Cells cultured on rough Ti discs showed reduced attachment, proliferation, and calcification ability suggesting that the surface inhibited osteoblast function. The findings can provide a basis for improving the biocompatibility of medical devices.

  8. Differentiation of bovine spermatogonial stem cells into osteoblasts.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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. Direct effects of casein phosphopeptides on growth and differentiation of in vitro cultured osteoblastic cells (MC3T3-E1).

    Science.gov (United States)

    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.

  13. Influences of magnetized hydroxyapatite on the growth behaviors of osteoblasts and the mechanism from molecular dynamics simulation

    International Nuclear Information System (INIS)

    Yang, Weihu; Xi, Xingfeng; Fang, Jiajia; Liu, Peng; Cai, Kaiyong

    2013-01-01

    To investigate the influence of magnetized hydroxyapatite on the growth and differentiation of osteoblasts, hydroxyapatite (HA) and magnetized hydroxyapatite (mHA) were synthesized and characterized. The cell viability, differentiation, and morphologies of osteoblasts were investigated in vitro, respectively. The results showed that compared to HA, cells cultured with mHA had better cell viability, and both HA and mHA were beneficial to the early differentiation of osteoblasts. Furthermore, the interaction mechanism between mHA and osteoblasts was elucidated using a molecular dynamics simulation. The simulation results indicated that when cultured with osteoblasts, HA adsorbed bovine serum protein onto its surface from the medium immediately, which was beneficial to the adhesion and proliferation of osteoblasts. The main driving force for the adsorption of bovine serum was the electronic properties of HA crystal faces. The (211) crystal face of HA had the highest electron density among its all crystal faces, thus mainly contributing to the protein adsorption of HA. Nevertheless, the (211) crystal face of mHA still had a relatively higher electron density than that of HA, thus possessing better protein adsorption than that of HA, and in turn promoting the biological functions of osteoblasts. - Highlights: • Both HA and magnetized HA were beneficial for the early osteogenesis of osteoblasts. • Magnetized HA had better cell viability than HA. • The HA–protein interaction mechanism was investigated by computation simulation. • The main driving force for protein adsorption came from the electronic properties. • The (211) face of mHA has higher electron density and better biological functions

  14. Cuscuta chinensis extract promotes osteoblast differentiation and mineralization in human osteoblast-like MG-63 cells.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  16. The Effects of Orbital Spaceflight on Human Osteoblastic Cell Physiology and Gene Expression

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  18. Low-intensity pulsed ultrasound regulates proliferation and differentiation of osteoblasts through osteocytes

    International Nuclear Information System (INIS)

    Li, Lei; Yang, Zheng; Zhang, Hai; Chen, Wenchuan; Chen, Mengshi; Zhu, Zhimin

    2012-01-01

    Highlights: ► CM from LIPUS-stimulated osteocytes inhibits proliferation of osteoblasts. ► CM from LIPUS-stimulated osteocytes enhances differentiation of osteoblasts. ► LIPUS stimulates MLO-Y4 cells to secrete PGE 2 and NO. -- Abstract: Low-intensity pulsed ultrasound (LIPUS) has been used as a safe and effective modality to enhance fracture healing. As the most abundant cells in bone, osteocytes orchestrate biological activities of effector cells via direct cell-to-cell contacts and by soluble factors. In this study, we have used the osteocytic MLO-Y4 cells to study the effects of conditioned medium from LIPUS-stimulated MLO-Y4 cells on proliferation and differentiation of osteoblastic MC3T3-E1 cells. Conditioned media from LIPUS-stimulated MLO-Y4 cells (LIPUS-Osteocyte-CM) were collected and added on MC3T3-E1 cell cultures. MC3T3-E1 cells cultured in LIPUS-Osteocyte-CM demonstrated a significant inhibition of proliferation and an increased alkaline phosphatase activity. The results of PGE 2 and NO assay showed that LIPUS could enhance PGE 2 and NO secretion from MLO-Y4 cells at all time points within 24 h after LIPUS stimulation. We conclude that LIPUS regulates proliferation and differentiation of osteoblasts through osteocytes in vitro. Increased secretion of PGE 2 from osteocytes may play a role in this effect.

  19. MEK5 suppresses osteoblastic differentiation

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-07-31

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

  20. Responses of human normal osteoblast cells and osteoblast-like cell line, MG-63 cells, to pulse electromagnetic field (PEMF

    Directory of Open Access Journals (Sweden)

    Suttatip Kamolmatyakul

    2008-01-01

    Full Text Available The objective of this in vitro study is to investigate the effect of pulsed electromagnetic field (PEMF on cellular proliferation and osteocalcin production of osteoblast-like cell line, MG-63 cells, and human normal osteoblast cells (NHOC obtained from surgical bone specimens. The cells were placed in 24-well culture plates in the amount of 3x104 cell/wells with 2 ml αMEM media supplemented with 10% FBS. The experimental plates were placed between a pair of Helmoltz coils powered by a pulse generator (PEMF, 50 Hz, 1.5 mV/cm in the upper compartment of a dual incubator (Forma. The control plates were placed in the lower compartment of the incubator without Helmotz coils. After three days, the cell proliferation was measured by the method modified from Mossman (J. Immunol Methods 1983; 65: 55-63. Other sets of plates were used for osteocalcin production assessment. Media from these sets were collected after 6 days and assessed for osteocalcin production using ELISA kits. The data were analyzed using a one-way analysis of variance (ANOVA. The results showed that MG-63 cells from the experimental group proliferated significantly more than those from the control group (20% increase, p<0.05. No significant difference in osteocalcin production was detected between the two groups. On the other hand, NHOC from the experimental group produced larger amount of osteocalcin (25% increase, p<0.05 and proliferated significantly more than those from the control group (100% increase, p<0.05. In conclusion, PEMF effect on osteoblasts might depend on their cell type of origin. For osteoblast-like cell line, MG-63 cells, PEMF increased proliferation rate but not osteocalcin production of the cells. However, PEMF stimulation effect on human normal osteoblast cells was most likely associated with enhancement of both osteocalcin production and cell proliferation.

  1. Platelet-poor plasma stimulates the proliferation but inhibits the differentiation of rat osteoblastic cells in vitro.

    Science.gov (United States)

    Hamdan, Ahmad Abdel-Salam; Loty, Sabine; Isaac, Juliane; Bouchard, Philippe; Berdal, Ariane; Sautier, Jean-Michel

    2009-06-01

    Recent studies have shown that the use of platelet preparations in bone and implant surgery might stimulate bone formation. However, the biological mechanisms are not well understood. Moreover, few studies have attempted to evaluate the effect of platelet-poor plasma (PPP), which is a product of the platelet-rich plasma preparation process. Thus, this study investigated the behavior of osteoblasts isolated from fetal rat calvaria cultivated in the presence of homologous PPP. PPP was obtained by centrifugation of the rat mother's blood and used in replacement of fetal calf serum, which is classically used in primary culture procedures. Proliferation was measured by an MTT assay at 24, 48, and 72 h. Real-time PCR was performed to study the expression of Runx2, Dlx5, and osteocalcin (OC) on days 0 (4 h), 1, 3, 7, and 12. Alkaline phosphatase (ALP) biochemical activity was evaluated on days 0 (4 h), 1, 3, 7, and 12. Observations by phase-contrast microscopy showed that osteoblasts were able to differentiate until the mineralization of the matrix in the presence of PPP. PPP enhanced the proliferation significantly compared with the control group (Pexpressed by cells in the experimental group at lower levels compared with the control group. Biochemical assay of ALP showed a lower activity in the experimental group compared with the control group (P<0.001). These results suggest that, in the presence of homologous PPP, rat osteoblastic cells are able to maintain their phenotype, with a higher rate of proliferation. However, PPP seems to inhibit osteoblastic differentiation.

  2. Inhibition of osteoclastogenesis by osteoblast-like cells genetically engineered to produce interleukin-10.

    Science.gov (United States)

    Fujioka, Kazuki; Kishida, Tsunao; Ejima, Akika; Yamamoto, Kenta; Fujii, Wataru; Murakami, Ken; Seno, Takahiro; Yamamoto, Aihiro; Kohno, Masataka; Oda, Ryo; Yamamoto, Toshiro; Fujiwara, Hiroyoshi; Kawahito, Yutaka; Mazda, Osam

    2015-01-16

    Bone destruction at inflamed joints is an important complication associated with rheumatoid arthritis (RA). Interleukin-10 (IL-10) may suppress not only inflammation but also induction of osteoclasts that play key roles in the bone destruction. If IL-10-producing osteoblast-like cells are induced from patient somatic cells and transplanted back into the destructive bone lesion, such therapy may promote bone remodeling by the cooperative effects of IL-10 and osteoblasts. We transduced mouse fibroblasts with genes for IL-10 and Runx2 that is a crucial transcription factor for osteoblast differentiation. The IL-10-producing induced osteoblast-like cells (IL-10-iOBs) strongly expressed osteoblast-specific genes and massively produced bone matrix that were mineralized by calcium phosphate in vitro and in vivo. Culture supernatant of IL-10-iOBs significantly suppressed induction of osteoclast from RANKL-stimulated Raw264.7 cells as well as LPS-induced production of inflammatory cytokine by macrophages. The IL-10-iOBs may be applicable to novel cell-based therapy against bone destruction associated with RA. Copyright © 2014 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    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

  4. Biocompatibility evaluation of HDPE-UHMWPE reinforced β-TCP nanocomposites using highly purified human osteoblast cells.

    Science.gov (United States)

    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.

  5. Low-intensity pulsed ultrasound regulates proliferation and differentiation of osteoblasts through osteocytes

    Energy Technology Data Exchange (ETDEWEB)

    Li, Lei, E-mail: geraldleelei@163.com [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu (China); Yang, Zheng [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu (China); Zhang, Hai [Department of Restorative Dentistry, School of Dentistry, University of Washington, Seattle, WA (United States); Chen, Wenchuan [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu (China); Chen, Mengshi [Department of Biomechanics, Sichuan University, Chengdu (China); Zhu, Zhimin, E-mail: hxzhimin@163.com [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu (China)

    2012-02-10

    Highlights: Black-Right-Pointing-Pointer CM from LIPUS-stimulated osteocytes inhibits proliferation of osteoblasts. Black-Right-Pointing-Pointer CM from LIPUS-stimulated osteocytes enhances differentiation of osteoblasts. Black-Right-Pointing-Pointer LIPUS stimulates MLO-Y4 cells to secrete PGE{sub 2} and NO. -- Abstract: Low-intensity pulsed ultrasound (LIPUS) has been used as a safe and effective modality to enhance fracture healing. As the most abundant cells in bone, osteocytes orchestrate biological activities of effector cells via direct cell-to-cell contacts and by soluble factors. In this study, we have used the osteocytic MLO-Y4 cells to study the effects of conditioned medium from LIPUS-stimulated MLO-Y4 cells on proliferation and differentiation of osteoblastic MC3T3-E1 cells. Conditioned media from LIPUS-stimulated MLO-Y4 cells (LIPUS-Osteocyte-CM) were collected and added on MC3T3-E1 cell cultures. MC3T3-E1 cells cultured in LIPUS-Osteocyte-CM demonstrated a significant inhibition of proliferation and an increased alkaline phosphatase activity. The results of PGE{sub 2} and NO assay showed that LIPUS could enhance PGE{sub 2} and NO secretion from MLO-Y4 cells at all time points within 24 h after LIPUS stimulation. We conclude that LIPUS regulates proliferation and differentiation of osteoblasts through osteocytes in vitro. Increased secretion of PGE{sub 2} from osteocytes may play a role in this effect.

  6. The Degradation Interface of Magnesium Based Alloys in Direct Contact with Human Primary Osteoblast Cells.

    Science.gov (United States)

    Ahmad Agha, Nezha; Willumeit-Römer, Regine; Laipple, Daniel; Luthringer, Bérengère; Feyerabend, Frank

    2016-01-01

    Magnesium alloys have been identified as a new generation material of orthopaedic implants. In vitro setups mimicking physiological conditions are promising for material / degradation analysis prior to in vivo studies however the direct influence of cell on the degradation mechanism has never been investigated. For the first time, the direct, active, influence of human primary osteoblasts on magnesium-based materials (pure magnesium, Mg-2Ag and Mg-10Gd alloys) is studied for up to 14 days. Several parameters such as composition of the degradation interface (directly beneath the cells) are analysed with a scanning electron microscope equipped with energy dispersive X-ray and focused ion beam. Furthermore, influence of the materials on cell metabolism is examined via different parameters like active mineralisation process. The results are highlighting the influences of the selected alloying element on the initial cells metabolic activity.

  7. Potential of Resveratrol Analogues as Antagonists of Osteoclasts and Promoters of Osteoblasts

    DEFF Research Database (Denmark)

    Kupisiewicz, Katarzyna; Boissy, Patrice; Abdallah, Basem M

    2010-01-01

    The plant phytoalexin resveratrol was previously demonstrated to inhibit the differentiation and bone resorbing activity of osteoclasts, to promote the formation of osteoblasts from mesenchymal precursors in cultures, and inhibit myeloma cell proliferation, when used at high concentrations....... In the current study, we screened five structurally modified resveratrol analogues for their ability to modify the differentiation of osteoclasts and osteoblasts and proliferation of myeloma cells. Compared to resveratrol, analogues showed an up to 5,000-fold increased potency to inhibit osteoclast...... differentiation. To a lesser extent, resveratrol analogues also promoted osteoblast maturation. However, they did not antagonize the proliferation of myeloma cells. The potency of the best-performing candidate in vitro was tested in vivo in an ovariectomy-induced model of osteoporosis, but an effect on bone loss...

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

    International Nuclear Information System (INIS)

    Torricelli, Paola; Fini, Milena; Giavaresi, Gianluca; Rimondini, Lia; Tschon, Matilde; Rimondini, Roberto; Carrassi, Antonio; Giardino, Roberto

    2008-01-01

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

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

    Science.gov (United States)

    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.

  10. Osteoblast role in osteoarthritis pathogenesis.

    Science.gov (United States)

    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.

  11. Myeloma cells suppress osteoblasts through sclerostin secretion

    Energy Technology Data Exchange (ETDEWEB)

    Colucci, S; Brunetti, G; Oranger, A [Department of Human Anatomy and Histology, University of Bari Medical School, Bari (Italy); Mori, G [Department of Biomedical Science, University of Foggia, Foggia (Italy); Sardone, F [Department of Human Anatomy and Histology, University of Bari Medical School, Bari (Italy); Specchia, G; Rinaldi, E; Curci, P; Liso, V [Department of Emergency and Organ Transplantation, Hematology Section, Bari University Medical School, Bari (Italy); Passeri, G [Department of Internal Medicine and Biomedical Sciences, Center for Metabolic Bone Diseases, University of Parma, Parma (Italy); Zallone, A [Department of Human Anatomy and Histology, University of Bari Medical School, Bari (Italy); Rizzi, R [Department of Emergency and Organ Transplantation, Hematology Section, Bari University Medical School, Bari (Italy); Grano, M [Department of Human Anatomy and Histology, University of Bari Medical School, Bari (Italy)

    2011-06-01

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

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

    Science.gov (United States)

    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

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

    International Nuclear Information System (INIS)

    Kawano, Michinao; Ariyoshi, Wataru; Iwanaga, Kenjiro; Okinaga, Toshinori; Habu, Manabu; Yoshioka, Izumi; Tominaga, Kazuhiro; Nishihara, Tatsuji

    2011-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-02-25

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

  15. Ihh/Gli2 signaling promotes osteoblast differentiation by regulating Runx2 expression and function.

    Science.gov (United States)

    Shimoyama, Atsuko; Wada, Masahiro; Ikeda, Fumiyo; Hata, Kenji; Matsubara, Takuma; Nifuji, Akira; Noda, Masaki; Amano, Katsuhiko; Yamaguchi, Akira; Nishimura, Riko; Yoneda, Toshiyuki

    2007-07-01

    Genetic and cell biological studies have indicated that Indian hedgehog (Ihh) plays an important role in bone development and osteoblast differentiation. However, the molecular mechanism by which Ihh regulates osteoblast differentiation is complex and remains to be fully elucidated. In this study, we investigated the role of Ihh signaling in osteoblast differentiation using mesenchymal cells and primary osteoblasts. We observed that Ihh stimulated alkaline phosphatase (ALP) activity, osteocalcin expression, and calcification. Overexpression of Gli2- but not Gli3-induced ALP, osteocalcin expression, and calcification of these cells. In contrast, dominant-negative Gli2 markedly inhibited Ihh-dependent osteoblast differentiation. Ihh treatment or Gli2 overexpression also up-regulated the expression of Runx2, an essential transcription factor for osteoblastogenesis, and enhanced the transcriptional activity and osteogenic action of Runx2. Coimmunoprecipitation analysis demonstrated a physical interaction between Gli2 and Runx2. Moreover, Ihh or Gli2 overexpression failed to increase ALP activity in Runx2-deficient mesenchymal cells. Collectively, these results suggest that Ihh regulates osteoblast differentiation of mesenchymal cells through up-regulation of the expression and function of Runx2 by Gli2.

  16. Antioxidant and bone repair properties of quercetin-functionalized hydroxyapatite: An in vitro osteoblast-osteoclast-endothelial cell co-culture study.

    Science.gov (United States)

    Forte, Lucia; Torricelli, Paola; Boanini, Elisa; Gazzano, Massimo; Rubini, Katia; Fini, Milena; Bigi, Adriana

    2016-03-01

    functionalized with different amounts of quercetin and obtain new composite materials which display both the good bioactivity of the inorganic phase and the therapeutic properties of the flavonoid. The innovative in vitro model developed in this study, which involves co-culture of osteoblast, osteoclast and endothelial cells, allows to state that the new materials exert a beneficial action onto bone repair microenvironment, stimulating osteoblast proliferation and activity, downregulating osteoclastogenesis, and supporting microangiogenetic processes necessary for new bone formation. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

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

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

  18. The Degradation Interface of Magnesium Based Alloys in Direct Contact with Human Primary Osteoblast Cells.

    Directory of Open Access Journals (Sweden)

    Nezha Ahmad Agha

    Full Text Available Magnesium alloys have been identified as a new generation material of orthopaedic implants. In vitro setups mimicking physiological conditions are promising for material / degradation analysis prior to in vivo studies however the direct influence of cell on the degradation mechanism has never been investigated. For the first time, the direct, active, influence of human primary osteoblasts on magnesium-based materials (pure magnesium, Mg-2Ag and Mg-10Gd alloys is studied for up to 14 days. Several parameters such as composition of the degradation interface (directly beneath the cells are analysed with a scanning electron microscope equipped with energy dispersive X-ray and focused ion beam. Furthermore, influence of the materials on cell metabolism is examined via different parameters like active mineralisation process. The results are highlighting the influences of the selected alloying element on the initial cells metabolic activity.

  19. Effect of thorium, cerium and lanthanium metals on the radiosensitivity of human osteoblasts

    International Nuclear Information System (INIS)

    Iwahara, Lucas Kiyoshi da Fonseca

    2016-01-01

    This work analyzed the effects of Th, Ce and La combinations on the human osteoblast proliferation. Due to the osteotropic potential of actinides and lanthanides, a human osteoblast cell line was used to evaluate the effects of metals on cell radiosensitivity using cell proliferation and total proteins as indicators. Assays were performed using cultures exposed to metal alone and in combination and to ionising radiation. It was not observed effects on proliferation for cultures exposed to the metals alone. Concerning the influence of the three elements on the radiosensitivity, it was seen that all three metals were able to interfere on this indicator, in a concentration dependent manner. Evaluating cultures exposed to binary mixtures (Th-Ce and Th-La) and a ternary mixture (Th-Ce-La), it was verified that there were chemical interactions between the metals, for the combinations tested. The results showed very strong antagonism on the inhibition of cell proliferation in cultures exposed to Th-La and Th- Ce-La combinations. Regarding the osteoblasts exposed to mixtures and to radiation it was seen an antagonistic effect on the cell proliferation in all tested combinations, and the Th-Ce combination with a higher degree. These results show that metal mixtures containing thorium, in association with ionising radiation, induced different effects on cell proliferation, regarding the exposure to the metals alone, suggesting the possibility that the combinations interfere on osteoblast radiosensitivity expressing the increase of the occupational hazard among workers involved with monazite sands. The results also indicate that the analysis of the effects of metal mixtures on human cells is a more realistic risk assessment in comparison with the analysis of risk for single elements. The work displays the need to development of risk assessment models that include the study of mixtures obtained in the work environment for the evaluation of cytotoxic and radiotoxic effects in

  20. The effect of globin scaffold on osteoblast adhesion and phenotype expression in vitro.

    Science.gov (United States)

    Hamdan, Ahmad A; Loty, Sabine; Isaac, Juliane; Tayot, Jean-Louis; Bouchard, Philippe; Khraisat, Ameen; Bedral, Ariane; Sautier, Jean-Michel

    2012-01-01

    Different synthetic and natural biomaterials have been used in bone tissue regeneration. However, several limitations are associated with the use of synthetic as well as allogenous or xenogenous natural materials. This study evaluated, in an in vitro model, the behavior of rat osteoblastic cells cultured on a human globin scaffold. Rat osteoblastic cells were isolated from the calvaria of 21-day-old fetal Sprague-Dawley rats. They were then grown in the presence of globin. Real-time polymerase chain reaction (RT-PCR) was performed to study the expression of cyclin D1, integrin Β1, Msx2, Dlx5, Runx2, and osteocalcin on days 1, 5, and 9. Moreover, alkaline phosphatase activity was measured on days 1, 3, 5, and 7. Alizarin red staining was performed on day 9 to observe calcium deposition. Cells were able to adhere, proliferate, and differentiate on globin scaffolds. Moreover, RT-PCR showed that globin may stimulate some key genes of osteoblastic differentiation (Runx2, osteocalcin, Dlx5). Globin had an inhibitory effect on alkaline phosphatase activity. Calcium deposits were seen after 9 days of culture. These results indicate that purified human globin might be a suitable scaffold for bone tissue regeneration.

  1. In vitro studies of osteoblasts response onto zinc aluminate ceramic films

    Directory of Open Access Journals (Sweden)

    Marco Antonio Alvarez-Pérez

    2009-01-01

    Full Text Available Zinc based or doped ceramics have shown to be capable of increasing osteoblasts proliferation, biomineralization and bone formation. However, studies regarding the biological applications processes in ZnAl2O4 ceramic films are very scarce. For this reason, the objective of this in vitro study was to investigate the response of osteoblasts cells cultured onto ZnAl2O4 films. Our results showed a good biological response related to attachment and viability, with good cell morphology attached to the semi-spherical grains of the ceramic and the analysis of mineral-like tissue showed a high quantity of mineral deposited and organized as tiny spherical-like nodules attached to nanostructure surface of ZnAl2O4 material films. Based in our results, ZnAl2O4 films stimulated the bioactivity of osteoblasts cells and provide a microenvironment that favors cell differentiation and mineralization processes, suggesting their potential use as osteoconductive coating onto currently orthopedic and dental implants.

  2. Cooperative effects in differentiation and proliferation between PDGF-BB and matrix derived synthetic peptides in human osteoblasts

    Directory of Open Access Journals (Sweden)

    Vordemvenne Thomas

    2011-11-01

    Full Text Available Abstract Background Enhancing osteogenic capabilities of bone matrix for the treatment of fractures and segmental defects using growth factors is an active area of research. Recently, synthetic peptides like AC- 100, TP508 or p-15 corresponding to biologically active sequences of matrix proteins have been proven to stimulate bone formation. The platelet-derived growth factor (PDGF BB has been identified as an important paracrine factor in early bone healing. We hypothesized that the combined use of PDGF-BB with synthetic peptides could result in an increase in proliferation and calcification of osteoblast-like cells. Methods Osteoblast-like cell cultures were treated with PDGF and synthetic peptides, singly and as combinations, and compared to non-treated control cell cultures. The cultures were evaluated at days 2, 5, and 10 in terms of cell proliferation, calcification and gene expression of alkaline phosphate, collagen I and osteocalcin. Results Experimental findings revealed that the addition of PDGF, p-15 and TP508 and combinations of PDGF/AC-100, PDGF/p-15 and PDGF/TP508 resulted in an increase in proliferating osteoblasts, especially in the first 5 days of cultivation. Proliferation did not significantly differ between single factors and factor combinations (p > 0.05. The onset of calcification in osteoblasts occurred earlier and was more distinct compared to the corresponding control or PDGF stimulation alone. Significant difference was found for the combined use of PDGF/p-15 and PDGF/AC-100 (p Conclusions Our findings indicate that PDGF exhibits cooperative effects with synthetic peptides in differentiation and proliferation. These cooperative effects cause a significant early calcification of osteoblast-like cells (p

  3. 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

    Science.gov (United States)

    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

  4. Bone culture research

    Science.gov (United States)

    Partridge, Nicola C.

    1993-01-01

    The experiments described are aimed at exploring PTH regulation of production of collagenase and protein inhibitors of collagenase (tissue inhibitors of metalloproteases, TIMP-1 and -2) by osteoblast-like osteosarcoma cells under conditions of weightlessness. The results of this work will contribute to information as to whether a microgravity environment alters the functions and responsiveness of the osteoblast. The objectives of the Bone Culture Research (BCR) experiment are: to observe the effects of microgravity on the morphology, rate of proliferation, and behavior of the osteoblastic cells, UMR 106-01; to determine whether microgravy affects the hormonal sensitivity of osteroblastic cells; and to measure the secretion of collagenase and its inhibitors into the medium under conditions of microgravity. The methods employed will consist of the following: the osteoblast-like cells, UMR-106-01, will be cultured in four NASDA cell culture chambers; two chambers will be subjected to microgravity on SL-J; two chambers will remain on the ground at KSC as ground controls but subjected to an identical set of culture conditions as on the shuttle; media will be changed four times; twice the cells will receive the hormone parathyroid hormone-related protein (PTHrP) and media collected; cells will be photographed under conditions of microgravity; and media and photographs will be analyzed upon return to determine whether functions of the cells changed.

  5. Osteoblastic cells: differentiation and trans-differentiation

    DEFF Research Database (Denmark)

    Kassem, Moustapha; Abdallah, Basem; Saeed, Hamid

    2008-01-01

    The osteoblast is the bone forming cell and is derived from mesenchymal stem cells (MSC) present among the bone marrow stroma. MSC are capable of multi-lineage differentiation into mesoderm-type cells such as osteoblasts and adipocytes. Understanding the mechanisms underlying osteoblast different...

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

    Directory of Open Access Journals (Sweden)

    Xiaoxue Yin

    2012-08-01

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

  7. Gene profile analysis of osteoblast genes differentially regulated by histone deacetylase inhibitors

    Directory of Open Access Journals (Sweden)

    Lamblin Anne-Francoise

    2007-10-01

    Full Text Available Abstract Background Osteoblast differentiation requires the coordinated stepwise expression of multiple genes. Histone deacetylase inhibitors (HDIs accelerate the osteoblast differentiation process by blocking the activity of histone deacetylases (HDACs, which alter gene expression by modifying chromatin structure. We previously demonstrated that HDIs and HDAC3 shRNAs accelerate matrix mineralization and the expression of osteoblast maturation genes (e.g. alkaline phosphatase, osteocalcin. Identifying other genes that are differentially regulated by HDIs might identify new pathways that contribute to osteoblast differentiation. Results To identify other osteoblast genes that are altered early by HDIs, we incubated MC3T3-E1 preosteoblasts with HDIs (trichostatin A, MS-275, or valproic acid for 18 hours in osteogenic conditions. The promotion of osteoblast differentiation by HDIs in this experiment was confirmed by osteogenic assays. Gene expression profiles relative to vehicle-treated cells were assessed by microarray analysis with Affymetrix GeneChip 430 2.0 arrays. The regulation of several genes by HDIs in MC3T3-E1 cells and primary osteoblasts was verified by quantitative real-time PCR. Nine genes were differentially regulated by at least two-fold after exposure to each of the three HDIs and six were verified by PCR in osteoblasts. Four of the verified genes (solute carrier family 9 isoform 3 regulator 1 (Slc9a3r1, sorbitol dehydrogenase 1, a kinase anchor protein, and glutathione S-transferase alpha 4 were induced. Two genes (proteasome subunit, beta type 10 and adaptor-related protein complex AP-4 sigma 1 were suppressed. We also identified eight growth factors and growth factor receptor genes that are significantly altered by each of the HDIs, including Frizzled related proteins 1 and 4, which modulate the Wnt signaling pathway. Conclusion This study identifies osteoblast genes that are regulated early by HDIs and indicates pathways that

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

    Science.gov (United States)

    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.

  9. Human osteoblast cells: isolation, characterization, and growth on polymers for musculoskeletal tissue engineering.

    Science.gov (United States)

    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.

  10. Select polyphenolic fractions from dried plum enhance osteoblast activity through BMP-2 signaling.

    Science.gov (United States)

    Graef, Jennifer L; Rendina-Ruedy, Elizabeth; Crockett, Erica K; Ouyang, Ping; King, Jarrod B; Cichewicz, Robert H; Lucas, Edralin A; Smith, Brenda J

    2018-05-01

    Dried plum supplementation has been shown to enhance bone formation while suppressing bone resorption. Evidence from previous studies has demonstrated that these responses can be attributed in part to the fruit's polyphenolic compounds. The purpose of this study was to identify the most bioactive polyphenolic fractions of dried plum with a focus on their osteogenic activity and to investigate their mechanisms of action under normal and inflammatory conditions. Utilizing chromatographic techniques, six fractions of polyphenolic compounds were prepared from a crude extract of dried plum. Initial screening assays revealed that two fractions (DP-FrA and DP-FrB) had the greatest osteogenic potential. Subsequent experiments using primary bone-marrow-derived osteoblast cultures demonstrated these two fractions enhanced extracellular alkaline phosphatase (ALP), an indicator of osteoblast activity, and mineralized nodule formation under normal conditions. Both fractions enhanced bone morphogenetic protein (BMP) signaling, as indicated by increased Bmp2 and Runx2 gene expression and protein levels of phosphorylated Smad1/5. DP-FrB was most effective at up-regulating Tak1 and Smad1, as well as protein levels of phospho-p38. Under inflammatory conditions, TNF-α suppressed ALP and tended to decrease nodule formation (P=.0674). This response coincided with suppressed gene expression of Bmp2 and the up-regulation of Smad6, an inhibitor of BMP signaling. DP-FrA and DP-FrB partially normalized these responses. Our results show that certain fractions of polyphenolic compounds in dried plum up-regulate osteoblast activity by enhancing BMP signaling, and when this pathway is inhibited by TNF-α, the osteogenic response is attenuated. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Osteoblast Differentiation and Bone Formation Gene Expression in Strontium-inducing Bone Marrow Mesenchymal Stem Cell

    OpenAIRE

    SILA-ASNA, MONNIPHA; BUNYARATVEJ, AHNOND; Maeda, Sakan; Kitaguchi, Hiromichi; BUNYARATAVEJ, NARONG

    2007-01-01

    Osteoblastic differentiation from human mesenchymal stem cell (hMSCs) is animportant step of bone formation. We studied the in vitro induction of hMSCs byusing strontium ranelate, a natural trace amount in water, food and human skeleton.The mRNA synthesis of various osteoblast specific genes was assessed by means ofreverse transcription polymerase chain reaction (RT-PCR). In the hMSCs culture,strontium ranelate could enhance the induction of hMSCs to differentiate intoosteoblasts. Cbfa1 gene ...

  12. The angiogenic behaviors of human umbilical vein endothelial cells (HUVEC) in co-culture with osteoblast-like cells (MG-63) on different titanium surfaces.

    Science.gov (United States)

    Shi, Bin; Andrukhov, Oleh; Berner, Simon; Schedle, Andreas; Rausch-Fan, Xiaohui

    2014-08-01

    Interaction between osteogenesis and angiogenesis plays an important role in implant osseointegration. In the present study we investigated the influence of titanium surface properties on the angiogenic behaviors of endothelial cells grown in direct contact co-culture with osteoblasts. Human umbilical vein endothelial cells (HUVECs) and osteoblast-like cells (MG-63 cells) were grown in direct co-culture on the following titanium surfaces: acid-etched (A), hydrophilic A (modA), coarse-gritblasted and acid-etched (SLA) and hydrophilic SLA (SLActive). Cell proliferation was evaluated by cell counting combined with flow cytometry. The expression of von Willebrand Factor (vWF), thrombomodulin (TM), endothelial cell protein C receptor (EPCR), E-Selectin, as well as vascular endothelial growth factor (VEGF) receptors Flt-1 and KDR in HUVECs and VEGF in MG-63 were measured by qPCR. The dynamic behavior of endothelial cells was recorded by time-lapse microscopy. Proliferation of HUVECs was highest on A, followed by SLA, modA and SLActive surfaces. The expression of vWF, TM, EPCR, E-Selectin and Flt-1 in HUVECs was significantly higher on A than on all other surfaces. The expression of KDR in HUVECs grown on A surface was below detection limit. VEGF expression in MG-63 cells was significantly higher on SLActive vs SLA and modA vs A surfaces. Time-lapse microscopy revealed that HUVECs moved quickest and formed cell clusters earlier on A surface, followed by SLA, modA and SLActive surface. In co-culture conditions, proliferation and expression of angiogenesis associated genes in HUVECs are promoted by smooth hydrophobic Ti surface, which is in contrast to previous mono-culture studies. Copyright © 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

  13. Nanoparticles prepared from the water extract of Gusuibu (Drynaria fortunei J. Sm. protects osteoblasts against insults and promotes cell maturation

    Directory of Open Access Journals (Sweden)

    Hsu C-K

    2011-07-01

    Full Text Available Chung-King Hsu1,2, Mei-Hsiu Liao3, Yu-Tyng Tai4, Shing-Hwa Liu5, Keng-Liang Ou6, Hsu-Wei Fang7, I-Jung Lee8, Ruei-Ming Chen2,31Institute of Materials Science and Engineering, National Taipei University of Technology, 2Cell Physiology and Molecular Image Research Center, Taipei Medical University-Wan Fang Medical Center, 3Graduate Institute of Medical Sciences, Taipei Medical University, 4Department of Anesthesiology, Taipei Medical University-Wan Fang Medical Center, 5Institute of Toxicology, College of Medicine, National Taiwan University, 6Graduate Institute of Biomedical Materials and Engineering, Taipei Medical University, 7Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, 8Division of Information and Herbarium, National Research Institute of Chinese Medicine, Taipei, TaiwanAbstract: Our previous study showed that Gusuibu (Drynaria fortunei J. Sm. can stimulate osteoblast maturation. This study was further designed to evaluate the effects of nanoparticles prepared from the water extract of Gusuibu (WEG on osteoblast survival and maturation. Primary osteoblasts were exposed to 1, 10, 100, and 1000 µg/mL nanoparticles of WEG (nWEG for 24, 48, and 72 hours did not affect morphologies, viability, or apoptosis of osteoblasts. In comparison, treatment of osteoblasts with 1000 µg/mL WEG for 72 hours decreased cell viability and induced DNA fragmentation and cell apoptosis. nWEG had better antioxidant bioactivity in protecting osteoblasts from oxidative and nitrosative stress-induced apoptosis than WEG. In addition, nWEG stimulated greater osteoblast maturation than did WEG. Therefore, this study shows that WEG nanoparticles are safer to primary osteoblasts than are normal-sized products, and may promote better bone healing by protecting osteoblasts from apoptotic insults, and by promoting osteogenic maturation.Keywords: Gusuibu, nanoparticles, cell protection, osteoblast maturation

  14. Estrogen binding, receptor mRNA, and biologic response in osteoblast-like osteosarcoma cells

    International Nuclear Information System (INIS)

    Komm, B.S.; Terpening, C.M.; Benz, D.J.; Graeme, K.A.; Gallegos, A.; Korc, M.; Greene, G.L.; O'Malley, B.W.; Haussler, M.R.

    1988-01-01

    High specific activity estradiol labeled with iodine-125 was used to detect approximately 200 saturable, high-affinity (dissociation constant approximately equal to 1.0 nM) nuclear binding sites in rat (ROS 17/2.8) and human (HOS TE85) clonal osteoblast-like osteosarcoma cells. Of the steroids tested, only testosterone exhibited significant cross-reactivity with estrogen binding. RNA blot analysis with a complementary DNA probe to the human estrogen receptor revealed putative receptor transcripts of 6 to 6.2 kilobases in both rat and human osteosarcoma cells. Type I procollagen and transforming growth factor-beta messenger RNA levels were enhanced in cultured human osteoblast-like cells treated with 1 nM estradiol. Thus, estrogen can act directly on osteoblasts by a receptor-mediated mechanism and thereby modulate the extracellular matrix and other proteins involved in the maintenance of skeletal mineralization and remodeling

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

    Science.gov (United States)

    Bell, Bryan Frederick, Jr.

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

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

    Directory of Open Access Journals (Sweden)

    Thomas Lind

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

  17. Osteoblast-targeted overexpression of TAZ increases bone mass in vivo.

    Directory of Open Access Journals (Sweden)

    Jae-Yeon Yang

    Full Text Available Osteoblasts are derived from mesenchymal progenitors. Differentiation to osteoblasts and adipocytes is reciprocally regulated. Transcriptional coactivator with a PDZ-binding motif (TAZ is a transcriptional coactivator that induces differentiation of mesenchymal cells into osteoblasts while blocking differentiation into adipocytes. To investigate the role of TAZ on bone metabolism in vivo, we generated transgenic mice that overexpress TAZ under the control of the procollagen type 1 promoter (Col1-TAZ. Whole body bone mineral density (BMD of 6- to 19-week-old Col-TAZ mice was 4% to 7% higher than that of their wild-type (WT littermates, whereas no difference was noticed in Col.1-TAZ female mice. Microcomputed tomography analyses of proximal tibiae at 16 weeks of age demonstrated a significant increase in trabecular bone volume (26.7% and trabecular number (26.6% with a reciprocal decrease in trabecular spacing (14.2% in Col1-TAZ mice compared with their WT littermates. In addition, dynamic histomorphometric analysis of the lumbar spine revealed increased mineral apposition rate (42.8% and the serum P1NP level was also significantly increased (53% in Col.1-TAZ mice. When primary calvaria cells were cultured in osteogenic medium, alkaline phosphatase (ALP activity was significantly increased and adipogenesis was significantly suppressed in Col1-TAZ mice compared with their WT littermates. Quantitative real-time polymerase chain reaction analyses showed that expression of collagen type 1, bone sialoprotein, osteocalcin, ALP, osterix, and Runx2 was significantly increased in calvaria cells from Col1-TAZ mice compared to their WT littermates. In vitro, TAZ enhanced Runx2-mediated transcriptional activity while suppressing the peroxisome proliferator-activated receptor gamma signaling pathway. TAZ also enhanced transcriptional activity from 3TP-Lux, which reflects transforming growth factor-beta (TGF-β-mediated signaling. In addition, TAZ enhanced TGF

  18. Confocal laser Raman microspectroscopy of biomineralization foci in UMR 106 osteoblastic cultures reveals temporally synchronized protein changes preceding and accompanying mineral crystal deposition.

    Science.gov (United States)

    Wang, Chuanyi; Wang, Yong; Huffman, Nichole T; Cui, Chaoying; Yao, Xiaomei; Midura, Sharon; Midura, Ronald J; Gorski, Jeff P

    2009-03-13

    Mineralization in UMR 106-01 osteoblastic cultures occurs within extracellular biomineralization foci (BMF) within 12 h after addition of beta-glycerol phosphate to cells at 64 h after plating. BMF are identified by their enrichment with an 85-kDa glycoprotein reactive with Maackia amurensis lectin. Laser Raman microspectroscopic scans were made on individual BMF at times preceding (64-76 h) and following the appearance of mineral crystals (76-88 h). The range of variation between spectra for different BMF in the same culture was rather small. In contrast, significant differences were observed for spectral bands at 957-960, 1004, and 1660 cm(-1) when normalized BMF spectra at different times were compared. Protein-dependent spectral bands at 1004 and 1660 cm(-1) increased and then decreased preceding the detection of hydroxyapatite crystals via the phosphate stretching peak at 959-960 cm(-1). When sodium phosphate was substituted for beta-glycerol phosphate, mineralization occurred 3-6 h earlier. Irrespective of phosphate source, the Raman full peak width at half-maximum ratio for 88 h cultures was similar to that for 10-day-old marrow ablation primary bone. However, if mineralization was blocked with serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, 64-88-h BMF spectra remained largely invariant. In summary, Raman spectral data demonstrate for the first time that formation of hydroxyapatite crystals within individual BMF is a multistep process. Second, changes in protein-derived signals at 1004 and 1660 cm(-1) reflect events within BMFs that precede or accompany mineral crystal production because they are blocked by mineralization inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride. Finally, the low extent of spectral variability detected among different BMF at the same time point indicates that mineralization of individual BMF within a culture is synchronized.

  19. Confocal Laser Raman Microspectroscopy of Biomineralization Foci in UMR 106 Osteoblastic Cultures Reveals Temporally Synchronized Protein Changes Preceding and Accompanying Mineral Crystal Deposition*

    Science.gov (United States)

    Wang, Chuanyi; Wang, Yong; Huffman, Nichole T.; Cui, Chaoying; Yao, Xiaomei; Midura, Sharon; Midura, Ronald J.; Gorski, Jeff P.

    2009-01-01

    Mineralization in UMR 106-01 osteoblastic cultures occurs within extracellular biomineralization foci (BMF) within 12 h after addition of β-glycerol phosphate to cells at 64 h after plating. BMF are identified by their enrichment with an 85-kDa glycoprotein reactive with Maackia amurensis lectin. Laser Raman microspectroscopic scans were made on individual BMF at times preceding (64–76 h) and following the appearance of mineral crystals (76–88 h). The range of variation between spectra for different BMF in the same culture was rather small. In contrast, significant differences were observed for spectral bands at 957–960, 1004, and 1660 cm-1 when normalized BMF spectra at different times were compared. Protein-dependent spectral bands at 1004 and 1660 cm-1 increased and then decreased preceding the detection of hydroxyapatite crystals via the phosphate stretching peak at 959–960 cm-1. When sodium phosphate was substituted for β-glycerol phosphate, mineralization occurred 3–6 h earlier. Irrespective of phosphate source, the Raman full peak width at half-maximum ratio for 88 h cultures was similar to that for 10-day-old marrow ablation primary bone. However, if mineralization was blocked with serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, 64–88-h BMF spectra remained largely invariant. In summary, Raman spectral data demonstrate for the first time that formation of hydroxyapatite crystals within individual BMF is a multistep process. Second, changes in protein-derived signals at 1004 and 1660 cm-1 reflect events within BMFs that precede or accompany mineral crystal production because they are blocked by mineralization inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride. Finally, the low extent of spectral variability detected among different BMF at the same time point indicates that mineralization of individual BMF within a culture is synchronized. PMID:19116206

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

    Science.gov (United States)

    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.

  1. Scanning electron microscopical observation of an osteoblast/osteoclast co-culture on micropatterned orthopaedic ceramics.

    Science.gov (United States)

    Halai, Mansur; Ker, Andrew; Meek, Rm Dominic; Nadeem, Danish; Sjostrom, Terje; Su, Bo; McNamara, Laura E; Dalby, Matthew J; Young, Peter S

    2014-01-01

    In biomaterial engineering, the surface of an implant can influence cell differentiation, adhesion and affinity towards the implant. On contact with an implant, bone marrow-derived mesenchymal stromal cells demonstrate differentiation towards bone forming osteoblasts, which can improve osteointegration. The process of micropatterning has been shown to improve osteointegration in polymers, but there are few reports surrounding ceramics. The purpose of this study was to establish a co-culture of bone marrow-derived mesenchymal stromal cells with osteoclast progenitor cells and to observe the response to micropatterned zirconia toughened alumina ceramics with 30 µm diameter pits. The aim was to establish whether the pits were specifically bioactive towards osteogenesis or were generally bioactive and would also stimulate osteoclastogenesis that could potentially lead to osteolysis. We demonstrate specific bioactivity of micropatterns towards osteogenesis, with more nodule formation and less osteoclastogenesis compared to planar controls. In addition, we found that that macrophage and osteoclast-like cells did not interact with the pits and formed fewer full-size osteoclast-like cells on the pitted surfaces. This may have a role when designing ceramic orthopaedic implants.

  2. Human immunodeficiency virus envelope protein Gp120 induces proliferation but not apoptosis in osteoblasts at physiologic concentrations.

    Directory of Open Access Journals (Sweden)

    Nathan W Cummins

    Full Text Available 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.

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

    Directory of Open Access Journals (Sweden)

    Gretel G. Pellegrini

    2016-07-01

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

  4. In vitro evaluation of osteoblastic cells on bacterial cellulose modified with multi-walled carbon nanotubes as scaffold for bone regeneration

    Energy Technology Data Exchange (ETDEWEB)

    Gutiérrez-Hernández, José Manuel [Coordination for Innovation and Application of Science and Technology, Autonomous University San Luis Potosi, 78000 San Luis Potosi (Mexico); Department of Wood, Cellulose and Paper Research, University Guadalajara, 45110 Guadalajara (Mexico); Escobar-García, Diana María [Laboratory of Basic Sciences, Faculty of Dentistry, Autonomous University San Luis Potosi, 78000 San Luis Potosi (Mexico); Escalante, Alfredo [Department of Wood, Cellulose and Paper Research, University Guadalajara, 45110 Guadalajara (Mexico); Flores, Hector [Laboratory of Basic Sciences, Faculty of Dentistry, Autonomous University San Luis Potosi, 78000 San Luis Potosi (Mexico); González, Francisco Javier [Coordination for Innovation and Application of Science and Technology, Autonomous University San Luis Potosi, 78000 San Luis Potosi (Mexico); Gatenholm, Paul [Chalmers University of Technology, Department of Chemistry and Chemical Engineering, Biopolymer Technology, SE-412 96 Göteborg (Sweden); Toriz, Guillermo, E-mail: gtoriz@dmcyp.cucei.udg.mx [Department of Wood, Cellulose and Paper Research, University Guadalajara, 45110 Guadalajara (Mexico); Chalmers University of Technology, Department of Chemistry and Chemical Engineering, Biopolymer Technology, SE-412 96 Göteborg (Sweden)

    2017-06-01

    In this paper we explore the use of native bacterial cellulose (BC) in combination with functionalized multi-walled carbon nanotubes (MWNTs) as an original biomaterial, suitable three-dimensional (3D) scaffold for osteoblastic cell culture. Functionalized MWNTs were mixed with native BC (secreted by Gluconacetobacter xylinus) with the aim of reinforcing the mechanical properties of BC. The results indicate that BC-MWNTs scaffolds support osteoblast viability, adhesion and proliferation at higher levels as compared to traditional culture substrates. Chemically functionalized MWNTs are also an excellent material to be used as scaffold because these did not affect cell viability and showed an enhanced osteoblast adhesion. These results suggest the potential for this combination of biomaterials, i.e. BC and carbon nanomaterials, as scaffolds for bone regeneration. - Highlights: • Functionalization of multiwalled carbon nanotubes with carboxyl groups for reduces their toxicity against osteoblastic cells. • Use of native bacterial cellulose with functionalized multi-walled carbon nanotubes as scaffolds for tissue engineering. • Bacterial cellulose with multi-walled carbon nanotubes as scaffolds give an excellent option to be used in bone regeneration.

  5. In vitro evaluation of osteoblastic cells on bacterial cellulose modified with multi-walled carbon nanotubes as scaffold for bone regeneration

    International Nuclear Information System (INIS)

    Gutiérrez-Hernández, José Manuel; Escobar-García, Diana María; Escalante, Alfredo; Flores, Hector; González, Francisco Javier; Gatenholm, Paul; Toriz, Guillermo

    2017-01-01

    In this paper we explore the use of native bacterial cellulose (BC) in combination with functionalized multi-walled carbon nanotubes (MWNTs) as an original biomaterial, suitable three-dimensional (3D) scaffold for osteoblastic cell culture. Functionalized MWNTs were mixed with native BC (secreted by Gluconacetobacter xylinus) with the aim of reinforcing the mechanical properties of BC. The results indicate that BC-MWNTs scaffolds support osteoblast viability, adhesion and proliferation at higher levels as compared to traditional culture substrates. Chemically functionalized MWNTs are also an excellent material to be used as scaffold because these did not affect cell viability and showed an enhanced osteoblast adhesion. These results suggest the potential for this combination of biomaterials, i.e. BC and carbon nanomaterials, as scaffolds for bone regeneration. - Highlights: • Functionalization of multiwalled carbon nanotubes with carboxyl groups for reduces their toxicity against osteoblastic cells. • Use of native bacterial cellulose with functionalized multi-walled carbon nanotubes as scaffolds for tissue engineering. • Bacterial cellulose with multi-walled carbon nanotubes as scaffolds give an excellent option to be used in bone regeneration.

  6. STUDY OF FUTURE PRIMARY SCHOOL TEACHERS’ CULTURAL TRAINING WITHIN THE INFORMATION CULTURE OF SOCIETY

    Directory of Open Access Journals (Sweden)

    Tatiana Vinnyk

    2016-05-01

    Full Text Available The article presents the results of scientific studies and experimental approbation of pedagogical conditions of future primary school teachers’ cultural training taking into account the information culture of society. The nature and structure of the notion «future primary school teachers’ cultural training» are clarified. The indicated phenomenon is considered as the structure of four levels, the core of which is personality’s humanistic orientation, the totality of psychological-pedagogical and cultural knowledge and skills, the complex of professionally significant personal qualities. The author pointed out the criteria and related indicators of cultural proficiency, they are: value-motivational (vocational and humanistic orientation; the presence of values and professional motives; motivation for success; substantial and procedural (knowledge and skills in psycho-pedagogical disciplines; the body of knowledge regarding the content and components of cultural training, cultural skills; assessment and behavioral (the existence of communicative qualities, ability to empathy, tolerance. Levels of future primary school teachers’ cultural readiness: high, average and low are characterized. The experience of ICT using in students’ cultural training is presented. Pedagogical conditions of future primary school teachers’ cultural training in University are identified, their effectiveness is proved by experimental testing

  7. Generation of Directly Converted Human Osteoblasts That Are Free of Exogenous Gene and Xenogenic Protein.

    Science.gov (United States)

    Yamamoto, Kenta; Sato, Yoshiki; Honjo, Kenichi; Ichioka, Hiroaki; Oseko, Fumishige; Sowa, Yoshihiro; Yamamoto, Toshiro; Kanamura, Narisato; Kishida, Tsunao; Mazda, Osam

    2016-11-01

    Generation of osteoblasts from human somatic cells may be applicable in an effective transplantation therapy against bone diseases. Recently we established a procedure to directly convert human fibroblasts into osteoblasts by transducing some transcription factor genes via retroviral vectors. However, retroviral vector-mediated transduction may potentially cause tumor formation from the infected cells, thus a non-viral gene transfection method may be more preferable for preparation of osteoblasts to be used for transplantation therapy. Here, we constructed a plasmid vector encoding Oct4, Osterix, and L-Myc that were an appropriate combination of transcription factors for this purpose. Osteoblast-like phenotypes including high alkaline phosphatase (ALP) activity, bone matrix production and osteoblast-specific gene expression were induced in normal human fibroblasts that were transfected with the plasmid followed by culturing in osteogenic medium. The plasmid-driven directly converted osteoblasts (p-dOBs) were obtained even in the absence of a xenogenic protein. The plasmid vector sequence had fallen out of the p-dOBs. The cells formed deposition of calcified bodies in situ after transplantation into mice. These results strongly suggest that p-dOBs can be put into practical use for a novel cell-based therapy against bone diseases. J. Cell. Biochem. 117: 2538-2545, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  8. Thermo-responsive poly(N-isopropylacrylamide)-grafted hollow fiber membranes for osteoblasts culture and non-invasive harvest

    Energy Technology Data Exchange (ETDEWEB)

    Zhuang, Meiling, E-mail: zhuangmeiling2006@126.com; Liu, Tianqing, E-mail: liutq@dlut.edu.cn; Song, Kedong, E-mail: kedongsong@dlut.edu.cn; Ge, Dan, E-mail: gedan@dlut.edu.cn; Li, Xiangqin, E-mail: xiangqinli@163.com

    2015-10-01

    Hollow fiber membrane (HFM) culture system is one of the most important bioreactors for the large-scale culture and expansion of therapeutic cells. However, enzymatic and mechanical treatments are traditionally applied to harvest the expanded cells from HFMs, which inevitably causes harm to the cells. In this study, thermo-responsive cellulose acetate HFMs for cell culture and non-invasive harvest were prepared for the first time via free radical polymerization in the presence of cerium (IV). ATR-FTIR and elemental analysis results indicated that the poly(N-isopropylacrylamide) (PNIPAAm) was covalently grafted on HFMs successfully. Dynamic contact angle measurements at different temperatures revealed that the magnitude of volume phase transition was decreased with increasing grafted amount of PNIPAAm. And the amount of serum protein adsorbed on HFMs surface also displayed the same pattern. Meanwhile osteoblasts adhered and spread well on the surface of PNIPAAm-grafted HFMs at 37 °C. And Calcein-AM/PI staining, AB assay, ALP activity and OCN protein expression level all showed that PNIPAAm-grafted HFMs had good cell compatibility. After incubation at 20 °C for 120 min, the adhering cells on PNIPAAm-grafted HFMs turned to be round and detached after being gently pipetted. These results suggest that thermo-responsive HFMs are attractive cell culture substrates which enable cell culture, expansion and the recovery without proteolytic enzyme treatment for the application in tissue engineering and regenerative medicine. - Highlights: • PNIPAAm-grafted HFMs exhibited thermoresponsive characteristic. • The OB cells could adhere and spread well on the surface of PNIPAAm-grafted HFMs. • PNIPAAm-grafted HFMs do not significantly impact ALP activity and OCN protein expression level of OB cells. • Cell could be detached from PNIPAAm-grafted HFMs when temperature decreased from 37 °C to 20 °C.

  9. Chondrocytic Atf4 regulates osteoblast differentiation and function via Ihh.

    Science.gov (United States)

    Wang, Weiguang; Lian, Na; Ma, Yun; Li, Lingzhen; Gallant, Richard C; Elefteriou, Florent; Yang, Xiangli

    2012-02-01

    Atf4 is a leucine zipper-containing transcription factor that activates osteocalcin (Ocn) in osteoblasts and indian hedgehog (Ihh) in chondrocytes. The relative contribution of Atf4 in chondrocytes and osteoblasts to the regulation of skeletal development and bone formation is poorly understood. Investigations of the Atf4(-/-);Col2a1-Atf4 mouse model, in which Atf4 is selectively overexpressed in chondrocytes in an Atf4-null background, demonstrate that chondrocyte-derived Atf4 regulates osteogenesis during development and bone remodeling postnatally. Atf4 overexpression in chondrocytes of the Atf4(-/-);Col2a1-Atf4 double mutants corrects the reduction in stature and limb in Atf4(-/-) embryos and rectifies the decrease in Ihh expression, Hh signaling, proliferation and accelerated hypertrophy that characterize the Atf4(-/-) developing growth plate cartilages. Unexpectedly, this genetic manipulation also restores the expression of osteoblastic marker genes, namely Ocn and bone sialoprotein, in Atf4(-/-) developing bones. In Atf4(-/-);Col2a1-Atf4 adult mice, all the defective bone parameters found in Atf4(-/-) mice, including bone volume, trabecular number and thickness, and bone formation rate, are rescued. In addition, the conditioned media of ex vivo cultures from wild-type or Atf4(-/-);Col2a1-Atf4, but not Atf4(-/-) cartilage, corrects the differentiation defects of Atf4(-/-) bone marrow stromal cells and Ihh-blocking antibody eliminates this effect. Together, these data indicate that Atf4 in chondrocytes is required for normal Ihh expression and for its paracrine effect on osteoblast differentiation. Therefore, the cell-autonomous role of Atf4 in chondrocytes dominates the role of Atf4 in osteoblasts during development for the control of early osteogenesis and skeletal growth.

  10. The Influence of Primary Microenvironment on Prostate Cancer Osteoblastic Bone LesionDevelopment

    Science.gov (United States)

    2016-11-01

    their normal niche . INTEGRINS IN PCa PROGRESSION Integrins are a large family of cell-surface glycoproteins, which form heterodimeric adhesion...might be induc- ing the CXCR4/CXCL12 axis and thus promoting PCa metastasis. PCa cells home toward areas in the bone marrow rich in osteoblasts where the...hematopoietic stem cell (HSC) niche resides. In fact, PCa cells can bind to and displace mouse HSCs from the niche . Furthermore, the cancer cells

  11. Bone marrow-derived osteoblast progenitor cells in circulating blood contribute to ectopic bone formation in mice

    International Nuclear Information System (INIS)

    Otsuru, Satoru; Tamai, Katsuto; Yamazaki, Takehiko; Yoshikawa, Hideki; Kaneda, Yasufumi

    2007-01-01

    Recent studies have suggested the existence of osteoblastic cells in the circulation, but the origin and role of these cells in vivo are not clear. Here, we examined how these cells contribute to osteogenesis in a bone morphogenetic protein (BMP)-induced model of ectopic bone formation. Following lethal dose-irradiation and subsequent green fluorescent protein-transgenic bone marrow cell-transplantation (GFP-BMT) in mice, a BMP-2-containing collagen pellet was implanted into muscle. Three weeks later, a significant number of GFP-positive osteoblastic cells were present in the newly generated ectopic bone. Moreover, peripheral blood mononuclear cells (PBMNCs) from the BMP-2-implanted mouse were then shown to include osteoblast progenitor cells (OPCs) in culture. Passive transfer of the PBMNCs isolated from the BMP-2-implanted GFP-mouse to the BMP-2-implanted nude mouse led to GFP-positive osteoblast accumulation in the ectopic bone. These data provide new insight into the mechanism of ectopic bone formation involving bone marrow-derived OPCs in circulating blood

  12. Establishment of primary keratinocyte culture from horse tissue biopsates

    Directory of Open Access Journals (Sweden)

    Jernej OGOREVC

    2015-12-01

    Full Text Available Primary cell lines established from skin tissue can be used in immunological, proteomic and genomic studies as in vitro skin models. The goal of our study was to establish a primary keratinocyte cell culture from tissue biopsates of two horses. The primary keratinocyte cell culture was obtained by mechanical and enzymatic dissociation and with explant culture method. The result was a heterogeneous primary culture comprised of keratinocytes and fibroblasts. To distinguish epithelial and mesenchymal cells immunofluorescent characterisation was performed, using antibodies against cytokeratin 14 and vimentin. We successfully at attained a primary cell line of keratinocytes, which could potentially be used to study equine skin diseases, as an animal model for human diseases, and for cosmetic and therapeutic product testing.

  13. Migration assay on primary culture isolated from patient's primary breast cancer tissue

    Directory of Open Access Journals (Sweden)

    ED Yuliana

    2014-12-01

    Full Text Available Background: Migration is an essential component of breast cancer metastasis, which studyhas been concentrated on culture of established breast cancer cell lines that do not accuratelyrepresent the sophistication and heterogeneity of patient's breast cancer. An attempt toperform migration assay using Boyden Chamber Assay (BCA on primary culture originatingfrom patient's breast cancer tissue was developed to accommodate upcoming study of breastcancer migration in lndonesian patients.Methods: Pathologically proven primary breast cancer tissue samples were obtained fromCiptomangunkusumo Hospital during core (n=4 and incisional (n=3 biopsies of stage llAup to stage lllA breast cancer patients. Following biopsy, the breast cancer tissue samplesunderwent processings to isolate the cancer cells. These cancer cells were -then resuspendedwithin Dulbecco's modified Eagle's medium (DMEM ahd cultured in 12-well plate. The growthof primary culture were observed and compared between the core biopsy and the incisionalbiopsy specimens. Optimization of BCA method was later performed to investigate themigration of the breast cancer primary culture towards different experirnental conditions, whichwere control, Fetal Bovine Serum (FBS, and Stromal Derived Factor-l (SDF-1. Two differentnumber of breast cancer cells were tested for the optimization of the BCA, which were 1 x 105and3x105cells.Results: None of the culture performed on core biopsy specimens grew, while one out ofthree incisional biopsy specimens grew until confluence. The one primary culture that grewwas later assesed using BCA to assess its migration index towards different experimentalconditions. Using 1 x 10s breast cancer cells in the BCA , the result of the absorbance level ofmigrated cells showed that the migration towards SDF-1 (0.529 nearly doubled the migrationtowards controlmedium (0.239 and FBS (0.209. Meanwhile, the absorbance levelwas simiiarbetween the control medium (1.050, FBS (1 .103

  14. Anti-osteoporotic activity of harpagide by regulation of bone formation in osteoblast cell culture and ovariectomy-induced bone loss mouse models.

    Science.gov (United States)

    Chung, Hwa-Jin; Kyung Kim, Won; Joo Park, Hyen; Cho, Lan; Kim, Me-Riong; Kim, Min Jeong; Shin, Joon-Shik; Ho Lee, Jin; Ha, In-Hyuk; Kook Lee, Sang

    2016-02-17

    Harpagide, an iridoid glucoside, is a constituent of the root of Harpagophytum procumbens var. sublobatum (Engl.) Stapf, Devil's claw which has been used in patients with osteoarthritis (OA). In the present study, we investigated the anti-osteoporotic potential of harpagide and its underlying mechanism of action in in vitro cell culture and in vivo bone loss animal models. Harpagide was obtained from the alkalic hydrolysis of harpagoside, a major constituent of H. procumbens var. sublobatum Analysis of biomarkers for bone formation in osteoblastic MC3T3-E1 cells and bone resorption in osteoclast cells derived from mouse bone marrow cells was performed to evaluate the mechanism of action. The protective activity of harpagide against bone loss was also evaluated in ovariectomized (OVX) mouse model. Harpagide improved bone properties by stimulating the process of differentiation and maturation of osteoblast cells and suppressing the process of RANKL-induced differentiation of osteoclast cells. In OVX-induced bone loss mouse model, oral administration of harpagide significantly improved recovery of bone mineral density, trabecular bone volume, and trabecular number in the femur. Harpagide also prevented increase of trabecular separation and structure model index induced by OVX. Harpagide effectively inhibited the serum levels of biochemical markers of bone loss, including alkaline phosphatase, osteocalcin, C-terminal telopeptide, and tartrate-resistant acid phosphatase. Taken together, the present study demonstrates that harpagide has a potential for prevention of bone loss in OVX mice by regulating the stimulation of osteoblast differentiation and the suppression of osteoclast formation. Therefore, these findings suggest that harpagide might serve as a bioactive compound derived from H. procumbens var. sublobatum for improvement of age-dependent bone destruction disease. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  15. Lithium doped calcium phosphate cement maintains physical mechanical properties and promotes osteoblast proliferation and differentiation.

    Science.gov (United States)

    Li, Li; Wang, Renchong; Li, Baichuan; Liang, Wei; Pan, Haobo; Cui, Xu; Tang, Jingli; Li, Bing

    2017-07-01

    Calcium phosphate cement (CPC) has been widely used in bone tissue repairing due to its physical mechanical properties and biocompatibility. Addition of trace element to CPC has shown promising evidence to improve the physical properties and biological activities of CPC. Lithium (Li) has effect on osteoblast proliferation and differentiation. In this study, we incorporated Li to CPC and examined the physical properties of Li/CPC and its effect on osteoblast proliferation and differentiation. We found that Li doped CPC maintained similar setting time, pore size distribution, compressive strength, composition, and morphology as CPC without Li. Additionally, Li doped CPC improved osteoblast proliferation and differentiation significantly compared to CPC without Li. To our knowledge, our results, for the first time, show that Li doped CPC has beneficial effect on osteoblast in cell culture while keeps the excellent physical-mechanical properties of CPC. This study will lead to potential application of Li doped CPC in bone tissue engineering. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 944-952, 2017. © 2016 Wiley Periodicals, Inc.

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

    International Nuclear Information System (INIS)

    Kihara, Tasuku; Ichikawa, Saki; Yonezawa, Takayuki; Lee, Ji-Won; Akihisa, Toshihiro; Woo, Je Tae; Michi, Yasuyuki; Amagasa, Teruo; Yamaguchi, Akira

    2011-01-01

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

  17. Ginsenoside Re Promotes Osteoblast Differentiation in Mouse Osteoblast Precursor MC3T3-E1 Cells and a Zebrafish Model

    Directory of Open Access Journals (Sweden)

    Hye-Min Kim

    2016-12-01

    Full Text Available Bone homeostasis is tightly regulated to balance bone formation and bone resorption. Many anabolic drugs are used as bone-targeted therapeutic agents for the promotion of osteoblast-mediated bone formation or inhibition of osteoclast-mediated bone resorption. Previous studies showed that ginsenoside Re has the effect of the suppression of osteoclast differentiation in mouse bone-marrow derived macrophages and zebrafish. Herein, we investigated whether ginsenoside Re affects osteoblast differentiation and mineralization in in vitro and in vivo models. Mouse osteoblast precursor MC3T3-E1 cells were used to investigate cell viability, alkaline phosphatase (ALP activity, and mineralization. In addition, we examined osteoblastic signaling pathways. Ginsenoside Re affected ALP activity without cytotoxicity, and we also observed the stimulation of osteoblast differentiation through the activation of osteoblast markers including runt-related transcription factor 2, type 1 collagen, ALP, and osteocalcin in MC3T3-E1 cells. Moreover, Alizarin red S staining indicated that ginsenoside Re increased osteoblast mineralization in MC3T3-E1 cells and zebrafish scales compared to controls. These results suggest that ginsenoside Re promotes osteoblast differentiation as well as inhibits osteoclast differentiation, and it could be a potential therapeutic agent for bone diseases.

  18. Patient safety culture in primary care

    NARCIS (Netherlands)

    Verbakel, N.J.

    2015-01-01

    Background A constructive patient safety culture is a main prerequisite for patient safety and improvement initiatives. Until now, patient safety culture (PSC) research was mainly focused on hospital care, however, it is of equal importance in primary care. Measuring PSC informs practices on their

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

    DEFF Research Database (Denmark)

    Saeed, Hamid; Qiu, Weimin; Li, Chen

    2015-01-01

    -regulation of several components of insulin-like growth factor (IGF) signaling. Specifically, a significant increase in IGF-induced AKT phosphorylation and alkaline phosphatase (ALP) activity were observed in hMSC-TERT. Enhanced ALP activity was reduced in presence of IGF1 receptor inhibitor: picropodophyllin....... In addition, telomerase deficiency caused significant reduction in IGF signaling proteins in osteoblastic cells cultured from telomerase deficient mice (Terc (-/-)). The low bone mass exhibited by Terc (-/-) mice was associated with significant reduction in serum levels of IGF1 and IGFBP3 as well as reduced...... skeletal mRNA expression of Igf1, Igf2, Igf2r, Igfbp5 and Igfbp6. IGF1-induced osteoblast differentiation was also impaired in Terc (-/-) MSC. In conclusion, our data demonstrate that impaired IGF/AKT signaling contributes to the observed decreased bone mass and bone formation exhibited by telomerase...

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

    Energy Technology Data Exchange (ETDEWEB)

    Okito, Asuka [Department of Cellular Physiological Chemistry, Tokyo Medical and Dental University, Tokyo (Japan); Department of Orthodontic Science, Tokyo Medical and Dental University, Tokyo (Japan); Nakahama, Ken-ichi, E-mail: nakacell@tmd.ac.jp [Department of Cellular Physiological Chemistry, Tokyo Medical and Dental University, Tokyo (Japan); Akiyama, Masako [Department of Cellular Physiological Chemistry, Tokyo Medical and Dental University, Tokyo (Japan); Ono, Takashi [Department of Orthodontic Science, Tokyo Medical and Dental University, Tokyo (Japan); Morita, Ikuo [Department of Cellular Physiological Chemistry, Tokyo Medical and Dental University, Tokyo (Japan)

    2015-03-06

    Osteoclast activity is enhanced in acidic environments following systemic or local inflammation. However, the regulatory mechanism of receptor activator of NF-κB ligand (RANKL) expression in osteoblasts under acidic conditions is not fully understood. In the present paper, we detected the mRNA expression of the G-protein-coupled receptor (GPR) proton sensors GPR4 and GPR65 (T-cell death-associated gene 8, TDAG8), in osteoblasts. RANKL expression and the cyclic AMP (cAMP) level in osteoblasts were up-regulated under acidic culture conditions. Acidosis-induced up-regulation of RANKL was abolished by the protein kinase A inhibitor H89. To clarify the role of GPR4 in RANKL expression, GPR4 gain and loss of function experiments were performed. Gene knockdown and forced expression of GPR4 caused reduction and induction of RANKL expression, respectively. These results suggested that, at least in part, RANKL expression by osteoblasts in an acidic environment was mediated by cAMP/PKA signaling resulting from GPR4 activation. A comprehensive microarray analysis of gene expression of osteoblasts revealed that, under acidic conditions, the phenotype of osteoblasts was that of an osteoclast supporting cell rather than that of a mineralizing cell. These findings will contribute to a molecular understanding of bone disruption in an acidic environment. - Highlights: • RANKL expression was increased in osteoblasts under acidosis via cAMP/PKA pathway. • GRP4 knockdown resulted in decrease of RANKL expression. • GRP4 overexpression resulted in increase of RANKL expression. • Osteoblast mineralization was reduced under acidic condition.

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

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Lingli; Fan Hongsong; Zhang Xingdong [National Engineering Research Center for Biomaterials, Sichuan University, Chengdu, Sichuan 610064 (China); Hanagata, Nobutaka; Ikoma, Toshiyuki [Biomaterials Center, National Institute for Materials Science, Tsukuba, Ibaraki 305-0047 (Japan); Maeda, Megumi; Minowa, Takashi, E-mail: HANAGATA.Nobutaka@nims.go.j [Nanotechnology Innovation Center, National Institute for Materials Science, Tsukuba, Ibaraki 305-0047 (Japan)

    2009-04-15

    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 ({beta}-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 {beta}-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 {beta}-TCP.

  2. Osteoblastic Metastases Mimickers on Contrast Enhanced CT

    Directory of Open Access Journals (Sweden)

    Fahad Al-Lhedan

    2017-01-01

    Full Text Available Secondary osseous involvement in lymphoma is more common compared to primary bone lymphoma. The finding of osseous lesion can be incidentally discovered during the course of the disease. However, osseous metastases are infrequently silent. Detection of osseous metastases is crucial for accurate staging and optimal treatment planning of lymphoma. The aim of imaging is to identify the presence and extent of osseous disease and to assess for possible complications such as pathological fracture of the load-bearing bones and cord compression if the lesion is spinal. We are presenting two patients with treated lymphoma who were in complete remission. On routine follow-up contrast enhanced CT, there were new osteoblastic lesions in the spine worrisome for metastases. Additional studies were performed for further evaluation of both of them which did not demonstrate any corresponding suspicious osseous lesion. The patients have a prior history of chronic venous occlusive thrombosis that resulted in collaterals formation. Contrast enhancement of the vertebral body marrow secondary to collaterals formation and venous flow through the vertebral venous plexus can mimic the appearance of spinal osteoblastic metastases.

  3. Influence of high glucose and advanced glycation end-products (ages) levels in human osteoblast-like cells gene expression.

    Science.gov (United States)

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

    2016-08-31

    Type 2 diabetes mellitus (T2DM) is associated with an increased risk of osteoporotic fracture. Several factors have been identified as being potentially responsible for this risk, such as alterations in bone remodelling that may have been induced by changes in circulating glucose or/and by the presence of non-oxidative end products of glycosylation (AGEs). The aim of this study is to assess whether such variations generate a change in the gene expression related to the differentiation and osteoblast activity (OPG, RANKL, RUNX2, OSTERIX, and AGE receptor) in primary cultures of human osteoblast-like cells (hOB). We recruited 32 patients; 10 patients had osteoporotic hip fractures (OP group), 12 patients had osteoporotic hip fractures with T2DM (T2DM group), and 10 patients had hip osteoarthritis (OA group) with no osteoporotic fractures and no T2DM. The gene expression was analyzed in hOB cultures treated with physiological glucose concentration (4.5 mM) as control, high glucose (25 mM), and high glucose plus AGEs (2 μg/ml) for 24 h. The hOB cultures from patients with hip fractures presented slower proliferation. Additionally, the hOB cultures from the T2DM group were the most negatively affected with respect to RUNX2 and OSX gene expression when treated solely with high glucose or with high glucose plus AGEs. Moreover, high levels of glucose induced a major decrease in the RANKL/OPG ratio when comparing the OP and the T2DM groups to the OA group. Our data indicates an altered bone remodelling rate in the T2DM group, which may, at least partially, explain the reduced bone strength and increased incidence of non-traumatic fractures in diabetic patients.

  4. [Low-frequency pulsed electromagnetic fields promotes rat osteoblast differentiation in vitro through cAMP/PKA signal pathway].

    Science.gov (United States)

    Fang, Qing-Qing; Li, Zhi-Zhong; Zhou, Jian; Shi, Wen-Gui; Yan, Juan-Li; Xie, Yan-Fang; Chen, Ke-Ming

    2016-11-20

    To study whether low-frequency pulsed electromagnetic fields promotes the differentiation of cultured rat osteoblasts through the cAMP/PKA signal pathway. Rat calvarial osteoblasts isolated by enzyme digestion were exposed to 50 Hz 0.6 mT low-frequency pulsed electromagnetic field for varying lengths of time, and the concentration of cAMP and levels of phosphorylated PKA in the cells were assayed. In cells treated with DDA to inhibit the activity of adenylate cyclase, the changes of ALP activity and transcription of osteogenic gene were detected after exposure to low-frequency pulsed electromagnetic field. The changes of osteogenic gene transcription and protein expression were tested in the osteoblasts pretreated with KT5720 in response to low-frequency pulsed electromagnetic field exposure. The intracellular cAMP concentration in the cells increased significantly at 20 min during exposure to low-frequency pulsed electromagnetic field, began to decrease at 40 min during the exposure, and increased again after a 2-h exposure; the same pattern of variation was also observed in p-PKA level. Application of DDA and KT5720 pretreatment both suppressed the increase in ALP activity and osteogenic gene transcription induced by electromagnetic field exposure. Low- frequency pulsed electromagnetic field exposure improves the differentiation of cultured rat osteoblasts by activating cAMP/PKA signal pathway.

  5. Silver oxide-containing hydroxyapatite coating supports osteoblast function and enhances implant anchorage strength in rat femur.

    Science.gov (United States)

    Eto, Shuichi; Miyamoto, Hiroshi; Shobuike, Takeo; Noda, Iwao; Akiyama, Takayuki; Tsukamoto, Masatsugu; Ueno, Masaya; Someya, Shinsuke; Kawano, Shunsuke; Sonohata, Motoki; Mawatari, Masaaki

    2015-09-01

    Antibacterial silver with hydroxyapatite (Ag-HA) is a promising coating material for imparting antibacterial properties to implants. We previously reported that 3% (w/w) silver with HA (3% Ag-HA) has both antibacterial activity and osteoconductivity. In this study, we investigated the effects of Ag-HA on the in vitro osteoblast function and the in vivo anchorage strength and osteoconductivity of implants. Production of the osteoblast marker alkaline phosphatase, but not cytotoxicity, was observed in cells of the osteoblast cell line MC3T3-E1 cultured on the 3% Ag-HA-coated surface. These results were similar to those observed with silver-free HA coating. In contrast, a significant high level of cytotoxicity was observed when the cells were cultured on a 50% Ag-HA-coated surface. The anchorage strength of implants inserted into the femur of Sprague-Dawley (SD) rats was enhanced by coating the implants with 3% Ag-HA. On the 3% Ag-HA-coated surface, both metaphyseal and diaphyseal areas were largely covered with new bone and had adequate osteoconductivity. These results suggest that 3% Ag-HA, like conventional HA, promotes osteogenesis by supporting osteoblast viability and function and thereby contributes to sufficient anchorage strength of implants. Application of 3% Ag-HA, which combines the osteoconductivity of HA and the antibacterial activity of silver, to prosthetic joints will help prevent postoperative infections. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  6. Magnetic nanohydroxyapatite/PVA composite hydrogels for promoted osteoblast adhesion and proliferation.

    Science.gov (United States)

    Hou, Ruixia; Zhang, Guohua; Du, Gaolai; Zhan, Danxia; Cong, Yang; Cheng, Yajun; Fu, Jun

    2013-03-01

    This paper reports on the systematic investigation of novel magnetic nano-hydroxyapatite/PVA composite hydrogels through cyclic freeze-thawing with controllable structure, mechanical properties, and cell adhesion and proliferation properties. The content of the magnetic nano-hydroxyapatite-coated γ-Fe(2)O(3) (m-nHAP) particles exhibited remarkable influence on the porous structures and compressive strength of the nanocomposite hydrogels. The average pore diameter of the nanocomposite hydrogels exhibited a minimum of 1.6 ± 0.3 μm whereas the compressive strength reached a maximum of about 29.6 ± 6.5 MPa with the m-nHAP content of around 10 wt% in the nanocomposite hydrogels. In order to elucidate the influence of the composite m-nHAP on the cell adhesion and proliferation on the composite hydrogels, the PVA, γ-Fe(2)O(3)/PVA, nHAP/PVA and m-nHAP/PVA hydrogels were seeded and cultured with osteoblasts. The results demonstrated that the osteoblasts preferentially adhered to and proliferated on the m-nHAP/PVA hydrogels, in comparison to the PVA and nHAP/PVA hydrogels, whereas the γ-Fe(2)O(3)/PVA hydrogels appeared most favorable to the osteoblasts. Moreover, with the increasing m-nHAP content in the composite hydrogels, the adhesion density and proliferation of the osteoblasts were significantly promoted, especially at the content of around 50 wt%. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. Biocompatibility and Inflammatory Potential of Titanium Alloys Cultivated with Human Osteoblasts, Fibroblasts and Macrophages

    Science.gov (United States)

    Markhoff, Jana; Krogull, Martin; Schulze, Christian; Rotsch, Christian; Hunger, Sandra; Bader, Rainer

    2017-01-01

    The biomaterials used to maintain or replace functions in the human body consist mainly of metals, ceramics or polymers. In orthopedic surgery, metallic materials, especially titanium and its alloys, are the most common, due to their excellent mechanical properties, corrosion resistance, and biocompatibility. Aside from the established Ti6Al4V alloy, shape memory materials such as nickel-titanium (NiTi) have risen in importance, but are also discussed because of the adverse effects of nickel ions. These might be reduced by specific surface modifications. In the present in vitro study, the osteoblastic cell line MG-63 as well as primary human osteoblasts, fibroblasts, and macrophages were cultured on titanium alloys (forged Ti6Al4V, additive manufactured Ti6Al4V, NiTi, and Diamond-Like-Carbon (DLC)-coated NiTi) to verify their specific biocompatibility and inflammatory potential. Additive manufactured Ti6Al4V and NiTi revealed the highest levels of metabolic cell activity. DLC-coated NiTi appeared as a suitable surface for cell growth, showing the highest collagen production. None of the implant materials caused a strong inflammatory response. In general, no distinct cell-specific response could be observed for the materials and surface coating used. In summary, all tested titanium alloys seem to be biologically appropriate for application in orthopedic surgery. PMID:28772412

  8. Biocompatibility and Inflammatory Potential of Titanium Alloys Cultivated with Human Osteoblasts, Fibroblasts and Macrophages

    Directory of Open Access Journals (Sweden)

    Jana Markhoff

    2017-01-01

    Full Text Available The biomaterials used to maintain or replace functions in the human body consist mainly of metals, ceramics or polymers. In orthopedic surgery, metallic materials, especially titanium and its alloys, are the most common, due to their excellent mechanical properties, corrosion resistance, and biocompatibility. Aside from the established Ti6Al4V alloy, shape memory materials such as nickel-titanium (NiTi have risen in importance, but are also discussed because of the adverse effects of nickel ions. These might be reduced by specific surface modifications. In the present in vitro study, the osteoblastic cell line MG-63 as well as primary human osteoblasts, fibroblasts, and macrophages were cultured on titanium alloys (forged Ti6Al4V, additive manufactured Ti6Al4V, NiTi, and Diamond-Like-Carbon (DLC-coated NiTi to verify their specific biocompatibility and inflammatory potential. Additive manufactured Ti6Al4V and NiTi revealed the highest levels of metabolic cell activity. DLC-coated NiTi appeared as a suitable surface for cell growth, showing the highest collagen production. None of the implant materials caused a strong inflammatory response. In general, no distinct cell-specific response could be observed for the materials and surface coating used. In summary, all tested titanium alloys seem to be biologically appropriate for application in orthopedic surgery.

  9. Stepwise Differentiation of Pluripotent Stem Cells into Osteoblasts Using Four Small Molecules under Serum-free and Feeder-free Conditions

    Directory of Open Access Journals (Sweden)

    Kosuke Kanke

    2014-06-01

    Full Text Available 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-methoxyphenylpyrido[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.

  10. The Role of Osteoblast-Derived Cytokines in Bone Metastatic Breast Cancer

    Science.gov (United States)

    2009-01-01

    transform infrared microspectroscopy [34]. The MC3T3-E1 cultures, however, contained material of an unknown origin that represented dystrophic...x- ray densitometer (GE Medical Systems, Fitchburg, WI) and scanned via a Dual Energy X- ray Absorptiometry (DEXA) scan. Company-provided software...including osteoporosis, rheumatoid arthritis , and skeletal metastases, resulting in osteoclast activity in excess of bone deposition by osteoblasts with

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

    Science.gov (United States)

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

    2013-01-01

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

  12. Palmitate attenuates osteoblast differentiation of fetal rat calvarial cells.

    Science.gov (United States)

    Yeh, Lee-Chuan C; Ford, Jeffery J; Lee, John C; Adamo, Martin L

    2014-07-18

    Aging is associated with the accumulation of ectopic lipid resulting in the inhibition of normal organ function, a phenomenon known as lipotoxicity. Within the bone marrow microenvironment, elevation in fatty acid levels may produce an increase in osteoclast activity and a decrease in osteoblast number and function, thus contributing to age-related osteoporosis. However, little is known about lipotoxic mechanisms in intramembraneous bone. Previously we reported that the long chain saturated fatty acid palmitate inhibited the expression of the osteogenic markers RUNX2 and osteocalcin in fetal rat calvarial cell (FRC) cultures. Moreover, the acetyl CoA carboxylase inhibitor TOFA blocked the inhibitory effect of palmitate on expression of these two markers. In the current study we have extended these observations to show that palmitate inhibits spontaneous mineralized bone formation in FRC cultures in association with reduced mRNA expression of RUNX2, alkaline phosphatase, osteocalcin, and bone sialoprotein and reduced alkaline phosphatase activity. The effects of palmitate on osteogenic marker expression were inhibited by TOFA. Palmitate also inhibited the mRNA expression of fatty acid synthase and PPARγ in FRC cultures, and as with osteogenic markers, this effect was inhibited by TOFA. Palmitate had no effect on FRC cell proliferation or apoptosis, but inhibited BMP-7-induced alkaline phosphatase activity. We conclude that palmitate accumulation may lead to lipotoxic effects on osteoblast differentiation and mineralization and that increases in fatty acid oxidation may help to prevent these lipotoxic effects. Copyright © 2014 Elsevier Inc. All rights reserved.

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

    International Nuclear Information System (INIS)

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

    2015-01-01

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

  14. Activation of the PI3K/Akt pathway by oxidative stress mediates high glucose-induced increase of adipogenic differentiation in primary rat osteoblasts.

    Science.gov (United States)

    Zhang, Yu; Yang, Jian-Hong

    2013-11-01

    Diabetes mellitus is associated with increased risk of osteopenia and bone fracture that may be related to hyperglycemia. However, the mechanisms accounting for diabetic bone disorder are unclear. Here, we showed that high glucose significantly promoted the production of reactive oxygen species (ROS) in rat primary osteoblasts. Most importantly, we reported for the first time that ROS induced by high glucose increased alkaline phosphatase activity, inhibited type I collagen (collagen I) protein level and cell mineralization, as well as gene expression of osteogenic markers including runt-related transcription factor 2 (Runx2), collagen I, and osteocalcin, but promoted lipid droplet formation and gene expression of adipogenic markers including peroxisome proliferator-activated receptor gamma, adipocyte fatty acid binding protein (aP2), and adipsin, which were restored by pretreatment with N-acetyl-L-cysteine (NAC), a ROS scavenger. Moreover, high glucose-induced oxidative stress activated PI3K/Akt pathway to inhibited osteogenic differentiation but stimulated adipogenic differentiation. In contrast, NAC and a PI3K inhibitor, LY-294002, reversed the down-regulation of osteogenic markers and the up-regulation of adipogenic markers as well as the activation of Akt under high glucose. These results indicated that oxidative stress played a key role in high glucose-induced increase of adipogenic differentiation, which contributed to the inhibition of osteogenic differentiation. This process was mediated by PI3K/Akt pathway in rat primary osteoblasts. Hence, suppression of oxidative stress could be a potential therapeutic approach for diabetic osteopenia. © 2013 Wiley Periodicals, Inc.

  15. Anhydride-functional silane immobilized onto titanium surfaces induces osteoblast cell differentiation and reduces bacterial adhesion and biofilm formation

    International Nuclear Information System (INIS)

    Godoy-Gallardo, Maria; Guillem-Marti, Jordi; Sevilla, Pablo; Manero, José M.; Gil, Francisco J.

    2016-01-01

    Bacterial infection in dental implants along with osseointegration failure usually leads to loss of the device. Bioactive molecules with antibacterial properties can be attached to titanium surfaces with anchoring molecules such as silanes, preventing biofilm formation and improving osseointegration. Properties of silanes as molecular binders have been thoroughly studied, but research on the biological effects of these coatings is scarce. The aim of the present study was to determine the in vitro cell response and antibacterial effects of triethoxysilypropyl succinic anhydride (TESPSA) silane anchored on titanium surfaces. X-ray photoelectron spectroscopy confirmed a successful silanization. The silanized surfaces showed no cytotoxic effects. Gene expression analyses of Sarcoma Osteogenic (SaOS-2) osteoblast-like cells cultured on TESPSA silanized surfaces reported a remarkable increase of biochemical markers related to induction of osteoblastic cell differentiation. A manifest decrease of bacterial adhesion and biofilm formation at early stages was observed on treated substrates, while favoring cell adhesion and spreading in bacteria–cell co-cultures. Surfaces treated with TESPSA could enhance a biological sealing on implant surfaces against bacteria colonization of underlying tissues. Furthermore, it can be an effective anchoring platform of biomolecules on titanium surfaces with improved osteoblastic differentiation and antibacterial properties. - Highlights: • TESPSA silane induces osteoblast differentiation. • TESPSA reduces bacterial adhesion and biofilm formation. • TESPSA is a promising anchoring platform of biomolecules onto titanium.

  16. Anhydride-functional silane immobilized onto titanium surfaces induces osteoblast cell differentiation and reduces bacterial adhesion and biofilm formation

    Energy Technology Data Exchange (ETDEWEB)

    Godoy-Gallardo, Maria, E-mail: maria.godoy.gallardo@upc.edu [Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgy, Technical University of Catalonia (UPC), ETSEIB, Av. Diagonal 647, 08028 Barcelona (Spain); Centre for Research in NanoEngineering (CRNE) — UPC, C/ Pascual i Vila 15, 08028 Barcelona (Spain); Guillem-Marti, Jordi, E-mail: jordi.guillem.marti@upc.edu [Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgy, Technical University of Catalonia (UPC), ETSEIB, Av. Diagonal 647, 08028 Barcelona (Spain); Centre for Research in NanoEngineering (CRNE) — UPC, C/ Pascual i Vila 15, 08028 Barcelona (Spain); Sevilla, Pablo, E-mail: psevilla@euss.es [Department of Mechanics, Escola Universitària Salesiana de Sarrià (EUSS), C/ Passeig de Sant Bosco, 42, 08017 Barcelona (Spain); Manero, José M., E-mail: jose.maria.manero@upc.edu [Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgy, Technical University of Catalonia (UPC), ETSEIB, Av. Diagonal 647, 08028 Barcelona (Spain); Centre for Research in NanoEngineering (CRNE) — UPC, C/ Pascual i Vila 15, 08028 Barcelona (Spain); Gil, Francisco J., E-mail: francesc.xavier.gil@upc.edu [Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgy, Technical University of Catalonia (UPC), ETSEIB, Av. Diagonal 647, 08028 Barcelona (Spain); Centre for Research in NanoEngineering (CRNE) — UPC, C/ Pascual i Vila 15, 08028 Barcelona (Spain); and others

    2016-02-01

    Bacterial infection in dental implants along with osseointegration failure usually leads to loss of the device. Bioactive molecules with antibacterial properties can be attached to titanium surfaces with anchoring molecules such as silanes, preventing biofilm formation and improving osseointegration. Properties of silanes as molecular binders have been thoroughly studied, but research on the biological effects of these coatings is scarce. The aim of the present study was to determine the in vitro cell response and antibacterial effects of triethoxysilypropyl succinic anhydride (TESPSA) silane anchored on titanium surfaces. X-ray photoelectron spectroscopy confirmed a successful silanization. The silanized surfaces showed no cytotoxic effects. Gene expression analyses of Sarcoma Osteogenic (SaOS-2) osteoblast-like cells cultured on TESPSA silanized surfaces reported a remarkable increase of biochemical markers related to induction of osteoblastic cell differentiation. A manifest decrease of bacterial adhesion and biofilm formation at early stages was observed on treated substrates, while favoring cell adhesion and spreading in bacteria–cell co-cultures. Surfaces treated with TESPSA could enhance a biological sealing on implant surfaces against bacteria colonization of underlying tissues. Furthermore, it can be an effective anchoring platform of biomolecules on titanium surfaces with improved osteoblastic differentiation and antibacterial properties. - Highlights: • TESPSA silane induces osteoblast differentiation. • TESPSA reduces bacterial adhesion and biofilm formation. • TESPSA is a promising anchoring platform of biomolecules onto titanium.

  17. Periostin inhibits mechanical stretch-induced apoptosis in osteoblast-like MG-63 cells.

    Science.gov (United States)

    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.

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

    International Nuclear Information System (INIS)

    Sharma, Sonali; Mahalingam, Chandrika D.; Das, Varsha; Jamal, Shazia; Levi, Edi; Rishi, Arun K.; Datta, Nabanita S.

    2013-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-07-12

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-11-01

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

  1. Time-lapse Raman imaging of osteoblast differentiation

    Science.gov (United States)

    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.

  2. Secreted Clusterin protein inhibits osteoblast differentiation of bone marrow mesenchymal stem cells by suppressing ERK1/2 signaling pathway.

    Science.gov (United States)

    Abdallah, Basem M; Alzahrani, Abdullah M; Kassem, Moustapha

    2018-05-01

    Secreted Clusterin (sCLU, also known as Apolipoprotein J) is an anti-apoptotic glycoprotein involved in the regulation of cell proliferation, lipid transport, extracellular tissue remodeling and apoptosis. sCLU is expressed and secreted by mouse bone marrow-derived skeletal (stromal or mesenchymal) stem cells (mBMSCs), but its functional role in MSC biology is not known. In this study, we demonstrated that Clusterin mRNA expression and protein secretion in conditioned medium increased during adipocyte differentiation and decreased during osteoblast differentiation of mBMSCs. Treatment of mBMSC cultures with recombinant sCLU protein increased cell proliferation and exerted an inhibitory effect on the osteoblast differentiation while stimulated adipocyte differentiation in a dose-dependent manner. siRNA-mediated silencing of Clu expression in mBMSCs reduced adipocyte differentiation and stimulated osteoblast differentiation of mBMSCs. Furthermore, the inhibitory effect of sCLU on the osteoblast differentiation of mBMSCs was mediated by the suppression of extracellular signal-regulated kinase (ERK1/2) phosphorylation. In conclusion, we identified sCLU as a regulator of mBMSCs lineage commitment to osteoblasts versus adipocytes through a mechanism mediated by ERK1/2 signaling. Inhibiting sCLU is a possible therapeutic approach for enhancing osteoblast differentiation and consequently bone formation. Copyright © 2018 Elsevier Inc. All rights reserved.

  3. A meta-ethnography of organisational culture in primary care medical practice.

    Science.gov (United States)

    Grant, Suzanne; Guthrie, Bruce; Entwistle, Vikki; Williams, Brian

    2014-01-01

    Over the past decade, there has been growing international interest in shaping local organisational cultures in primary healthcare. However, the contextual relevance of extant culture assessment instruments to the primary care context has been questioned. The aim of this paper is to derive a new contextually appropriate understanding of the key dimensions of primary care medical practice organisational culture and their inter-relationship through a synthesis of published qualitative research. A systematic search of six electronic databases followed by a synthesis using techniques of meta-ethnography involving translation and re-interpretation. A total of 16 papers were included in the meta-ethnography from the UK, the USA, Canada, Australia and New Zealand that fell into two related groups: those focused on practice organisational characteristics and narratives of practice individuality; and those focused on sub-practice variation across professional, managerial and administrative lines. It was found that primary care organisational culture was characterised by four key dimensions, i.e. responsiveness, team hierarchy, care philosophy and communication. These dimensions are multi-level and inter-professional in nature, spanning both practice and sub-practice levels. The research contributes to organisational culture theory development. The four new cultural dimensions provide a synthesized conceptual framework for researchers to evaluate and understand primary care cultural and sub-cultural levels. The synthesised cultural dimensions present a framework for practitioners to understand and change organisational culture in primary care teams. The research uses an innovative research methodology to synthesise the existing qualitative research and is one of the first to develop systematically a qualitative conceptual framing of primary care organisational culture.

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

    International Nuclear Information System (INIS)

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

    2016-01-01

    Initial cellular responses following implantation are important for inducing osteoconduction. We investigated cell adhesion, spreading, proliferation and differentiation of mouse MC3T3-E1 osteoblastic cells on untreated or fibronectin (Fn)-coated discs of hydroxyapatite (HAp) or alpha-type alumina (α-Al 2 O 3 ). Fn coating significantly enhanced adhesion and spreading of MC3T3-E1 cells on HAp, but did not affect MC3T3-E1 cell proliferation and differentiation on HAp or α-Al 2 O 3 . Fn-coated HAp likely does not stimulate pre-osteoblast cells to initiate the process of osteoconduction; however, Fn adsorption might affect the response of inflammatory cells to the implanted material or, in conjunction with other serum proteins, stimulate pre-osteoblast cell proliferation and differentiation. Further studies on the effect of serum proteins in cell culture and the efficacy of Fn-coated HAp and α-Al 2 O 3 in vivo are warranted. - Highlights: • We studied osteoblast-like MC3T3-E1 cell responses on fibronectin (Fn)-coated discs (HAp/α-Al 2 O 3 ). • Fn adsorption enhanced adhesion and spreading of MC3T3-E1 cells on HAp but not on α-Al 2 O 3 . • Fn adsorption hardly affected proliferation and differentiation of MC3T3-E1 cells on HAp and α-Al 2 O 3 . • Fn adsorption might stimulate osteoconduction on HAp along with other serum proteins.

  5. Retinoic acid receptor signalling directly regulates osteoblast and adipocyte differentiation from mesenchymal progenitor cells

    Energy Technology Data Exchange (ETDEWEB)

    Green, A.C. [St Vincent' s Institute, Fitzroy, Victoria 3065 (Australia); Department of Medicine at St. Vincent' s Hospital, The University of Melbourne, Victoria 3065 (Australia); Kocovski, P.; Jovic, T.; Walia, M.K. [St Vincent' s Institute, Fitzroy, Victoria 3065 (Australia); Chandraratna, R.A.S. [IO Therapeutics, Inc., Santa Ana, CA 92705 (United States); Martin, T.J.; Baker, E.K. [St Vincent' s Institute, Fitzroy, Victoria 3065 (Australia); Department of Medicine at St. Vincent' s Hospital, The University of Melbourne, Victoria 3065 (Australia); Purton, L.E., E-mail: lpurton@svi.edu.au [St Vincent' s Institute, Fitzroy, Victoria 3065 (Australia); Department of Medicine at St. Vincent' s Hospital, The University of Melbourne, Victoria 3065 (Australia)

    2017-01-01

    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 osteogenic 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.

  6. Mechanisms underlying the attachment and spreading of human osteoblasts: from transient interactions to focal adhesions on vitronectin-grafted bioactive surfaces.

    Science.gov (United States)

    Brun, Paola; Scorzeto, Michele; Vassanelli, Stefano; Castagliuolo, Ignazio; Palù, Giorgio; Ghezzo, Francesca; Messina, Grazia M L; Iucci, Giovanna; Battaglia, Valentina; Sivolella, Stefano; Bagno, Andrea; Polzonetti, Giovanni; Marletta, Giovanni; Dettin, Monica

    2013-04-01

    The features of implant devices and the reactions of bone-derived cells to foreign surfaces determine implant success during osseointegration. In an attempt to better understand the mechanisms underlying osteoblasts attachment and spreading, in this study adhesive peptides containing the fibronectin sequence motif for integrin binding (Arg-Gly-Asp, RGD) or mapping the human vitronectin protein (HVP) were grafted on glass and titanium surfaces with or without chemically induced controlled immobilization. As shown by total internal reflection fluorescence microscopy, human osteoblasts develop adhesion patches only on specifically immobilized peptides. Indeed, cells quickly develop focal adhesions on RGD-grafted surfaces, while HVP peptide promotes filopodia, structures involved in cellular spreading. As indicated by immunocytochemistry and quantitative polymerase chain reaction, focal adhesions kinase activation is delayed on HVP peptides with respect to RGD while an osteogenic phenotypic response appears within 24h on osteoblasts cultured on both peptides. Cellular pathways underlying osteoblasts attachment are, however, different. As demonstrated by adhesion blocking assays, integrins are mainly involved in osteoblast adhesion to RGD peptide, while HVP selects osteoblasts for attachment through proteoglycan-mediated interactions. Thus an interfacial layer of an endosseous device grafted with specifically immobilized HVP peptide not only selects the attachment and supports differentiation of osteoblasts but also promotes cellular migration. Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  7. Carnosol Inhibits Pro-Inflammatory and Catabolic Mediators of Cartilage Breakdown in Human Osteoarthritic Chondrocytes and Mediates Cross-Talk between Subchondral Bone Osteoblasts and Chondrocytes.

    Directory of Open Access Journals (Sweden)

    Christelle Sanchez

    Full Text Available The aim of this work was to evaluate the effects of carnosol, a rosemary polyphenol, on pro-inflammatory and catabolic mediators of cartilage breakdown in chondrocytes and via bone-cartilage crosstalk.Osteoarthritic (OA human chondrocytes were cultured in alginate beads for 4 days in presence or absence of carnosol (6 nM to 9 μM. The production of aggrecan, matrix metalloproteinase (MMP-3, tissue inhibitor of metalloproteinase (TIMP-1, interleukin (IL-6 and nitric oxide (NO and the expression of type II collagen and ADAMTS-4 and -5 were analyzed. Human osteoblasts from sclerotic (SC or non-sclerotic (NSC subchondral bone were cultured for 3 days in presence or absence of carnosol before co-culture with chondrocytes. Chondrocyte gene expression was analyzed after 4 days of co-culture.In chondrocytes, type II collagen expression was significantly enhanced in the presence of 3 μM carnosol (p = 0.008. MMP-3, IL-6, NO production and ADAMTS-4 expression were down-regulated in a concentration-dependent manner by carnosol (p<0.01. TIMP-1 production was slightly increased at 3 μM (p = 0.02 and ADAMTS-5 expression was decreased from 0.2 to 9 μM carnosol (p<0.05. IL-6 and PGE2 production was reduced in the presence of carnosol in both SC and NSC osteoblasts while alkaline phosphatase activity was not changed. In co-culture experiments preincubation of NSC and SC osteoblasts wih carnosol resulted in similar effects to incubation with anti-IL-6 antibody, namely a significant increase in aggrecan and decrease in MMP-3, ADAMTS-4 and -5 gene expression by chondrocytes.Carnosol showed potent inhibition of pro-inflammatory and catabolic mediators of cartilage breakdown in chondrocytes. Inhibition of matrix degradation and enhancement of formation was observed in chondrocytes cocultured with subchondral osteoblasts preincubated with carnosol indicating a cross-talk between these two cellular compartments, potentially mediated via inhibition of IL-6 in

  8. A 3D printed nano bone matrix for characterization of breast cancer cell and osteoblast interactions

    Science.gov (United States)

    Zhu, Wei; Castro, Nathan J.; Cui, Haitao; Zhou, Xuan; Boualam, Benchaa; McGrane, Robert; Glazer, Robert I.; Zhang, Lijie Grace

    2016-08-01

    Bone metastasis is one of the most prevalent complications of late-stage breast cancer, in which the native bone matrix components, including osteoblasts, are intimately involved in tumor progression. The development of a successful in vitro model would greatly facilitate understanding the underlying mechanism of breast cancer bone invasion as well as provide a tool for effective discovery of novel therapeutic strategies. In the current study, we fabricated a series of in vitro bone matrices composed of a polyethylene glycol hydrogel and nanocrystalline hydroxyapatite of varying concentrations to mimic the native bone microenvironment for the investigation of breast cancer bone metastasis. A stereolithography-based three-dimensional (3D) printer was used to fabricate the bone matrices with precisely controlled architecture. The interaction between breast cancer cells and osteoblasts was investigated in the optimized bone matrix. Using a Transwell® system to separate the two cell lines, breast cancer cells inhibited osteoblast proliferation, while osteoblasts stimulated breast cancer cell growth, whereas, both cell lines increased IL-8 secretion. Breast cancer cells co-cultured with osteoblasts within the 3D bone matrix formed multi-cellular spheroids in comparison to two-dimensional monolayers. These findings validate the use of our 3D printed bone matrices as an in vitro metastasis model, and highlights their potential for investigating breast cancer bone metastasis.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Teruhito Yamashita

    Full Text Available 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

  12. Hedgehog signaling in tumor cells facilitates osteoblast-enhanced osteolytic metastases.

    Directory of Open Access Journals (Sweden)

    Shamik Das

    Full Text Available The remodeling process in bone yields numerous cytokines and chemokines that mediate crosstalk between osteoblasts and osteoclasts and also serve to attract and support metastatic tumor cells. The metastatic tumor cells disturb the equilibrium in bone that manifests as skeletal complications. The Hedgehog (Hh pathway plays an important role in skeletogenesis. We hypothesized that the Hh pathway mediates an interaction between tumor cells and osteoblasts and influences osteoblast differentiation in response to tumor cells. We have determined that breast tumor cells have an activated Hh pathway characterized by upregulation of the ligand, IHH and transcription factor GLI1. Breast cancer cells interact with osteoblasts and cause an enhanced differentiation of pre-osteoblasts to osteoblasts that express increased levels of the osteoclastogenesis factors, RANKL and PTHrP. There is sustained expression of osteoclast-promoting factors, RANKL and PTHrP, even after the osteoblast differentiation ceases and apoptosis sets in. Moreover, tumor cells that are deficient in Hh signaling are compromised in their ability to induce osteoblast differentiation and consequently are inefficient in causing osteolysis. The stimulation of osteoblast differentiation sets the stage for osteoclast differentiation and overall promotes osteolysis. Thus, in the process of developing newer therapeutic strategies against breast cancer metastasis to bone it would worthwhile to keep in mind the role of the Hh pathway in osteoblast differentiation in an otherwise predominant osteolytic phenomenon.

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

    Energy Technology Data Exchange (ETDEWEB)

    Yin, Xinhua [Department of Spine Surgery, Xiangya Hospital of Central South University, Changsha (China); Wang, Xiaoyuan [Department of Nephrology, Xi An Honghui Hospital, Xi an (China); Hu, Xiongke; Chen, Yong; Zeng, Kefeng [Department of Spine Surgery, Xiangya Hospital of Central South University, Changsha (China); Zhang, Hongqi, E-mail: zhq9699@126.com [Department of Spine Surgery, Xiangya Hospital of Central South University, Changsha (China)

    2015-07-01

    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) expression 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.

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

    International Nuclear Information System (INIS)

    Yin, Xinhua; Wang, Xiaoyuan; Hu, Xiongke; Chen, Yong; Zeng, Kefeng; Zhang, Hongqi

    2015-01-01

    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) expression 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

  15. The role of osteoblast cells in the pathogenesis of unicameral bone cysts.

    Science.gov (United States)

    Aarvold, Alexander; Smith, James O; Tayton, Edward R; Edwards, Caroline J; Fowler, Darren J; Gent, Edward D; Oreffo, Richard O C

    2012-08-01

    The pathogenesis of unicameral bone cysts (UBCs) remains largely unknown. Osteoclasts have been implicated, but the role of osteoblastic cells has, to date, not been explored. This study investigated the pathophysiology of UBCs by examining the interactions between the cyst fluid and human bone marrow stromal cells (hBMSCs) and the effect of the fluid on osteogenesis. Fluid was aspirated from two UBCs and analysed for protein, electrolyte and cytokine levels. Graded concentrations of the fluid were used as culture media for hBMSCs to determine the effects of the fluid on hBMSC proliferation and osteogenic differentiation. The fibrocellular lining was analysed histologically and by electron microscopy. Alkaline phosphatase (ALP) staining of hBMSCs that were cultured in cyst fluid demonstrated increased cell proliferation and osteogenic differentiation compared to basal media controls. Biochemical analysis of these hBMSCs compared to basal controls confirmed a marked increase in DNA content (as a marker of proliferation) and ALP activity (as a marker of osteogenic differentiation) which was highly significant (p < 0.001). Osteoclasts were demonstrated in abundance in the cyst lining. The cyst fluid cytokine profile revealed levels of the pro-osteoclast cytokines IL-6, MIP-1α and MCP-1 that were 19×, 31× and 35× greater than those in reference serum. Cyst fluid promoted osteoblastic growth and differentiation. Despite appearing paradoxical that the cyst fluid promoted osteogenesis, osteoblastic cells are required for osteoclastogenesis through RANKL signalling. Three key cytokines in this pathway (IL-6, MIP-1α, MCP-1) were highly elevated in cyst fluid. These findings may hold the key to the pathogenesis of UBCs, with implications for treatment methods.

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

    Directory of Open Access Journals (Sweden)

    Karl F. Braun

    2011-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Kawashita, Masakazu, E-mail: m-kawa@ecei.tohoku.ac.jp [Graduate School of Biomedical Engineering, Tohoku University, Sendai 980-8579 (Japan); Hasegawa, Maki [Graduate School of Biomedical Engineering, Tohoku University, Sendai 980-8579 (Japan); Kudo, Tada-aki; Kanetaka, Hiroyasu [Graduate School of Dentistry, Tohoku University, Sendai 980-8575 (Japan); Miyazaki, Toshiki [Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, Kitakyushu 808-0196 (Japan); Hashimoto, Masami [Japan Fine Ceramics Center, Nagoya 456-8587 (Japan)

    2016-12-01

    Initial cellular responses following implantation are important for inducing osteoconduction. We investigated cell adhesion, spreading, proliferation and differentiation of mouse MC3T3-E1 osteoblastic cells on untreated or fibronectin (Fn)-coated discs of hydroxyapatite (HAp) or alpha-type alumina (α-Al{sub 2}O{sub 3}). Fn coating significantly enhanced adhesion and spreading of MC3T3-E1 cells on HAp, but did not affect MC3T3-E1 cell proliferation and differentiation on HAp or α-Al{sub 2}O{sub 3}. Fn-coated HAp likely does not stimulate pre-osteoblast cells to initiate the process of osteoconduction; however, Fn adsorption might affect the response of inflammatory cells to the implanted material or, in conjunction with other serum proteins, stimulate pre-osteoblast cell proliferation and differentiation. Further studies on the effect of serum proteins in cell culture and the efficacy of Fn-coated HAp and α-Al{sub 2}O{sub 3}in vivo are warranted. - Highlights: • We studied osteoblast-like MC3T3-E1 cell responses on fibronectin (Fn)-coated discs (HAp/α-Al{sub 2}O{sub 3}). • Fn adsorption enhanced adhesion and spreading of MC3T3-E1 cells on HAp but not on α-Al{sub 2}O{sub 3}. • Fn adsorption hardly affected proliferation and differentiation of MC3T3-E1 cells on HAp and α-Al{sub 2}O{sub 3}. • Fn adsorption might stimulate osteoconduction on HAp along with other serum proteins.

  18. The role of surface microtopography in the modulation of osteoblast differentiation

    Directory of Open Access Journals (Sweden)

    JS Hayes

    2010-07-01

    Full Text Available The osteoinductive and conductive capabilities of commercially pure titanium and its alloys is well documented, as is their ability to provide long-term stability for permanent implantable devices. Fracture fixation in paediatric and trauma patients generally requires transient fixation after which the implant becomes redundant and requires removal. Removal can be complicated due to excessive bony over-growth which is encouraged by the standard micro-rough implant surface. We have shown in vivo that removal related morbidity can be significantly reduced with surface polishing, a technique which reduces the micro-roughness of clinically available materials. However, tissue integration at the bone-implant interface requires activation of key regulatory pathways which influences osteoblastic differentiation and maturation therefore we do not believe this effect to be purely mechanical. To elucidate potential mechanisms by which surface polishing exerts its effect on bone regeneration this study assessed in vitro the effect of surface polishing commercially pure titanium on cell growth, morphology and on the regulation of core binding factor 1, osterix, collagen I, alkaline phosphatase, bone sialoprotein and osteocalcin for primary rat calvarial osteoblasts. Results indicate that polishing differentially influences osteoblast differentiation in a surface dependent manner and that these changes are potentially linked to surface dependent morphology, but not to differences in cell proliferation.

  19. Deduction of Novel Genes Potentially Involved in Osteoblasts of Rheumatoid Arthritis Using Next-Generation Sequencing and Bioinformatic Approaches

    Directory of Open Access Journals (Sweden)

    Yi-Jen Chen

    2017-11-01

    Full Text Available The role of osteoblasts in peri-articular bone loss and bone erosion in rheumatoid arthritis (RA has gained much attention, and microRNAs are hypothesized to play critical roles in the regulation of osteoblast function in RA. The aim of this study is to explore novel microRNAs differentially expressed in RA osteoblasts and to identify genes potentially involved in the dysregulated bone homeostasis in RA. RNAs were extracted from cultured normal and RA osteoblasts for sequencing. Using the next generation sequencing and bioinformatics approaches, we identified 35 differentially expressed microRNAs and 13 differentially expressed genes with potential microRNA–mRNA interactions in RA osteoblasts. The 13 candidate genes were involved mainly in cell–matrix adhesion, as classified by the Gene Ontology. Two genes of interest identified from RA osteoblasts, A-kinase anchoring protein 12 (AKAP12 and leucin rich repeat containing 15 (LRRC15, were found to express more consistently in the related RA synovial tissue arrays in the Gene Expression Omnibus database, with the predicted interactions with miR-183-5p and miR-146a-5p, respectively. The Ingenuity Pathway Analysis identified AKAP12 as one of the genes involved in protein kinase A signaling and the function of chemotaxis, interconnecting with molecules related to neovascularization. The findings indicate new candidate genes as the potential indicators in evaluating therapies targeting chemotaxis and neovascularization to control joint destruction in RA.

  20. The dependence of MG63 osteoblast responses to (meth)acrylate-based networks on chemical structure and stiffness.

    Science.gov (United States)

    Smith, Kathryn E; Hyzy, Sharon L; Sunwoo, Moonhae; Gall, Ken A; Schwartz, Zvi; Boyan, Barbara D

    2010-08-01

    The cell response to an implant is regulated by the implant's surface properties including topography and chemistry, but less is known about how the mechanical properties affect cell behavior. The objective of this study was to evaluate how the surface stiffness and chemistry of acrylate-based copolymer networks affect the in vitro response of human MG63 pre-osteoblast cells. Networks comprised of poly(ethylene glycol) dimethacrylate (PEGDMA; Mn approximately 750) and diethylene glycol dimethacrylate (DEGDMA) were photopolymerized at different concentrations to produce three compositions with moduli ranging from 850 to 60 MPa. To further decouple chemistry and stiffness, three networks comprised of 2-hydroxyethyl methacrylate (2HEMA) and PEGDMA or DEGDMA were also designed that exhibited a range of moduli similar to the PEGDMA-DEGDMA networks. MG63 cells were cultured on each surface and tissue culture polystyrene (TCPS), and the effect of copolymer composition on cell number, osteogenic markers (alkaline phosphatase specific activity and osteocalcin), and local growth factor production (OPG, TGF-beta1, and VEGF-A) were assessed. Cells exhibited a more differentiated phenotype on the PEGDMA-DEGDMA copolymers compared to the 2HEMA-PEGDMA copolymers. On the PEGDMA-DEGDMA system, cells exhibited a more differentiated phenotype on the stiffest surface indicated by elevated osteocalcin compared with TCPS. Conversely, cells on 2HEMA-PEGDMA copolymers became more differentiated on the less stiff 2HEMA surface. Growth factors were regulated in a differential manner. These results indicate that copolymer chemistry is the primary regulator of osteoblast differentiation, and the effect of stiffness is secondary to the surface chemistry. 2010 Elsevier Ltd. All rights reserved.

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

    International Nuclear Information System (INIS)

    Mizoshiri, N.; Kishida, T.; Yamamoto, K.; Shirai, T.; Terauchi, R.; Tsuchida, S.; Mori, Y.; Ejima, A.; Sato, Y.; Arai, Y.; Fujiwara, H.; Yamamoto, T.; Kanamura, N.; Mazda, O.; Kubo, T.

    2015-01-01

    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 genes 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Mizoshiri, N. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto (Japan); Kishida, T. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Yamamoto, K. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Department of Dental Medicine, Kyoto Prefectural University of Medicine, Kyoto (Japan); Shirai, T.; Terauchi, R.; Tsuchida, S. [Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto (Japan); Mori, Y. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto (Japan); Ejima, A. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Sato, Y. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Department of Dental Medicine, Kyoto Prefectural University of Medicine, Kyoto (Japan); Arai, Y.; Fujiwara, H. [Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto (Japan); Yamamoto, T.; Kanamura, N. [Department of Dental Medicine, Kyoto Prefectural University of Medicine, Kyoto (Japan); Mazda, O., E-mail: mazda@koto.kpu-m.ac.jp [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Kubo, T. [Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto (Japan)

    2015-11-27

    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 genes 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.

  3. Functional response of osteoblasts in functionally gradient titanium alloy mesh arrays processed by 3D additive manufacturing.

    Science.gov (United States)

    Nune, K C; Kumar, A; Misra, R D K; Li, S J; Hao, Y L; Yang, R

    2017-02-01

    We elucidate here the osteoblasts functions and cellular activity in 3D printed interconnected porous architecture of functionally gradient Ti-6Al-4V alloy mesh structures in terms of cell proliferation and growth, distribution of cell nuclei, synthesis of proteins (actin, vinculin, and fibronectin), and calcium deposition. Cell culture studies with pre-osteoblasts indicated that the interconnected porous architecture of functionally gradient mesh arrays was conducive to osteoblast functions. However, there were statistically significant differences in the cellular response depending on the pore size in the functionally gradient structure. The interconnected porous architecture contributed to the distribution of cells from the large pore size (G1) to the small pore size (G3), with consequent synthesis of extracellular matrix and calcium precipitation. The gradient mesh structure significantly impacted cell adhesion and influenced the proliferation stage, such that there was high distribution of cells on struts of the gradient mesh structure. Actin and vinculin showed a significant difference in normalized expression level of protein per cell, which was absent in the case of fibronectin. Osteoblasts present on mesh struts formed a confluent sheet, bridging the pores through numerous cytoplasmic extensions. The gradient mesh structure fabricated by electron beam melting was explored to obtain fundamental insights on cellular activity with respect to osteoblast functions. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Ectopic bone formation in nude rats using human osteoblasts seeded poly(3)hydroxybutyrate embroidery and hydroxyapatite-collagen tapes constructs.

    Science.gov (United States)

    Mai, Ronald; Hagedorn, Manolo Gunnar; Gelinsky, Michael; Werner, Carsten; Turhani, Dritan; Späth, Heike; Gedrange, Tomas; Lauer, Günter

    2006-09-01

    The aim of this study was to evaluate the ectopic bone formation using tissue engineered cell-seeded constructs with two different scaffolds and primary human maxillary osteoblasts in nude rats over an implantation period of up to 96 days. Collagen I-coated Poly(3)hydroxybutyrate (PHB) embroidery and hydroxyapatite (HAP) collagen tapes were seeded with primary human maxillary osteoblasts (hOB) and implanted into athymic rnu/run rats. A total of 72 implants were placed into the back muscles of 18 rats. 24, 48 and 96 days after implantation, histological and histomorphometric analyses were made. The osteoblastic character of the cells was confirmed by immunocytochemistry and RT-PCR for osteocalcin. Histological analysis demonstrated that all cell-seeded constructs induced ectopic bone formation after 24, 48 and 96 days of implantation. There was more mineralized tissue in PHB constructs than in HAP-collagen tapes (at day 24; p embroidery or HAP-collagen tapes can induce ectopic bone formation. However, the amount of bone formed decreased with increasing length of implantation.

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

    Science.gov (United States)

    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.

  6. The effect of inorganic additives to calcium phosphate on in vitro behavior of osteoblasts and osteoclasts

    NARCIS (Netherlands)

    Yang, Liang; Perez-Amodio, Soledad; Barrère, F.; Everts, Vincent; van Blitterswijk, Clemens; Habibovic, Pamela

    2010-01-01

    This study describes a medium-throughput system based on deposition of calcium phosphate films in multi-well tissue culture plates that can be used to study the effect of inorganic additives on the behavior of osteoblasts and osteoclasts in a standardized manner. All tested elements, copper, zinc,

  7. The effects of inorganic additives to calcium phosphate on in vitro behavior of osteoblasts and osteoclasts

    NARCIS (Netherlands)

    Yang, L.; Perez-Amodio, S.; Barrere-de Groot, F.Y.F.; Everts, V.; van Blitterswijk, C.A.; Habibovic, P.

    2010-01-01

    This study describes a medium-throughput system based on deposition of calcium phosphate films in multi-well tissue culture plates that can be used to study the effect of inorganic additives on the behavior of osteoblasts and osteoclasts in a standardized manner. All tested elements, copper, zinc,

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

    LENUS (Irish Health Repository)

    O'Neill, S C

    2013-08-01

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

  9. Substrate Stiffness Controls Osteoblastic and Chondrocytic Differentiation of Mesenchymal Stem Cells without Exogenous Stimuli.

    Directory of Open Access Journals (Sweden)

    Rene Olivares-Navarrete

    Full Text Available Stem cell fate has been linked to the mechanical properties of their underlying substrate, affecting mechanoreceptors and ultimately leading to downstream biological response. Studies have used polymers to mimic the stiffness of extracellular matrix as well as of individual tissues and shown mesenchymal stem cells (MSCs could be directed along specific lineages. In this study, we examined the role of stiffness in MSC differentiation to two closely related cell phenotypes: osteoblast and chondrocyte. We prepared four methyl acrylate/methyl methacrylate (MA/MMA polymer surfaces with elastic moduli ranging from 0.1 MPa to 310 MPa by altering monomer concentration. MSCs were cultured in media without exogenous growth factors and their biological responses were compared to committed chondrocytes and osteoblasts. Both chondrogenic and osteogenic markers were elevated when MSCs were grown on substrates with stiffness <10 MPa. Like chondrocytes, MSCs on lower stiffness substrates showed elevated expression of ACAN, SOX9, and COL2 and proteoglycan content; COMP was elevated in MSCs but reduced in chondrocytes. Substrate stiffness altered levels of RUNX2 mRNA, alkaline phosphatase specific activity, osteocalcin, and osteoprotegerin in osteoblasts, decreasing levels on the least stiff substrate. Expression of integrin subunits α1, α2, α5, αv, β1, and β3 changed in a stiffness- and cell type-dependent manner. Silencing of integrin subunit beta 1 (ITGB1 in MSCs abolished both osteoblastic and chondrogenic differentiation in response to substrate stiffness. Our results suggest that substrate stiffness is an important mediator of osteoblastic and chondrogenic differentiation, and integrin β1 plays a pivotal role in this process.

  10. Proton concentrations can be a major contributor to the modification of osteoclast and osteoblast differentiation, working independently of extracellular bicarbonate ions.

    Science.gov (United States)

    Kato, Kohtaro; Matsushita, Misao

    2014-01-01

    We established a system to separately analyze the role of protons and bicarbonate ions in vitro in which the pH of the medium was controlled by HEPES at various concentrations of sodium bicarbonate (NaHCO3) in the absence of carbon dioxide (CO2). Using this system, we demonstrated that acidosis promoted osteoclast formation independently of extracellular NaHCO3 in a short-term culture. Protons and bicarbonate ions acted on osteoclast differentiation with opposite effects, the former positively and the latter negatively. The HEPES-based system maintained pH in the absence of extracellular NaHCO3 without CO2. Therefore, we could demonstrate that osteoblast differentiation was promoted at higher pH in a long-term culture system without NaHCO3 in which ALP activity and nodule mineralization were enhanced. This finding indicates that protons negatively control osteoblast differentiation independently of extracellular bicarbonate ions. However, the difference in the concentration of NaHCO3 did not have any influence on nodule mineralization. The opposite effects of protons, the promotion of osteoclast formation and the inhibition of osteoblast differentiation, were suppressed in the presence of 5 mM N-acetyl cysteine, a reagent activating the scavenging of reactive oxygen species (ROS), implying that ROS act on both systems, the promotion of large osteoclast formation and the deterioration of osteoblast formation under acidosis.

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

    Science.gov (United States)

    Mizoshiri, N; Kishida, T; Yamamoto, K; Shirai, T; Terauchi, R; Tsuchida, S; Mori, Y; Ejima, A; Sato, Y; Arai, Y; Fujiwara, H; Yamamoto, T; Kanamura, N; Mazda, O; Kubo, T

    2015-11-27

    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. We examined which Oct family genes and Myc family genes are capable of inducing osteoblast-like phenotypic conversion. 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. 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Nukbone® promotes proliferation and osteoblastic differentiation of mesenchymal stem cells from human amniotic membrane

    Energy Technology Data Exchange (ETDEWEB)

    Rodríguez-Fuentes, Nayeli; Rodríguez-Hernández, Ana G. [Depto. Microbiología y Parasitología, Facultad de Medicina, Universidad Nacional Autónoma de México (UNAM), Mexico City 04510 (Mexico); Enríquez-Jiménez, Juana [Depto. Biología de la Reproducción, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán (INCMNSZ), México City 14000 (Mexico); Alcántara-Quintana, Luz E. [Subd. de Investigación, Centro Nacional de la Transfusión Sanguínea, Secretaria de Salud, Mexico City 07370 (Mexico); Fuentes-Mera, Lizeth [Depto. Biología Molecular e Histocompatibilidad, Hospital General “Dr. Manuel Gea González”, México City 4800 (Mexico); Piña-Barba, María C. [Depto. Materiales Metálicos y Cerámicos, Instituto de Investigaciones en Materiales, Universidad Nacional Autónoma de México (UNAM), México City 04510 (Mexico); Zepeda-Rodríguez, Armando [Depto. Biología Celular y Tisular, Facultad de Medicina, Universidad Nacional Autónoma de México (UNAM), México City 04510 (Mexico); and others

    2013-05-10

    Highlights: •Nukbone showed to be a good scaffold for adhesion, proliferation and differentiation of stem cells. •Nukbone induced osteoblastic differentiation of human mesenchymal stem cells. •Results showed that Nukbone offer an excellent option for bone tissue regeneration due to properties. -- Abstract: Bovine bone matrix Nukbone® (NKB) is an osseous tissue-engineering biomaterial that retains its mineral and organic phases and its natural bone topography and has been used as a xenoimplant for bone regeneration in clinics. There are not studies regarding its influence of the NKB in the behavior of cells during the repairing processes. The aim of this research is to demonstrate that NKB has an osteoinductive effect in human mesenchymal stem cells from amniotic membrane (AM-hMSCs). Results indicated that NKB favors the AM-hMSCs adhesion and proliferation up to 7 days in culture as shown by the scanning electron microscopy and proliferation measures using an alamarBlue assay. Furthermore, as demonstrated by reverse transcriptase polymerase chain reaction, it was detected that two gene expression markers of osteoblastic differentiation: the core binding factor and osteocalcin were higher for AM-hMSCs co-cultured with NKB in comparison with cultivated cells in absence of the biomaterial. As the results indicate, NKB possess the capability for inducing successfully the osteoblastic differentiation of AM-hMSC, so that, NKB is an excellent xenoimplant option for repairing bone tissue defects.

  13. Nukbone® promotes proliferation and osteoblastic differentiation of mesenchymal stem cells from human amniotic membrane

    International Nuclear Information System (INIS)

    Rodríguez-Fuentes, Nayeli; Rodríguez-Hernández, Ana G.; Enríquez-Jiménez, Juana; Alcántara-Quintana, Luz E.; Fuentes-Mera, Lizeth; Piña-Barba, María C.; Zepeda-Rodríguez, Armando

    2013-01-01

    Highlights: •Nukbone showed to be a good scaffold for adhesion, proliferation and differentiation of stem cells. •Nukbone induced osteoblastic differentiation of human mesenchymal stem cells. •Results showed that Nukbone offer an excellent option for bone tissue regeneration due to properties. -- Abstract: Bovine bone matrix Nukbone® (NKB) is an osseous tissue-engineering biomaterial that retains its mineral and organic phases and its natural bone topography and has been used as a xenoimplant for bone regeneration in clinics. There are not studies regarding its influence of the NKB in the behavior of cells during the repairing processes. The aim of this research is to demonstrate that NKB has an osteoinductive effect in human mesenchymal stem cells from amniotic membrane (AM-hMSCs). Results indicated that NKB favors the AM-hMSCs adhesion and proliferation up to 7 days in culture as shown by the scanning electron microscopy and proliferation measures using an alamarBlue assay. Furthermore, as demonstrated by reverse transcriptase polymerase chain reaction, it was detected that two gene expression markers of osteoblastic differentiation: the core binding factor and osteocalcin were higher for AM-hMSCs co-cultured with NKB in comparison with cultivated cells in absence of the biomaterial. As the results indicate, NKB possess the capability for inducing successfully the osteoblastic differentiation of AM-hMSC, so that, NKB is an excellent xenoimplant option for repairing bone tissue defects

  14. Sr-substituted bone cements direct mesenchymal stem cells, osteoblasts and osteoclasts fate.

    Directory of Open Access Journals (Sweden)

    Monica Montesi

    Full Text Available Strontium-substituted apatitic bone cements enriched with sodium alginate were developed as a potential modulator of bone cells fate. The biological impact of the bone cement were investigated in vitro through the study of the effect of the nanostructured apatitic composition and the doping of strontium on mesenchymal stem cells, pre-osteoblasts and osteoclasts behaviours. Up to 14 days of culture the bone cells viability, proliferation, morphology and gene expression profiles were evaluated. The results showed that different concentrations of strontium were able to evoke a cell-specific response, in fact an inductive effect on mesenchymal stem cells differentiation and pre-osteoblasts proliferation and an inhibitory effect on osteoclasts activity were observed. Moreover, the apatitic structure of the cements provided a biomimetic environment suitable for bone cells growth. Therefore, the combination of biological features of this bone cement makes it as promising biomaterials for tissue regeneration.

  15. Irisin Enhances Osteoblast Differentiation In Vitro

    Directory of Open Access Journals (Sweden)

    Graziana Colaianni

    2014-01-01

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

  16. Dual Effect of Chrysanthemum indicum Extract to Stimulate Osteoblast Differentiation and Inhibit Osteoclast Formation and Resorption In Vitro

    Directory of Open Access Journals (Sweden)

    Jong Min Baek

    2014-01-01

    Full Text Available The risk of bone-related diseases increases due to the imbalance between bone resorption and bone formation by osteoclasts and osteoblasts, respectively. The goal in the development of antiosteoporotic treatments is an agent that will improve bone through simultaneous osteoblast stimulation and osteoclast inhibition without undesirable side effects. To achieve this goal, numerous studies have been performed to identify novel approaches using natural oriental herbs to treat bone metabolic diseases. In the present study, we investigated the effect of Chrysanthemum indicum extract (CIE on the differentiation of osteoclastic and osteoblastic cells. CIE inhibited the formation of TRAP-positive mature osteoclasts and of filamentous-actin rings and disrupted the bone-resorbing activity of mature osteoclasts in a dose-dependent manner. CIE strongly inhibited Akt, GSK3β, and IκB phosphorylation in RANKL-stimulated bone marrow macrophages and did not show any effects on MAP kinases, including p38, ERK, and JNK. Interestingly, CIE also enhanced primary osteoblast differentiation via upregulation of the expression of alkaline phosphatase and the level of extracellular calcium concentrations during the early and terminal stages of differentiation, respectively. Our results revealed that CIE could have a potential therapeutic role in bone-related disorders through its dual effects on osteoclast and osteoblast differentiation.

  17. Surface modification of TiO{sub 2} nanotubes with osteogenic growth peptide to enhance osteoblast differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Lai, Min, E-mail: minlai@jsnu.edu.cn [School of Life Science, Jiangsu Normal University, Xuzhou, Jiangsu 221116 (China); Jin, Ziyang [School of Life Science, Jiangsu Normal University, Xuzhou, Jiangsu 221116 (China); Su, Zhiguo [Department of Pharmacy, The Affiliated hospital of Qingdao University, Qingdao, Shandong 266555 (China)

    2017-04-01

    To investigate the influence of surface-biofunctionalized substrates on osteoblast behavior, a layer of aligned TiO{sub 2} nanotubes with a diameter of around 70 nm was fabricated on titanium surface by anodization, and then osteogenic growth peptide (OGP) was conjugated onto TiO{sub 2} nanotubes through the intermediate layer of polydopamine. The morphology, composition and wettability of different surfaces were characterized by field-emission scanning electron microscopy (FE-SEM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and contact angle measurements, respectively. The effects of OGP-modified TiO{sub 2} nanotube substrates on the morphology, proliferation and differentiation of osteoblasts were examined in vitro. Immunofluorescence staining revealed that the OGP-functionalized TiO{sub 2} nanotubes were favorable for cell spreading. However, there was no significant difference in cell proliferation observed among the different groups. Cells grown onto OGP-functionalized TiO{sub 2} nanotubes showed significantly higher (p < 0.05 or p < 0.01) levels of alkaline phosphatase (ALP) and mineralization after 4, 7 and 14 days of culture, respectively. Cells grown on OGP-functionalized TiO{sub 2} nanotubes had significantly higher (p < 0.05 or p < 0.01) expression of osteogenic-related genes including runt related transcription factor 2 (Runx2), ALP, collagen type I (Col I), osteopontin (OPN) and osteocalcin (OC) after 14 days of culture. These data suggest that surface functionalization of TiO{sub 2} nanotubes with OGP was beneficial for cell spreading and differentiation. This study provides a novel platform for the development and fabrication of titanium-based implants that enhance the propensity for osseointegration between the native tissue and implant interface. - Highlights: • The OGP functionalized TiO{sub 2} nanotube substrates were successfully fabricated through a direct and effective method. • The OGP functionalized substrates

  18. Nano rough micron patterned titanium for directing osteoblast morphology and adhesion

    Directory of Open Access Journals (Sweden)

    Sabrina Puckett

    2008-06-01

    Full Text Available Sabrina Puckett, Rajesh Pareta, Thomas J WebsterDivision of Engineering, Brown University, Providence, RI, USAAbstract: Previous studies have demonstrated greater functions of osteoblasts (bone-forming cells on nanophase compared with conventional metals. Nanophase metals possess a biologically inspired nanostructured surface that mimics the dimensions of constituent components in bone, including collagen and hydroxyapatite. Not only do these components possess dimensions on the nanoscale, they are aligned in a parallel manner creating a defined orientation in bone. To date, research has yet to evaluate the effect that organized nanosurface features can have on the interaction of osteoblasts with material surfaces. Therefore, to determine if surface orientation of features can mediate osteoblast adhesion and morphology, this study investigated osteoblast function on patterned titanium substrates containing alternating regions of micron rough and nano rough surfaces prepared by novel electron beam evaporation techniques. This study was also interested in determining whether or not the size of the patterned regions had an effect on osteoblast behavior and alignment. Results indicated early controlled osteoblast alignment on these patterned materials as well as greater osteoblast adhesion on the nano rough regions of these patterned substrates. Interestingly, decreasing the width of the nano rough regions (from 80 µm to 22 µm on these patterned substrates resulted in a decreased number of osteoblasts adhering to these areas. Changes in the width of the nano rough regions also resulted in changes in osteoblast morphology, thus, suggesting there is an optimal pattern dimension that osteoblasts prefer. In summary, results of this study provided evidence that aligned nanophase metal features on the surface of titanium improved early osteoblast functions (morphology and adhesion promising for their long term functions, criteria necessary to improve

  19. Temporal Profiling and Pulsed SILAC Labeling Identify Novel Secreted Proteins during ex vivo Osteoblast Differentiation of Human Stromal Stem Cells

    DEFF Research Database (Denmark)

    Kristensen, Lars P; Chen, Li; Nielsen, Maria Overbeck

    2012-01-01

    , 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...... 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...... 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...

  20. The roles of P2Y2 purinergic receptors in osteoblasts and mechanotransduction.

    Directory of Open Access Journals (Sweden)

    Yanghui Xing

    Full Text Available We previously demonstrated, using osteoblastic MC3T3-E1 cells, that P2Y2 purinergic receptors are involved in osteoblast mechanotransduction. In this study, our objective was to further investigate, using a knockout mouse model, the roles of P2Y2 receptors in bone mechanobiology. We first examined bone structure with micro-CT and measured bone mechanical properties with three point bending experiments in both wild type mice and P2Y2 knockout mice. We found that bones from P2Y2 knockout mice have significantly decreased bone volume, bone thickness, bone stiffness and bone ultimate breaking force at 17 week old age. In order to elucidate the mechanisms by which P2Y2 receptors contribute to bone biology, we examined differentiation and mineralization of bone marrow cells from wild type and P2Y2 knockout mice. We found that P2Y2 receptor deficiency reduces the differentiation and mineralization of bone marrow cells. Next, we compared the response of primary osteoblasts, from both wild type and P2Y2 knockout mice, to ATP and mechanical stimulation (oscillatory fluid flow, and found that osteoblasts from wild type mice have a stronger response, in terms of ERK1/2 phosphorylation, to both ATP and fluid flow, relative to P2Y2 knockout mice. However, we did not detect any difference in ATP release in response to fluid flow between wild type and P2Y2 knock out osteoblasts. Our findings suggest that P2Y2 receptors play important roles in bone marrow cell differentiation and mineralization as well as in bone cell mechanotransduction, leading to an osteopenic phenotype in P2Y2 knockout mice.

  1. Osteoblast recruitment routes in human cancellous bone remodeling

    DEFF Research Database (Denmark)

    Kristensen, Helene Bjørg; Andersen, Thomas Levin; Marcussen, Niels

    2014-01-01

    It is commonly proposed that bone forming osteoblasts recruited during bone remodeling originate from bone marrow perivascular cells, bone remodeling compartment canopy cells, or bone lining cells. However, an assessment of osteoblast recruitment during adult human cancellous bone remodeling...... is lacking. We addressed this question by quantifying cell densities, cell proliferation, osteoblast differentiation markers, and capillaries in human iliac crest biopsy specimens. We found that recruitment occurs on both reversal and bone-forming surfaces, as shown by the cell density and osterix levels...

  2. Osteoblast-specific deletion of Pkd2 leads to low-turnover osteopenia and reduced bone marrow adiposity.

    Directory of Open Access Journals (Sweden)

    Zhousheng Xiao

    Full Text Available Polycystin-1 (Pkd1 interacts with polycystin-2 (Pkd2 to form an interdependent signaling complex. Selective deletion of Pkd1 in the osteoblast lineage reciprocally regulates osteoblastogenesis and adipogenesis. The role of Pkd2 in skeletal development has not been defined. To this end, we conditionally inactivated Pkd2 in mature osteoblasts by crossing Osteocalcin (Oc-Cre;Pkd2+/null mice with floxed Pkd2 (Pkd2flox/flox mice. Oc-Cre;Pkd2flox/null (Pkd2Oc-cKO mice exhibited decreased bone mineral density, trabecular bone volume, cortical thickness, mineral apposition rate and impaired biomechanical properties of bone. Pkd2 deficiency resulted in diminished Runt-related transcription factor 2 (Runx2 expressions in bone and impaired osteoblastic differentiation ex vivo. Expression of osteoblast-related genes, including, Osteocalcin, Osteopontin, Bone sialoprotein (Bsp, Phosphate-regulating gene with homologies to endopeptidases on the X chromosome (Phex, Dentin matrix protein 1 (Dmp1, Sclerostin (Sost, and Fibroblast growth factor 23 (FGF23 were reduced proportionate to the reduction of Pkd2 gene dose in bone of Oc-Cre;Pkd2flox/+ and Oc-Cre;Pkd2flox/null mice. Loss of Pkd2 also resulted in diminished peroxisome proliferator-activated receptor γ (PPARγ expression and reduced bone marrow fat in vivo and reduced adipogenesis in osteoblast culture ex vivo. Transcriptional co-activator with PDZ-binding motif (TAZ and Yes-associated protein (YAP, reciprocally acting as co-activators and co-repressors of Runx2 and PPARγ, were decreased in bone of Oc-Cre;Pkd2flox/null mice. Thus, Pkd1 and Pkd2 have coordinate effects on osteoblast differentiation and opposite effects on adipogenesis, suggesting that Pkd1 and Pkd2 signaling pathways can have independent effects on mesenchymal lineage commitment in bone.

  3. The Src inhibitor dasatinib accelerates the differentiation of human bone marrow-derived mesenchymal stromal cells into osteoblasts

    International Nuclear Information System (INIS)

    Id Boufker, Hichame; Lagneaux, Laurence; Najar, Mehdi; Piccart, Martine; Ghanem, Ghanem; Body, Jean-Jacques; Journé, Fabrice

    2010-01-01

    The proto-oncogene Src is an important non-receptor protein tyrosine kinase involved in signaling pathways that control cell adhesion, growth, migration and differentiation. It negatively regulates osteoblast activity, and, as such, its inhibition is a potential means to prevent bone loss. Dasatinib is a new dual Src/Bcr-Abl tyrosine kinase inhibitor initially developed for the treatment of chronic myeloid leukemia. It has also shown promising results in preclinical studies in various solid tumors. However, its effects on the differentiation of human osteoblasts have never been examined. We evaluated the effects of dasatinib on bone marrow-derived mesenchymal stromal cells (MSC) differentiation into osteoblasts, in the presence or absence of a mixture of dexamethasone, ascorbic acid and β-glycerophosphate (DAG) for up to 21 days. The differentiation kinetics was assessed by evaluating mineralization of the extracellular matrix, alkaline phosphatase (ALP) activity, and expression of osteoblastic markers (receptor activator of nuclear factor kappa B ligand [RANKL], bone sialoprotein [BSP], osteopontin [OPN]). Dasatinib significantly increased the activity of ALP and the level of calcium deposition in MSC cultured with DAG after, respectively, 7 and 14 days; it upregulated the expression of BSP and OPN genes independently of DAG; and it markedly downregulated the expression of RANKL gene and protein (decrease in RANKL/OPG ratio), the key factor that stimulates osteoclast differentiation and activity. Our results suggest a dual role for dasatinib in both (i) stimulating osteoblast differentiation leading to a direct increase in bone formation, and (ii) downregulating RANKL synthesis by osteoblasts leading to an indirect inhibition of osteoclastogenesis. Thus, dasatinib is a potentially interesting candidate drug for the treatment of osteolysis through its dual effect on bone metabolism

  4. The Role of Moderate Static Magnetic Fields on Biomineralization of Osteoblasts on Sulfonated Polystryene Films

    Energy Technology Data Exchange (ETDEWEB)

    X Ba; M Hadjiargyrou; E DiMasi; Y Meng; M Simon; Z Tan; M Rafailovich

    2011-12-31

    We have investigated the effects of moderate static magnetic fields (SMFs) on murine MC3T3-E1 osteoblasts, and found that they enhance proliferations and promote differentiation. The increase in proliferation rates in response to SMFs was greater in cultures grown on partially sulfonated polytstyrene (SPS, degree of sulfonation: 33%) than in cultures grown on tissue culture plastic. We have previously shown that when the degree of sulfonation exceeded a critical value (12%) [1], spontaneous fibrillogenesis occured which allowed for direct observation of the ECM fibrillar organization under the influence of external fields. We found that the ECM produced in cultures grown on the SPS in the presence of the SMFs assembled into a lattice with larger dimensions than the ECM of the cultures grown in the absence of SMFs. During the early stages of the biomineralization process (day 7), the SMF exposed cultures also templated mineral deposition more rapidly than the control cultures. The rapid response is attributed to orientation of diamagnetic ECM proteins already present in the serum, which could then initiate further cellular signaling. SMFs also influenced late stage osteoblast differentiation as measured by the increased rate of osteocalcin secretion and gene expression beginning 15 days after SFM exposure. This correlated with a large increase in mineral deposition, and in cell modulus. GIXD and EDXS analysis confirmed early deposition of crystalline hydroxyapatite. Previous studies on the effects of moderate SMF had focused on cellular gene and protein expression, but did not consider the organization of the ECM fibers. Our ability to form these fibers has allowed us explore this additional effect and highlight its significance in the initiation of the biomineralization process.

  5. [Characterization of epithelial primary culture from human conjunctiva].

    Science.gov (United States)

    Rivas, L; Blázquez, A; Muñoz-Negrete, F J; López, S; Rebolleda, G; Domínguez, F; Pérez-Esteban, A

    2014-01-01

    To evaluate primary cultures from human conjunctiva supplemented with fetal bovine serum, autologous serum, and platelet-rich autologous serum, over human amniotic membrane and lens anterior capsules. One-hundred and forty-eight human conjunctiva explants were cultured in CnT50(®) supplemented with 1, 2.5, 5 and 10% fetal bovine serum, autologous serum and platelet-rich autologous serum. Conjunctival samples were incubated at 37°C, 5% CO2 and 95% HR, for 3 weeks. The typical phenotype corresponding to conjunctival epithelial cells was present in all primary cultures. Conjunctival cultures had MUC5AC-positive secretory cells, K19-positive conjunctival cells, and MUC4-positive non-secretory conjunctival cells, but were not corneal phenotype (cytokeratin K3-negative) and fibroblasts (CD90-negative). Conjunctiva epithelial progenitor cells were preserved in all cultures; thus, a cell culture in CnT50(®) supplemented with 1 to 5% autologous serum over human amniotic membrane can provide better information of epithelial cell differentiation for the conjunctival surface reconstruction. Copyright © 2013 Sociedad Española de Oftalmología. Published by Elsevier Espana. All rights reserved.

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

    DEFF Research Database (Denmark)

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

    2006-01-01

    to be independent of the differential production of soluble RANKL and OPG and, instead, due to a decrease in osteoblast maturation accompanied by increase in osteoblastic proliferation. In addition to the imbalance between differentiation and proliferation, there was a differential decrease in secretory leukocyte......Bone mass is maintained by a fine balance between bone formation by osteoblasts and bone resorption by osteoclasts. Although osteoblasts and osteoclasts have different developmental origins, it is generally believed that the differentiation, function, and survival of osteoclasts are regulated...... by osteogenic cells. We have previously shown that the extracellular matrix protein, biglycan (Bgn), plays an important role in the differentiation of osteoblast precursors. In this paper, we showed that Bgn is involved in regulating osteoclast differentiation through its effect on osteoblasts...

  7. Biphasic Response to Luteolin in MG-63 Osteoblast-Like Cells under High Glucose‑Induced Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Naser Abbasi

    2016-03-01

    Full Text Available Background: Clinical evidence indicates the diabetes-induced impairment of osteogenesis caused by a decrease in osteoblast activity. Flavonoids can increase the differentiation and mineralization of osteoblasts in a high-glucose state. However, some flavonoids such as luteolin may have the potential to induce cytotoxicity in osteoblast-like cells. This study was performed to investigate whether a cytoprotective concentration range of luteolin could be separated from a cytotoxic concentration range in human MG-63 osteoblast-like cells in high-glucose condition. Methods: Cells were cultured in a normal- or high-glucose medium. Cell viability was determined with the MTT assay. The formation of intracellular reactive oxygen species (ROS was measured using probe 2’,7’ -dichlorofluorescein diacetate, and osteogenic differentiation was evaluated with an alkaline phosphatase bioassay. Results: ROS generation, reduction in alkaline phosphatase activity, and cell death induced by high glucose were inhibited by lower concentrations of luteolin (EC50, 1.29±0.23 µM. Oxidative stress mediated by high glucose was also overcome by N-acetyl-L-cysteine. At high concentrations, luteolin caused osteoblast cell death in normal- and high-glucose states (IC50, 34±2.33 and 27±2.42 µM, respectively, as represented by increased ROS and decreased alkaline phosphatase activity. Conclusion: Our results indicated that the cytoprotective action of luteolin in glucotoxic condition was manifested in much lower concentrations, by a factor of approximately 26 and 20, than was its cytotoxic activity, which occurred under normal or glucotoxic condition, respectively.

  8. Immunocytochemical characterization of primary cell culture in canine transmissible venereal tumor

    Directory of Open Access Journals (Sweden)

    Luis M.M. Flórez

    Full Text Available Abstract: Immunochemistry with anti-vimentin, anti-lysozyme, anti-alpha 1 antitrypsin, anti-CD3 and anti-CD79α antibodies has been used for characterization of primary cell culture in the transmissible venereal tumor (TVT. Samples for primary cell culture and immunohistochemistry assays were taken from eight dogs with cytological and clinical diagnosis of TVT. To validate the immunochemical results in the primary cell culture of TVT, a chromosome count was performed. For the statistical analysis, the Mann-Whitney test with p<0.05 was used. TVT tissues and culture cells showed intense anti-vimentin immunoreactivity, lightly to moderate immunoreactivity for anti-lysozyme, and mild for anti-alpha-antitrypsin. No marking was achieved for CD3 and CD79α. All culture cells showed chromosomes variable number of 56 to 68. This is the first report on the use of immunocytochemical characterization in cell culture of TVT. Significant statistic difference between immunochemistry in tissue and culture cell was not established, what suggests that the use of this technique may provide greater certainty for the confirmation of tumors in the primary culture. This fact is particularly important because in vitro culture of tumor tissues has been increasingly used to provide quick access to drug efficacy and presents relevant information to identify potential response to anticancer medicine; so it is possible to understand the behavior of the tumor.

  9. Cancer Metastases to Bone: Concepts, Mechanisms, and Interactions with Bone Osteoblasts

    Directory of Open Access Journals (Sweden)

    Alison B. Shupp

    2018-06-01

    Full Text Available The skeleton is a unique structure capable of providing support for the body. Bone resorption and deposition are controlled in a tightly regulated balance between osteoblasts and osteoclasts with no net bone gain or loss. However, under conditions of disease, the balance between bone resorption and deposition is upset. Osteoblasts play an important role in bone homeostasis by depositing new bone osteoid into resorption pits. It is becoming increasingly evident that osteoblasts additionally play key roles in cancer cell dissemination to bone and subsequent metastasis. Our laboratory has evidence that when osteoblasts come into contact with disseminated breast cancer cells, the osteoblasts produce factors that initially reduce breast cancer cell proliferation, yet promote cancer cell survival in bone. Other laboratories have demonstrated that osteoblasts both directly and indirectly contribute to dormant cancer cell reactivation in bone. Moreover, we have demonstrated that osteoblasts undergo an inflammatory stress response in late stages of breast cancer, and produce inflammatory cytokines that are maintenance and survival factors for breast cancer cells and osteoclasts. Advances in understanding interactions between osteoblasts, osteoclasts, and bone metastatic cancer cells will aid in controlling and ultimately preventing cancer cell metastasis to bone.

  10. Culture-bound syndromes in Hispanic primary care patients.

    Science.gov (United States)

    Bayles, Bryan P; Katerndahl, David A

    2009-01-01

    We sought to document Hispanic primary care patients' knowledge and experience of five culture-bound syndromes (CBS), as well as the basic socio-cultural correlates of these disorders. A convenience sample of 100 adult Hispanic patients presenting in an urban South Texas primary care clinic was recruited to complete a brief cross-sectional survey, presented in an oral format. Interviews sought information concerning five culture-bound syndromes--susto, empacho, nervios, mal de ojo, and ataques de nervios. Additional demographic, socio-economic, and acculturation data was collected. Descriptive and bivariate statistics (chi square, Fisher's) were used to assess relationships among variables and experience with each CBS. A multivariate logistic analysis was conducted to determine the possible contributions of age, gender, acculturation, and education to the personal experience of a culture-bound syndrome. Results indicate that 77% of respondents had knowledge of all five syndromes, with 42% reporting having personally experienced at least one CBS. Nervios was the most commonly suffered disorder, being reported by 30 respondents. This was followed, in declining order ofprevalence, by susto, mal de ojo, empacho, and ataques de nervios. Multivariate logistic regression analysis found that higher education beyond high school was associated with a slightly decreased likelihood of reporting having suffered from any culture-bound syndrome. While co-occurrence among these disorders occurred, the patterns of predictors suggest that the co-occurrence is not a reflection of mislabeling of one common syndrome. Knowledge of and experience with culture-bound syndromes is common among Hispanic primary care patients in South Texas. Healthcare providers ought to consider discussing these illnesses in a non-judgmental manner with patients who present with symptoms that are consistent with these syndromes. Future studies, with larger sample sizes, are warranted to elucidate the nature

  11. Radiosensitivity of primary cultured fish cells with different ploidy

    International Nuclear Information System (INIS)

    Mitani, Hiroshi; Egami, Nobuo; Kobayashi, Hiromu.

    1986-01-01

    The radiosensitivity of primary cultured goldfish cells (Carassius auratus) was investigated by colony formation assay. The radiosensitivity of cells from two varieties of goldfish, which show different sensitivity to lethal effect of ionizing radiation in vivo, was almost identical. Primary cultured cells from diploid, triploid and tetraploid fish retained their DNA content as measured by microfluorometry, and the nuclear size increases as ploidy increases. However, radiosensitivity was not related to ploidy. (author)

  12. Mycobacterium leprae downregulates the expression of PHEX in Schwann cells and osteoblasts

    Directory of Open Access Journals (Sweden)

    Sandra R Boiça Silva

    2010-08-01

    Full Text Available Neuropathy and bone deformities, lifelong sequelae of leprosy that persist after treatment, result in significant impairment to patients and compromise their social rehabilitation. Phosphate-regulating gene with homologies to endopeptidase on the X chromosome (PHEX is a Zn-metalloendopeptidase, which is abundantly expressed in osteoblasts and many other cell types, such as Schwann cells, and has been implicated in phosphate metabolism and X-linked rickets. Here, we demonstrate that Mycobacterium leprae stimulation downregulates PHEX transcription and protein expression in a human schwannoma cell line (ST88-14 and human osteoblast lineage. Modulation of PHEX expression was observed to a lesser extent in cells stimulated with other species of mycobacteria, but was not observed in cultures treated with latex beads or with the facultative intracellular bacterium Salmonella typhimurium. Direct downregulation of PHEX by M. leprae could be involved in the bone resorption observed in leprosy patients. This is the first report to describe PHEX modulation by an infectious agent.

  13. Nano-hydroxyapatite modulates osteoblast lineage commitment by stimulation of DNA methylation and regulation of gene expression

    Science.gov (United States)

    Ha, Shin-Woo; Jang, Hae Lin; Nam, Ki Tae; Beck, George R.

    2015-01-01

    Hydroxyapatite (HA) is the primary structural component of the skeleton and dentition. Under biological conditions, HA does not occur spontaneously and therefore must be actively synthesized by mineralizing cells such as osteoblasts. The mechanism(s) by which HA is actively synthesized by cells and deposited to create a mineralized matrix are not fully understood and the consequences of mineralization on cell function are even less well understood. HA can be chemically synthesized (HAp) and is therefore currently being investigated as a promising therapeutic biomaterial for use as a functional scaffold and implant coating for skeletal repair and dental applications. Here we investigated the biological effects of nano-HAp (10×100 nm) on the lineage commitment and differentiation of bone forming osteoblasts. Exposure of early stage differentiating osteoblasts resulted in dramatic and sustained changes in gene expression, both increased and decreased, whereas later stage osteoblasts were much less responsive. Analysis of the promoter region one of the most responsive genes, alkaline phosphatase, identified the stimulation of DNA methylation following cell exposure to nano-HAp. Collectively, the results reveal the novel epigenetic regulation of cell function by nano-HAp which has significant implication on lineage determination as well as identifying a novel potential therapeutic use of nanomaterials. PMID:26141836

  14. Osteoblast Adhesion on Cathodic Arc Plasma Deposited Nano-Multilayered TiCrAlSiN Thin Films

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sun Kyu [University of Ulsan, Ulsan (Korea, Republic of); Pham, Vuong Hung [Hanoi University of Science and Technology (HUST), Hanoi (Viet Nam)

    2014-03-15

    Adhesion of osteoblast cells to TiCrAlSiN thin films was evaluated in vitro. Ti and TiCrAlSiN thin films were deposited on glass substrates by cathodic arc deposition. Surface roughness and chemistry of the TiCrAlSiN thin films was characterized by AFM and EPMA, respectively. Ti and TiCrAlSiN thin films and glass coverslips were cultured with human osteoblast cells (hFOB 1.19). The cell cytoskeleton was analyzed by observing the organization of actin stress fibers and microtubules. Cell proliferation was investigated by MTT assay and visualization. Focal contact adhesion was studied by observing the vinculin density. The results indicated that the TiCrAlSiN coating significantly influenced the actin cytoskeleton and microtubule organization. Human osteoblasts hFOB attached and proliferated better on TiCrAlSiN thin films with more focal contact adhesions than on Ti thin films or glass surfaces. These results suggest that TiCrAlSiN thin films can be an implantable material where the maximum cell adhesion is required.

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

    Science.gov (United States)

    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.

  16. Application of pulsed-magnetic field enhances non-viral gene delivery in primary cells from different origins

    Energy Technology Data Exchange (ETDEWEB)

    Kamau Chapman, Sarah W. [Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, Winterthurerstr. 190, 8057 Zurich (Switzerland); Hassa, Paul O. [Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, Winterthurerstr. 190, 8057 Zurich (Switzerland); European Molecular Biology Laboratory (EMBL) Heidelberg, Meyerhofstrasse 1, 69117 Heidelberg (Germany); Koch-Schneidemann, Sabine; Rechenberg, Brigitte von [Musculoskeletal Research Unit, Equine Hospital, Vetsuisse Faculty Zurich, University of Zurich, Winterthurerstr. 260, 8057 Zurich (Switzerland); Hofmann-Amtenbrink, Margarethe [MatSearch, Chemin Jean Pavillard 14, 1009 Pully (Switzerland); Steitz, Benedikt; Petri-Fink, Alke; Hofmann, Heinrich [Laboratory of Powder Technology, Ecole Polytechnique Federale de Lausanne (EPFL), Lausanne (Switzerland); Hottiger, Michael O. [Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, Winterthurerstr. 190, 8057 Zurich (Switzerland)], E-mail: hottiger@vetbio.uzh.ch

    2008-04-15

    Primary cell lines are more difficult to transfect when compared to immortalized/transformed cell lines, and hence new techniques are required to enhance the transfection efficiency in these cells. We isolated and established primary cultures of synoviocytes, chondrocytes, osteoblasts, melanocytes, macrophages, lung fibroblasts, and embryonic fibroblasts. These cells differed in several properties, and hence were a good representative sample of cells that would be targeted for expression and delivery of therapeutic genes in vivo. The efficiency of gene delivery in all these cells was enhanced using polyethylenimine-coated polyMAG magnetic nanoparticles, and the rates (17-84.2%) surpassed those previously achieved using other methods, especially in cells that are difficult to transfect. The application of permanent and pulsating magnetic fields significantly enhanced the transfection efficiencies in synoviocytes, chondrocytes, osteoblasts, melanocytes and lung fibroblasts, within 5 min of exposure to these magnetic fields. This is an added advantage for future in vivo applications, where rapid gene delivery is required before systemic clearance or filtration of the gene vectors occurs.

  17. Therapeutic Doses of Nonsteroidal Anti-Inflammatory Drugs Inhibit Osteosarcoma MG-63 Osteoblast-Like Cells Maturation, Viability, and Biomineralization Potential

    Directory of Open Access Journals (Sweden)

    E. De Luna-Bertos

    2013-01-01

    Full Text Available Nonsteroidal anti-inflammatory drugs (NSAIDs are frequently used to reduce pain and inflammation. However, their effect on bone metabolisms is not well known, and results in the literature are contradictory. The present study focusses on the effect of dexketoprofen, ketorolac, metamizole, and acetylsalicylic acid, at therapeutic doses, on different biochemical and phenotypic pathways in human osteoblast-like cells. Osteoblasts (MG-63 cell line were incubated in culture medium with 1–10 μM of dexketoprofen, ketorolac, metamizole, and acetylsalicylic acid. Flow cytometry was used to study antigenic profile and phagocytic activity. The osteoblastic differentiation was evaluated by mineralization and synthesis of collagen fibers by microscopy and alkaline phosphatase activity (ALP by spectrophotometric assay. Short-term treatment with therapeutic doses of NSAIDs modulated differentiation, antigenic profile, and phagocyte activity of osteoblast-like cells. The treatment reduced ALP synthesis and matrix mineralization. However, nonsignificant differences were observed on collagen syntheses after treatments. The percentage of CD54 expression was increased with all treatments. CD80, CD86, and HLA-DR showed a decreased expression, which depended on NSAID and the dose applied. The treatments also decreased phagocyte activity in this cellular population. The results of this paper provide evidences that NSAIDs inhibit the osteoblast differentiation process thus reducing their ability to produce new bone mineralized extracellular matrix.

  18. Therapeutic Doses of Nonsteroidal Anti-Inflammatory Drugs Inhibit Osteosarcoma MG-63 Osteoblast-Like Cells Maturation, Viability, and Biomineralization Potential

    Science.gov (United States)

    De Luna-Bertos, E.; Ramos-Torrecillas, J.; García-Martínez, O.; Guildford, A.; Santin, M.; Ruiz, C.

    2013-01-01

    Nonsteroidal anti-inflammatory drugs (NSAIDs) are frequently used to reduce pain and inflammation. However, their effect on bone metabolisms is not well known, and results in the literature are contradictory. The present study focusses on the effect of dexketoprofen, ketorolac, metamizole, and acetylsalicylic acid, at therapeutic doses, on different biochemical and phenotypic pathways in human osteoblast-like cells. Osteoblasts (MG-63 cell line) were incubated in culture medium with 1–10 μM of dexketoprofen, ketorolac, metamizole, and acetylsalicylic acid. Flow cytometry was used to study antigenic profile and phagocytic activity. The osteoblastic differentiation was evaluated by mineralization and synthesis of collagen fibers by microscopy and alkaline phosphatase activity (ALP) by spectrophotometric assay. Short-term treatment with therapeutic doses of NSAIDs modulated differentiation, antigenic profile, and phagocyte activity of osteoblast-like cells. The treatment reduced ALP synthesis and matrix mineralization. However, nonsignificant differences were observed on collagen syntheses after treatments. The percentage of CD54 expression was increased with all treatments. CD80, CD86, and HLA-DR showed a decreased expression, which depended on NSAID and the dose applied. The treatments also decreased phagocyte activity in this cellular population. The results of this paper provide evidences that NSAIDs inhibit the osteoblast differentiation process thus reducing their ability to produce new bone mineralized extracellular matrix. PMID:24170983

  19. Cord blood-derived macrophage-lineage cells rapidly stimulate osteoblastic maturation in mesenchymal stem cells in a glycoprotein-130 dependent manner.

    Directory of Open Access Journals (Sweden)

    Tania J Fernandes

    Full Text Available In bone, depletion of osteoclasts reduces bone formation in vivo, as does osteal macrophage depletion. How osteoclasts and macrophages promote the action of bone forming osteoblasts is, however, unclear. Since recruitment and differentiation of multi-potential stromal cells/mesenchymal stem cells (MSC generates new active osteoblasts, we investigated whether human osteoclasts and macrophages (generated from cord blood-derived hematopoietic progenitors induce osteoblastic maturation in adipose tissue-derived MSC. When treated with an osteogenic stimulus (ascorbate, dexamethasone and β-glycerophosphate these MSC form matrix-mineralising, alkaline phosphatase-expressing osteoblastic cells. Cord blood-derived progenitors were treated with macrophage colony stimulating factor (M-CSF to form immature proliferating macrophages, or with M-CSF plus receptor activator of NFκB ligand (RANKL to form osteoclasts; culture medium was conditioned for 3 days by these cells to study their production of osteoblastic factors. Both osteoclast- and macrophage-conditioned medium (CM greatly enhanced MSC osteoblastic differentiation in both the presence and absence of osteogenic medium, evident by increased alkaline phosphatase levels within 4 days and increased mineralisation within 14 days. These CM effects were completely ablated by antibodies blocking gp130 or oncostatin M (OSM, and OSM was detectable in both CM. Recombinant OSM very potently stimulated osteoblastic maturation of these MSC and enhanced bone morphogenetic protein-2 (BMP-2 actions on MSC. To determine the influence of macrophage activation on this OSM-dependent activity, CM was collected from macrophage populations treated with M-CSF plus IL-4 (to induce alternative activation or with GM-CSF, IFNγ and LPS to cause classical activation. CM from IL-4 treated macrophages stimulated osteoblastic maturation in MSC, while CM from classically-activated macrophages did not. Thus, macrophage-lineage cells

  20. Translationally controlled tumor protein supplemented chitosan modified glass ionomer cement promotes osteoblast proliferation and function

    Energy Technology Data Exchange (ETDEWEB)

    Sangsuwan, Jiraporn [Department of Molecular Biology and Bioinformatics, Center for Genomics and Bioinformatics Research, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112 (Thailand); Department of Oral Biology and Occlusion, Faculty of Dentistry, Prince of Songkla University, Hat Yai, Songkhla 90112 (Thailand); Wanichpakorn, Supreya; Kedjarune-Leggat, Ureporn [Department of Oral Biology and Occlusion, Faculty of Dentistry, Prince of Songkla University, Hat Yai, Songkhla 90112 (Thailand)

    2015-09-01

    The objective of this study was to evaluate the effect of translationally controlled tumor protein (TCTP) supplemented in a novel glass ionomer cement (BIO-GIC) on normal human osteoblasts (NHost cells). BIO-GIC was a glass ionomer cement (GIC) modified by adding chitosan and albumin to promote the release of TCTP. NHost cells were seeded on specimens of GIC, GIC + TCTP, BIO-GIC and BIO-GIC + TCTP. Cell proliferation was determined by BrdU assay. It was found that BIO-GIC + TCTP had significantly higher proliferation of cells than other specimens. Bone morphogenetic protein-2 (BMP-2) and osteopontin (OPN) gene expressions assessed by quantitative real time PCR and alkaline phosphatase (ALP) activity were used to determine cell differentiation. Bone cell function was investigated by calcium deposition using alizarin assay. Both BMP-2 and OPN gene expressions of cells cultured on specimens with added TCTP increased gradually up-regulation after day 1 and reached the highest on day 3 then down-regulation on day 7. The ALP activity of cells cultured on BIO-GIC + TCTP for 7 days and calcium content after 14 days were significantly higher than other groups. BIO-GIC + TCTP can promote osteoblast cells proliferation, differentiation and function. - Highlights: • Developed a new GIC by supplementing TCTP in BIO-GIC (GIC with chitosan and albumin) • BIO-GIC + TCTP released a higher amount of TCTP than GIC + TCTP. • BIO-GIC + TCTP promoted cell proliferation higher than other specimens and control. • BIO-GIC + TCTP promoted osteoblasts differentiation and function.

  1. Translationally controlled tumor protein supplemented chitosan modified glass ionomer cement promotes osteoblast proliferation and function

    International Nuclear Information System (INIS)

    Sangsuwan, Jiraporn; Wanichpakorn, Supreya; Kedjarune-Leggat, Ureporn

    2015-01-01

    The objective of this study was to evaluate the effect of translationally controlled tumor protein (TCTP) supplemented in a novel glass ionomer cement (BIO-GIC) on normal human osteoblasts (NHost cells). BIO-GIC was a glass ionomer cement (GIC) modified by adding chitosan and albumin to promote the release of TCTP. NHost cells were seeded on specimens of GIC, GIC + TCTP, BIO-GIC and BIO-GIC + TCTP. Cell proliferation was determined by BrdU assay. It was found that BIO-GIC + TCTP had significantly higher proliferation of cells than other specimens. Bone morphogenetic protein-2 (BMP-2) and osteopontin (OPN) gene expressions assessed by quantitative real time PCR and alkaline phosphatase (ALP) activity were used to determine cell differentiation. Bone cell function was investigated by calcium deposition using alizarin assay. Both BMP-2 and OPN gene expressions of cells cultured on specimens with added TCTP increased gradually up-regulation after day 1 and reached the highest on day 3 then down-regulation on day 7. The ALP activity of cells cultured on BIO-GIC + TCTP for 7 days and calcium content after 14 days were significantly higher than other groups. BIO-GIC + TCTP can promote osteoblast cells proliferation, differentiation and function. - Highlights: • Developed a new GIC by supplementing TCTP in BIO-GIC (GIC with chitosan and albumin) • BIO-GIC + TCTP released a higher amount of TCTP than GIC + TCTP. • BIO-GIC + TCTP promoted cell proliferation higher than other specimens and control. • BIO-GIC + TCTP promoted osteoblasts differentiation and function

  2. Liposomal clodronate selectively eliminates microglia from primary astrocyte cultures

    Directory of Open Access Journals (Sweden)

    Kumamaru Hiromi

    2012-05-01

    Full Text Available Abstract Background There is increasing interest in astrocyte biology because astrocytes have been demonstrated to play prominent roles in physiological and pathological conditions of the central nervous system, including neuroinflammation. To understand astrocyte biology, primary astrocyte cultures are most commonly used because of the direct accessibility of astrocytes in this system. However, this advantage can be hindered by microglial contamination. Although several authors have warned regarding microglial contamination in this system, complete microglial elimination has never been achieved. Methods The number and proliferative potential of contaminating microglia in primary astrocyte cultures were quantitatively assessed by immunocytologic and flow cytometric analyses. To examine the utility of clodronate for microglial elimination, primary astrocyte cultures or MG-5 cells were exposed to liposomal or free clodronate, and then immunocytologic, flow cytometric, and gene expression analyses were performed. The gene expression profiles of microglia-eliminated and microglia-contaminated cultures were compared after interleukin-6 (IL-6 stimulation. Results The percentage of contaminating microglia exceeded 15% and continued to increase because of their high proliferative activity in conventional primary astrocyte cultures. These contaminating microglia were selectively eliminated low concentration of liposomal clodronate. Although primary microglia and MG-5 cells were killed by both liposomal and free clodronate, free clodronate significantly affected the viability of astrocytes. In contrast, liposomal clodronate selectively eliminated microglia without affecting the viability, proliferation or activation of astrocytes. The efficacy of liposomal clodronate was much higher than that of previously reported methods used for decreasing microglial contamination. Furthermore, we observed rapid tumor necrosis factor-α and IL-1b gene induction in

  3. Transfection in Primary Cultured Neuronal Cells.

    Science.gov (United States)

    Marwick, Katie F M; Hardingham, Giles E

    2017-01-01

    Transfection allows the introduction of foreign nucleic acid into eukaryotic cells. It is an important tool in understanding the roles of NMDARs in neurons. Here, we describe using lipofection-mediated transfection to introduce cDNA encoding NMDAR subunits into postmitotic rodent primary cortical neurons maintained in culture.

  4. Enhancing Access to Primary Cultural Heritage Materials of India

    Science.gov (United States)

    Scharf, Peter M.; Hyman, Malcolm

    This chapter is about enhancing access to primary cultural heritage materials of India housed in academic libraries by integrating them with machine-readable texts, lexical resources, and linguistic software in a digital library. Integrating primary cultural materials with a digital library can enable broad use of Indic collections for research and education. For the purposes of illustrating this procedure, we outline here the development of a prototype using the collections of Sanskrit manuscripts in the libraries at Brown University and the University of Pennsylvania and integrating them with The Sanskrit Library. The result is extendable to collections of Indic materials throughout the world and can serve as a model for digitization projects of cultural materials in other major culture-bearing languages such as Greek, Latin, Arabic, Persian, and Chinese.

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

    Directory of Open Access Journals (Sweden)

    Eames B Frank

    2012-03-01

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

  6. Receptors and effects of gut hormones in three osteoblastic cell lines

    Directory of Open Access Journals (Sweden)

    Wilson Peter JM

    2011-07-01

    Full Text Available Abstract Background 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. Methods 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. Results 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

  7. 3D cell culture to determine in vitro biocompatibility of bioactive glass in association with chitosan.

    Science.gov (United States)

    Bédouin, Y; Pellen Mussi, P; Tricot-Doleux, S; Chauvel-Lebret, D; Auroy, P; Ravalec, X; Oudadesse, H; Perez, F

    2015-01-01

    This study reports the in vitro biocompatibility of a composite biomaterial composed of 46S6 bioactive glass in association with chitosan (CH) by using 3D osteoblast culture of SaOS2. The 46S6 and CH composite (46S6-CH) forms small hydroxyapatite crystals on its surface after only three days immersion in the simulated body fluid. For 2D osteoblast culture, a significant increase in cell proliferation was observed after three days of contact with 46S6 or 46S6-CH-immersed media. After six days, 46S6-CH led to a significant increase in cell proliferation (128%) compared with pure 46S6 (113%) and pure CH (122%). For 3D osteoblast culture, after six days of culture, there was an increase in gene expression of markers of the early osteoblastic differentiation (RUNX2, ALP, COL1A1). Geometric structures corresponding to small apatite clusters were observed by SEM on the surface of the spheroids cultivated with 46S6 or 46S6-CH-immersed media. We showed different cellular responses depending on the 2D and 3D cell culture model. The induction of osteoblast differentiation in the 3D cell culture explained the differences of cell proliferation in contact with 46S6, CH or 46S6-CH-immersed media. This study confirmed that the 3D cell culture model is a very promising tool for in vitro biological evaluation of bone substitutes' properties.

  8. Effect of water-soluble P-chitosan and S-chitosan on human primary osteoblasts and giant cell tumor of bone stromal cells

    Energy Technology Data Exchange (ETDEWEB)

    Tang, T; Zhang, G; PY Lau, Carol; Zheng, L Z; Xie, X H; Wang, X L; Patrick, Y; Qin, L; Kumta, Shekhar M [Department of Orthopaedics and Traumatology, Chinese University of Hong Kong (Hong Kong); Wang, X H; He, K, E-mail: kumta@cuhk.edu.hk [Department of Mechanical Engineering, Institute of Bio-manufacturing Engineering, Tsinghua University, Beijing (China)

    2011-02-15

    Water-soluble phosphorylated chitosan (P-chitosan) and disodium (1 {yields} 4)-2-deoxy-2-sulfoamino-{beta}-D-glucopyranuronan (S-chitosan) are two chemically modified chitosans. In this study, we found that P-chitosan significantly promotes cell proliferation of both human primary osteoblasts (OBs) and the OB like stromal cell component of the giant cell tumor of bone (GCTB) cells at the concentration from 125 to 1000 {mu}g ml{sup -1} at all time points of 1, 3, 5 and 7 days after treatment. Further investigation of the osteogenic effect of the P-chitosan suggested that it regulates the levels of osteoclastogenic factors, receptor activator of nuclear factor kappa B ligand and osteoprotegerin expression. An interesting finding is that S-chitosan at lower concentration (100 {mu}g ml{sup -1}) stimulates cell proliferation while a higher dose (1000 {mu}g ml{sup -1}) of S-chitosan inhibits it. The inhibitory effect of S-chitosan on human primary GCT stromal cells was greater than that of OBs (p < 0.05). Taken together, our findings elucidated the osteogenic effect of P-chitosan and the varying effects of S-chitosan on the proliferation of human primary OBs and GCT stromal cells and provided us the rationale for the construction of novel bone repair biomaterials with the dual properties of bone induction and bone tumor inhibition.

  9. Megakaryocytes promote murine osteoblastic HSC niche expansion and stem cell engraftment after radioablative conditioning.

    Science.gov (United States)

    Olson, Timothy S; Caselli, Anna; Otsuru, Satoru; Hofmann, Ted J; Williams, Richard; Paolucci, Paolo; Dominici, Massimo; Horwitz, Edwin M

    2013-06-27

    Successful hematopoietic stem cell (HSC) transplantation requires donor HSC engraftment within specialized bone marrow microenvironments known as HSC niches. We have previously reported a profound remodeling of the endosteal osteoblastic HSC niche after total body irradiation (TBI), defined as relocalization of surviving megakaryocytes to the niche site and marked expansion of endosteal osteoblasts. We now demonstrate that host megakaryocytes function critically in expansion of the endosteal niche after preparative radioablation and in the engraftment of donor HSC. We show that TBI-induced migration of megakaryocytes to the endosteal niche depends on thrombopoietin signaling through the c-MPL receptor on megakaryocytes, as well as CD41 integrin-mediated adhesion. Moreover, niche osteoblast proliferation post-TBI required megakaryocyte-secreted platelet-derived growth factor-BB. Furthermore, blockade of c-MPL-dependent megakaryocyte migration and function after TBI resulted in a significant decrease in donor HSC engraftment in primary and competitive secondary transplantation assays. Finally, we administered thrombopoietin to mice beginning 5 days before marrow radioablation and ending 24 hours before transplant to enhance megakaryocyte function post-TBI, and found that this strategy significantly enhanced donor HSC engraftment, providing a rationale for improving hematopoietic recovery and perhaps overall outcome after clinical HSC transplantation.

  10. Comparison of the Influence of Phospholipid-Coated Porous Ti-6Al-4V Material on the Osteosarcoma Cell Line Saos-2 and Primary Human Bone Derived Cells

    Directory of Open Access Journals (Sweden)

    Axel Deing

    2016-03-01

    Full Text Available Biomaterial surface functionalization remains of great interest in the promotion of cell osteogenic induction. Previous studies highlighted the positive effects of porous Ti-6Al-4V and phospholipid coating on osteoblast differentiation and bone remodeling. Therefore, the first objective of this study was to evaluate the potential synergistic effects of material porosity and phospholipid coating. Primary human osteoblasts and Saos-2 cells were cultured on different Ti-6Al-4V specimens (mirror-like polished or porous specimens and were coated or not with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE for three weeks or five weeks. Selected gene expressions (e.g., classical bone markers: alkaline phosphatase, osteocalcin, osteoprotegerin (OPG, receptor activator of nuclear factor kappa-β ligand (RANKL and runt-related transcription factor 2 were estimated in vitro. Furthermore, the expressions of osteocalcin and osteopontin were examined via fluorescent microscopy at five weeks (immunocytochemistry. Consequently, it was observed that phospholipid coating potentiates preferences for low and high porosities in Saos-2 and primary cells, respectively, at the gene and protein levels. Additionally, RANKL and OPG exhibited different gene expression patterns; primary cells showed dramatically increased RANKL expression, whereas OPG expression was decreased in the presence of POPE. A synergistic effect of increased porosity and phospholipid coating was observed in primary osteoblasts in bone remodeling. This study showed the advantage of primary cells over the standard bone cell model.

  11. Porphyromonas gingivalis decreases osteoblast proliferation through IL-6-RANKL/OPG and MMP-9/TIMPs pathways

    Directory of Open Access Journals (Sweden)

    Le Xuan

    2009-01-01

    Full Text Available Background: Porphyromonas gingivalis, an important periodontal pathogen, is closely associated with inflammatory alveolar bone resorption. This bacterium exerts its pathogenic effect indirectly through multiple virulence factors, such as lipopolysaccharides, fimbriae, and proteases. Another possible pathogenic path may be through a direct interaction with the host′s soft and hard tissues (e.g., alveolar bone, which could lead to periodontitis. Aims and Objectives: The aim of the present study was to investigate the direct effect of live and heat-inactivated P gingivalis on bone resorption, using an in vitro osteoblast culture model. Results: Optical microscopy and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide MTT assay revealed that live P gingivalis induced osteoblast detachment and reduced their proliferation. This effect was specific to live bacteria and was dependent on their concentration. Live P gingivalis increased IL-6 mRNA expression and protein production and downregulated RANKL and OPG mRNA expression. The effect of live P gingivalis on bone resorption was strengthened by an increase in MMP-9 expression and its activity. This increase was accompanied by an increase in TIMP-1 and TIMP-2 mRNA expression and protein production by osteoblasts infected with live P gingivalis. Conclusion: Overall, the results suggest that direct contact of P gingivalis with osteoblasts induces bone resorption through an inflammatory pathway that involves IL-6, RANKL/OPG, and MMP-9/TIMPs.

  12. Identification and proteomic analysis of osteoblast-derived exosomes

    Energy Technology Data Exchange (ETDEWEB)

    Ge, Min; Ke, Ronghu; Cai, Tianyi; Yang, Junyi; Mu, Xiongzheng, E-mail: cranio@vip.163.com

    2015-11-06

    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 786 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.

  13. Osteoblast and osteocyte: games without frontiers.

    Science.gov (United States)

    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.

  14. Examining School Culture in Flemish and Chinese Primary Schools

    Science.gov (United States)

    Zhu, Chang; Devos, Geert; Tondeur, Jo

    2014-01-01

    The aim of this research is to gain understanding about school culture characteristics of primary schools in the Flemish and Chinese context. The study was carried out in Flanders (Belgium) and China, involving a total of 44 Flemish schools and 40 Chinese schools. The School Culture Scales were used to measure five school culture dimensions with…

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

    Science.gov (United States)

    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.

  16. Maintenance of osteoblastic and adipocytic differentiation potential with age and osteoporosis in human marrow stromal cell cultures

    DEFF Research Database (Denmark)

    Justesen, J; Dokkedahl, Karin Stenderup; Eriksen, E F

    2002-01-01

    Osteoblasts and adipocytes share a common precursor cell in the bone marrow stroma, termed marrow stromal cell (MSC). As the volume of bone adipose tissue increases in vivo with age, we hypothesized that decreased bone formation observed during aging and in patients with osteoporosis (OP) is the ...

  17. MiR-9 is overexpressed in spontaneous canine osteosarcoma and promotes a metastatic phenotype including invasion and migration in osteoblasts and osteosarcoma cell lines.

    Science.gov (United States)

    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

  18. Predicting Organizational Commitment from Organizational Culture in Turkish Primary Schools

    Science.gov (United States)

    Ipek, Cemalettin

    2010-01-01

    This study aims to describe organizational culture and commitment and to predict organizational commitment from organizational culture in Turkish primary schools. Organizational Culture Scale (Ipek "1999") and Organizational Commitment Scale (Balay "2000") were used in the data gathering process. The data were collected from…

  19. The Effect of Exogenous Zinc Concentration on the Responsiveness of MC3T3-E1 Pre-Osteoblasts to Surface Microtopography: Part II (Differentiation

    Directory of Open Access Journals (Sweden)

    Kathryn Dorst

    2014-02-01

    Full Text Available Osseointegration of bone implants is a vital part of the recovery process. Numerous studies have shown that micropatterned geometries can promote cell-substrate associations and strengthen the bond between tissue and the implanted material. As demonstrated previously, exogenous zinc levels can influence the responsiveness of pre-osteoblasts to micropatterns and modify their migratory behavior. In this study, we sought to determine the effect of exogenous zinc on differentiation of osteoblasts cultured on micropatterned vs. planar substrates. Levels of activated metalloproteinase-2 (MMP-2 and transforming growth factor-beta 1 (TGF-β1, as well as early stage differentiation marker alkaline phosphatase, were altered with the addition of zinc. These results suggest that exogenous zinc concentration and micropatterning may interdependently modulate osteoblast differentiation.

  20. Adhesion and Growth of Human Osteoblast-Like Cell in Cultures on Nanocomposite Carbon-Based Materials

    Czech Academy of Sciences Publication Activity Database

    Bačáková, Lucie; Grausová, Ľubica; Vacík, Jiří; Lavrentiev, Vasyl; Blazewicz, S.; Fraczek, A.; Kromka, Alexander; Haenen, K.

    2011-01-01

    Roč. 3, č. 1 (2011), s. 99-109 ISSN 1941-4900 R&D Projects: GA AV ČR(CZ) IAAX00100902; GA MŠk(CZ) 2B06173; GA AV ČR(CZ) KAN400480701 Institutional research plan: CEZ:AV0Z50110509; CEZ:AV0Z10480505; CEZ:AV0Z10100521 Keywords : nanoscale surface roughness * electrical conductivity * osteoblasts * bone tissue engineering Subject RIV: EI - Biotechnology ; Bionics Impact factor: 0.528, year: 2011

  1. Adhesion of osteoblasts to a nanorough titanium implant surface

    Directory of Open Access Journals (Sweden)

    Gongadze E

    2011-08-01

    Full Text Available Ekaterina Gongadze1, Doron Kabaso2, Sebastian Bauer3, Tomaž Slivnik2, Patrik Schmuki3, Ursula van Rienen1, Aleš Iglič21Institute of General Electrical Engineering, University of Rostock, Rostock, Germany; 2Laboratory of Biophysics, Faculty of Electrical Engineering, University of Ljubljana, Ljubljana, Slovenia; 3Department of Materials Science, Friedrich-Alexander University of Erlangen-Nurenberg, Erlangen, GermanyAbstract: 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.Keywords: osteoblasts, nanostructures, adhesion, titanium implants, osteointegration

  2. In Vitro Evaluation the Influence of Glass-Ceramic Degradation Products on Osteoblast Cells.

    Directory of Open Access Journals (Sweden)

    Israa K. Sabree

    2016-03-01

    Full Text Available Regenerative medicine focuses on using biomaterials as three-dimensional (3D porous scaffolds, specifically designed to mimic the nature of host tissue and hence to promote cell growth and tissue regeneration. 3D bioactive glass-ceramic scaffolds are one of the most frequently studied types of scaffolds for bone tissue engineering because of their excellent bioactivity and potential for stimulating osteogenesis and angiogenesis. For such purposes, porous 3D 70%SiO2-30%CaO bioactive glass-ceramic scaffolds with three different pore sizes and identical porosity are used in present study to investigate In vitro, the effect of pore size on the degradation rate of scaffold which is achieved through examining changes in the composition of the immersion solution(SBF, simulated body fluid, and to investigate the action of released ions from the bioactive glass-ceramic scaffold during soaking process on osteoblast cells The results confirmed that all three scaffolds behaved in a similar manner and the ions release from the three scaffolds were of comparable concentration, which may be attributable to the identical porosity for all the scaffolds in addition to the using static immersion which delays ions diffusion. The pH of culture media increased from 7.6 to 8.2 after one day soaking. The optical microscopy images demonstrated that high ion concentration (Si, Ca, P in the culture medium could have a negative effect on the cells and induce cell death, while low concentration of ionic dissolution products induces osteoblast proliferation in dilute culture medium.

  3. Inhibition of fatty acid biosynthesis prevents adipocyte lipotoxicity on human osteoblasts in vitro

    Science.gov (United States)

    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

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

    International Nuclear Information System (INIS)

    Sato, Chieri; Iwasaki, Tsuyoshi; Kitano, Sachie; Tsunemi, Sachi; Sano, Hajime

    2012-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Shinsuke Kido

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

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

    NARCIS (Netherlands)

    Glass, D.A.; Bialek, P.; Ahn, J.D.; Starbuck, M.; Patel, M.S.; Clevers, J.C.; Taketo, M.M.; Long, F.; McMahon, A.P.; Lang, R.A.; Karsenty, G.

    2005-01-01

    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

  7. Beneficial Effects of Concentrated Growth Factors and Resveratrol on Human Osteoblasts In Vitro Treated with Bisphosphonates

    Directory of Open Access Journals (Sweden)

    Elisa Borsani

    2018-01-01

    Full Text Available Bisphosphonates are primary pharmacological agents against osteoclast-mediated bone loss and widely used in the clinical practice for prevention and treatment of a variety of skeletal conditions, such as low bone density and osteogenesis imperfecta, and pathologies, such as osteoporosis, malignancies metastatic to bone, Paget disease of bone, multiple myeloma, and hypercalcemia of malignancy. However, long-term bisphosphonate treatment is associated with pathologic conditions including osteonecrosis of the jaw, named BRONJ, which impaired bone regeneration process. Clinical management of BRONJ is controversy and one recent approach is the use of platelet concentrates, such as Concentrated Growth Factors, alone or together with biomaterials or antioxidants molecules, such as resveratrol. The aim of the present study was to investigate the in vitro effects of Concentrated Growth Factors and/or resveratrol on the proliferation and differentiation of human osteoblasts, treated or not with bisphosphonates. Human osteoblasts were stimulated for 3 days in complete medium and for 21 days in mineralization medium. At the end of the experimental period, the in vitro effect on osteoblast proliferation and differentiation was evaluated using different techniques such as MTT, ELISA for the quantification/detection of osteoprotegerin and bone morphogenetic protein-2, immunohistochemistry for sirtuin 1 and collagen type I, and the Alizarin Red S staining for the rate of mineralization. Results obtained showed that Concentrated Growth Factors and/or resveratrol significantly increased osteoblast proliferation and differentiation and that the cotreatment with Concentrated Growth Factors and resveratrol had a protective role on osteoblasts treated with bisphosphonates. In conclusion, these data suggest that this approach could be promised in the clinical management of BRONJ.

  8. Osteomacs interact with megakaryocytes and osteoblasts to regulate murine hematopoietic stem cell function.

    Science.gov (United States)

    Mohamad, Safa F; Xu, Linlin; Ghosh, Joydeep; Childress, Paul J; Abeysekera, Irushi; Himes, Evan R; Wu, Hao; Alvarez, Marta B; Davis, Korbin M; Aguilar-Perez, Alexandra; Hong, Jung Min; Bruzzaniti, Angela; Kacena, Melissa A; Srour, Edward F

    2017-12-12

    Networking between hematopoietic stem cells (HSCs) and cells of the hematopoietic niche is critical for stem cell function and maintenance of the stem cell pool. We characterized calvariae-resident osteomacs (OMs) and their interaction with megakaryocytes to sustain HSC function and identified distinguishing properties between OMs and bone marrow (BM)-derived macrophages. OMs, identified as CD45 + F4/80 + cells, were easily detectable (3%-5%) in neonatal calvarial cells. Coculture of neonatal calvarial cells with megakaryocytes for 7 days increased OM three- to sixfold, demonstrating that megakaryocytes regulate OM proliferation. OMs were required for the hematopoiesis-enhancing activity of osteoblasts, and this activity was augmented by megakaryocytes. Serial transplantation demonstrated that HSC repopulating potential was best maintained by in vitro cultures containing osteoblasts, OMs, and megakaryocytes. With or without megakaryocytes, BM-derived macrophages were unable to functionally substitute for neonatal calvarial cell-associated OMs. In addition, OMs differentiated into multinucleated, tartrate resistant acid phosphatase-positive osteoclasts capable of bone resorption. Nine-color flow cytometric analysis revealed that although BM-derived macrophages and OMs share many cell surface phenotypic similarities (CD45, F4/80, CD68, CD11b, Mac2, and Gr-1), only a subgroup of OMs coexpressed M-CSFR and CD166, thus providing a unique profile for OMs. CD169 was expressed by both OMs and BM-derived macrophages and therefore was not a distinguishing marker between these 2 cell types. These results demonstrate that OMs support HSC function and illustrate that megakaryocytes significantly augment the synergistic activity of osteoblasts and OMs. Furthermore, this report establishes for the first time that the crosstalk between OMs, osteoblasts, and megakaryocytes is a novel network supporting HSC function.

  9. Increased osteoblast density in the presence of novel calcium phosphate coated magnetic nanoparticles

    International Nuclear Information System (INIS)

    Pareta, Rajesh A; Webster, Thomas J; Taylor, Erik

    2008-01-01

    Bone diseases (including osteoporosis, osteoarthritis and bone cancer) are of great concern to the medical world. Drugs are available to treat such diseases, but often these drugs are not specifically targeted to the site of the disease and, thus, lack an immediate directed therapeutic effect. The optimal drug delivery system should enhance healthy bone growth with high specificity to the site of bone disease. It has been previously shown that magnetic nanoparticles can be directed in the presence of a magnetic field to any part of the body, allowing for site-specific drug delivery and possibly an immediate increase in bone density. The objective of the present study was to build off of this evidence and determine the density of osteoblasts (bone forming cells) in the presence of various uncoated and coated magnetic nanoparticles that could eventually be used in drug delivery applications. Results showed that some magnetic nanoparticles (specifically, γ-Fe 2 O 3 ) significantly promoted osteoblast density (that is, cells per well) after 5 and 8 days of culture compared to controls (no particles). These magnetic nanoparticles were further coated with calcium phosphate (CaP; the main inorganic component of bone) to tailor them for treating various bone diseases. The coatings were conducted in the presence of either bovine serum albumin (BSA) or citric acid (CA) to reduce magnetic nanoparticle agglomeration, a common problem resulting from the use of nanoparticles which decreases their effectiveness. Results with these coatings showed that magnetic nanoparticles, specifically (γ-Fe 2 O 3 ), coated in the presence of BSA significantly increased osteoblast density compared to controls after 1 day. In this manner, this study provided unexpected evidence that CaP-coated γ-Fe 2 O 3 magnetic nanoparticles increased osteoblast density (compared to no particles) and, thus, should be further studied to treat numerous bone diseases

  10. Understanding magnetic nanoparticle osteoblast receptor-mediated endocytosis using experiments and modeling

    International Nuclear Information System (INIS)

    Tran, Nhiem; Webster, Thomas J

    2013-01-01

    Iron oxide nanoparticles are promising candidates for controlling drug delivery through an external magnetic force to treat a wide range of diseases, including osteoporosis. Previous studies have demonstrated that in the presence of hydroxyapatite coated magnetite (Fe 3 O 4 ) nanoparticles, osteoblast (or bone forming cell) proliferation and long-term functions (such as calcium deposition) were significantly enhanced. Hydroxyapatite is the major inorganic component of bone. As a further attempt to understand why, in the current study, the uptake of such nanoparticles into osteoblasts was experimentally investigated and mathematically modeled. Magnetite nanoparticles were synthesized using a co-precipitation method and were coated with hydroxyapatite. A cellular uptake experiment at low temperatures indicated that receptor-mediated endocytosis contributed to the internalization of the magnetic nanoparticles into osteoblasts. A model was further developed to explain the uptake of magnetic nanoparticles into osteoblasts using receptor-mediated endocytosis. This model may explain the internalization of hydroxyapatite into osteoblasts to elevate intracellular calcium levels necessary to promote osteoblast functions to treat a wide range of orthopedic problems, including osteoporosis. (paper)

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Sophie Thiolloy

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

  13. Assessment of patient safety culture in primary care setting, Al-Mukala, Yemen.

    Science.gov (United States)

    Webair, Hana H; Al-Assani, Salwa S; Al-Haddad, Reema H; Al-Shaeeb, Wafa H; Bin Selm, Manal A; Alyamani, Abdulla S

    2015-10-13

    Patient safety culture in primary care is the first step to achieve high quality health care. This study aims to provide a baseline assessment of patient safety culture in primary care settings in Al-Mukala, Yemen as a first published study from a least developed country. A survey was conducted in primary healthcare centres and units in Al-Mukala District, Yemen. A comprehensive sample from the available 16 centres was included. An Arabic version of the Medical Office Survey on Patient Safety Culture was distributed to all health workers (110). Participants were physicians, nurses and administrative staff. The response rate from the participating centres was 71 %. (N = 78). The percent positive responses of the items is equal to the percentage of participants who answered positively. Composite scores were calculated by averaging the percent positive response on the items within a dimension. Positive safety culture was defined as 60 % or more positive responses on items or dimensions. Patient safety culture was perceived to be generally positive with the exception of the dimensions of 'Communication openness', 'Work pressure and pace' and 'Patient care tracking/follow-up', as the percent positive response of these dimensions were 58, 57, and 52 % respectively. Overall, positive rating on quality and patient safety were low (49 and 46 % respectively). Although patient safety culture in Al-Mukala primary care setting is generally positive, patient safety and quality rating were fairly low. Implementation of a safety and quality management system in Al-Mukala primary care setting are paramount. Further research is needed to confirm the applicability of the Medical Office Survey on Patient Safety Culture (MOSPSC) for Al-Mukala primary care.

  14. Potential of Osteoblastic Cells Derived from Bone Marrow and Adipose Tissue Associated with a Polymer/Ceramic Composite to Repair Bone Tissue.

    Science.gov (United States)

    Freitas, Gileade P; Lopes, Helena B; Almeida, Adriana L G; Abuna, Rodrigo P F; Gimenes, Rossano; Souza, Lucas E B; Covas, Dimas T; Beloti, Marcio M; Rosa, Adalberto L

    2017-09-01

    One of the tissue engineering strategies to promote bone regeneration is the association of cells and biomaterials. In this context, the aim of this study was to evaluate if cell source, either from bone marrow or adipose tissue, affects bone repair induced by osteoblastic cells associated with a membrane of poly(vinylidene-trifluoroethylene)/barium titanate (PVDF-TrFE/BT). Mesenchymal stem cells (MSC) were isolated from rat bone marrow and adipose tissue and characterized by detection of several surface markers. Also, both cell populations were cultured under osteogenic conditions and it was observed that MSC from bone marrow were more osteogenic than MSC from adipose tissue. The bone repair was evaluated in rat calvarial defects implanted with PVDF-TrFE/BT membrane and locally injected with (1) osteoblastic cells differentiated from MSC from bone marrow, (2) osteoblastic cells differentiated from MSC from adipose tissue or (3) phosphate-buffered saline. Luciferase-expressing osteoblastic cells derived from bone marrow and adipose tissue were detected in bone defects after cell injection during 25 days without difference in luciferin signal between cells from both sources. Corroborating the in vitro findings, osteoblastic cells from bone marrow combined with the PVDF-TrFE/BT membrane increased the bone formation, whereas osteoblastic cells from adipose tissue did not enhance the bone repair induced by the membrane itself. Based on these findings, it is possible to conclude that, by combining a membrane with cells in this rat model, cell source matters and that bone marrow could be a more suitable source of cells for therapies to engineer bone.

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

    Science.gov (United States)

    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.

  16. 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.

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Julia Matena

    2015-04-01

    Full Text Available 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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  20. A cultural congruence test for primary school students

    Directory of Open Access Journals (Sweden)

    Bayanova L. F.

    2016-12-01

    Full Text Available The study presented in this article relies on the principles of the cultural-historical theory, which defines cultural impact as the main driving force behind psychological development. Based on the assumption that culture is a set of normative situations, the study identifies rules that are typical for primary school students in big Russian cities. These rules are grouped into what we refer to as factors of cultural compliance, which ultimately can be seen as indicators of pupils’ cultural congruence. In specifying the cultural congruence of primary school students, we take into account not only the rules of school life but also the whole range of stable rules for children 7- to 10-years-old. Researchers at the Psychology Institute of the Higher University of the Chinese Academy of Science (Wang, Zhu, & Shi, 2011 call such rules usual or contextually usual. We include rules that govern the behavior of children who have cultural differences, so in this article we are talking about the rules that are typical for children of this age in Russia. The goal of the study was to develop a test to diagnose the level of cultural congruence. The test was exposed to psychometric evaluation for validity, reliability, and discriminatory power. Factor analysis by means of varimax rotation provided for calibration of the rules by consolidating them into factors. These factors underpin the test and include the categories social interaction, academic competence, regulation, obedience, self-service, and self-control. In accordance with the principles employed in psychology, the factors confirm the construct validity of the test in relation to children’s development when they are between 7 and 10 years old. The study confirms that learning is the main activity at this age by introducing a factor that brings together rules inherent in normative situations in the education process. The social setting for psychological development, viewed as a specific

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-06-22

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

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

    Science.gov (United States)

    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.

  3. Mechanisms involved in regulation of osteoclastic differentiation by mechanical stress-loaded osteoblasts

    International Nuclear Information System (INIS)

    Kaneuji, Takeshi; Ariyoshi, Wataru; Okinaga, Toshinori; Toshinaga, Akihiro; Takahashi, Tetsu; Nishihara, Tatsuji

    2011-01-01

    Highlights: → Effect of compressive force on osteoblasts were examined. → Compressive force induced OPG expression and suppressed osteoclastogenesis. → This enhancement of OPG is dependent on Wnt/Ca2+ signal pathway. -- Abstract: Mechanical stress is known to be important for regulation of bone turnover, though the detailed mechanisms are not fully understood. In the present study, we examined the effect of mechanical stress on osteoblasts using a novel compression model. Mouse osteoblastic MC3T3-E1 cells were embedded in three-dimensional (3D) gels and cultured with continuous compressive force (0-10.0 g/cm 2 ) for 48 h, and the conditioned medium were collected. RAW264.7 cells were then incubated with the conditioned medium for various times in the presence of receptor activator of nuclear factor-κB ligand (RANKL). Conditioned medium was found to inhibit the differentiation of RAW264.7 cells into osteoclasts induced by RANKL via down-regulation of the expression of tumor necrosis factor receptor-associated factor 6 (TRAF6), phosphorylation of IκBα, and nuclear translocation of p50 and p65. Interestingly, the conditioned medium also had a high level of binding activity to RANKL and blocked the binding of RANK to RANKL. Furthermore, the binding activity of conditioned medium to RANKL was reduced when the 3D gel was supplemented with KN-93, an inhibitor of non-canonical Wnt/Ca 2+ pathway. In addition, expression level of osteoprotegerin (OPG) mRNA was increased in time- and force-dependent manners, and remarkably suppressed by KN-93. These results indicate that osteoblastic cells subjected to mechanical stress produce OPG, which binds to RANKL. Furthermore, this binding activity strongly inhibited osteoclastogenesis through suppression of TRAF6 and the nuclear factor-kappa B (NF-κB) signaling pathway, suggesting that enhancement of OPG expression induced by mechanical stress is dependent on non-canonical Wnt/Ca 2+ pathway.

  4. Mechanisms involved in regulation of osteoclastic differentiation by mechanical stress-loaded osteoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Kaneuji, Takeshi [Division of Oral and Maxillofacial Reconstructive Surgery, Department of Oral and Maxillofacial Surgery, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu 803-8580 (Japan); Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu 803-8580 (Japan); Ariyoshi, Wataru; Okinaga, Toshinori; Toshinaga, Akihiro [Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu 803-8580 (Japan); Takahashi, Tetsu [Division of Oral and Maxillofacial Reconstructive Surgery, Department of Oral and Maxillofacial Surgery, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu 803-8580 (Japan); Oral Bioresearch Center, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu 803-8580 (Japan); Nishihara, Tatsuji, E-mail: tatsujin@kyu-dent.ac.jp [Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu 803-8580 (Japan); Oral Bioresearch Center, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu 803-8580 (Japan)

    2011-04-29

    Highlights: {yields} Effect of compressive force on osteoblasts were examined. {yields} Compressive force induced OPG expression and suppressed osteoclastogenesis. {yields} This enhancement of OPG is dependent on Wnt/Ca2+ signal pathway. -- Abstract: Mechanical stress is known to be important for regulation of bone turnover, though the detailed mechanisms are not fully understood. In the present study, we examined the effect of mechanical stress on osteoblasts using a novel compression model. Mouse osteoblastic MC3T3-E1 cells were embedded in three-dimensional (3D) gels and cultured with continuous compressive force (0-10.0 g/cm{sup 2}) for 48 h, and the conditioned medium were collected. RAW264.7 cells were then incubated with the conditioned medium for various times in the presence of receptor activator of nuclear factor-{kappa}B ligand (RANKL). Conditioned medium was found to inhibit the differentiation of RAW264.7 cells into osteoclasts induced by RANKL via down-regulation of the expression of tumor necrosis factor receptor-associated factor 6 (TRAF6), phosphorylation of I{kappa}B{alpha}, and nuclear translocation of p50 and p65. Interestingly, the conditioned medium also had a high level of binding activity to RANKL and blocked the binding of RANK to RANKL. Furthermore, the binding activity of conditioned medium to RANKL was reduced when the 3D gel was supplemented with KN-93, an inhibitor of non-canonical Wnt/Ca{sup 2+} pathway. In addition, expression level of osteoprotegerin (OPG) mRNA was increased in time- and force-dependent manners, and remarkably suppressed by KN-93. These results indicate that osteoblastic cells subjected to mechanical stress produce OPG, which binds to RANKL. Furthermore, this binding activity strongly inhibited osteoclastogenesis through suppression of TRAF6 and the nuclear factor-kappa B (NF-{kappa}B) signaling pathway, suggesting that enhancement of OPG expression induced by mechanical stress is dependent on non-canonical Wnt

  5. MiR-9 is overexpressed in spontaneous canine osteosarcoma and promotes a metastatic phenotype including invasion and migration in osteoblasts and osteosarcoma cell lines

    International Nuclear Information System (INIS)

    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.

    2016-01-01

    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

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

    Science.gov (United States)

    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.

  7. Differences in otosclerotic and normal human stapedial osteoblast properties are normalized by alendronate in vitro.

    Science.gov (United States)

    Gronowicz, Gloria; Richardson, Yvonne L; Flynn, John; Kveton, John; Eisen, Marc; Leonard, Gerald; Aronow, Michael; Rodner, Craig; Parham, Kourosh

    2014-10-01

    Identify and compare phenotypic properties of osteoblasts from patients with otosclerosis (OSO), normal bones (HOB), and normal stapes (NSO) to determine a possible cause for OSO hypermineralization and assess any effects of the bisphosphonate, alendronate. OSO (n = 11), NSO (n = 4), and HOB (n = 13) cultures were assayed for proliferation, adhesion, mineralization, and gene expression with and without 10(-10)M-10(-8)M alendronate. Academic hospital. Cultures were matched for age, sex, and passage number. Cell attachment and proliferation + alendronate were determined by Coulter counting cells and assaying tritiated thymidine uptake, respectively. At 7, 14, and 21 days of culture + alendronate, calcium content and gene expression by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were determined. OSO had significantly more cells adhere but less proliferation than NSO or HOB. Calcification was significantly increased in OSO compared to HOB and NSO. NSO and HOB had similar cell adhesion and proliferation rates. A dose-dependent effect of alendronate on OSO adhesion, proliferation, and mineralization was found, resulting in levels equal to NSO and HOB. All cultures expressed osteoblast-specific genes such as RUNX2, alkaline phosphatase, type I collagen, and osteocalcin. However, osteopontin was dramatically reduced, 9.4-fold at 14 days, in OSO compared to NSO. Receptor activator of nuclear factor κB ligand/osteoprotegerin (RANKL/OPG), important in bone resorption, was elevated in OSO with decreased levels of OPG levels. Alendronate had little effect on gene expression in HOB but in OSO increased osteopontin levels and decreased RANKL/OPG. OSO cultures displayed properties of hypermineralization due to decreased osteopontin (OPN) and also had increased RANKL/OPG, which were normalized by alendronate. © American Academy of Otolaryngology—Head and Neck Surgery Foundation 2014.

  8. Science-Based Thematic Cultural Art Learning in Primary School (2013 Curriculum

    Directory of Open Access Journals (Sweden)

    Warih Handayaningrum

    2016-12-01

    Full Text Available This study is aimed at discussing the development result of thematic cultural art subject’s learning material based on science for primary school (2013 curriculum. This study is expected to inspire teacher to develop learning material that may explore artworks exist in our living environment (based on the context of children’s environment. This study applies steps in developmental research collaboration by Borg & Gall (1989 and Puslitjaknov (2008 to create the product. The development stages comprise observation in several primary schools in Surabaya, Gresik, and Sidoarjo that has implemented 2013 curriculum that is followed up by stages of development. Furthermore, prototype of cultural and art thematic learning material development results are verified by learning material experts, material expert, primary school teacher, and revised afterwards. The result of this research development is a set of teacher and student books. Science-based cultural art here means cultural art learning as the main medium to introduce local culture products (music, drawing, dance, and drama by integrating mathematics, sciences, Bahasa Indonesia, and local language subjects. Cultural art products in the form of dance, music, drawing, dramas will help children to understand a simple mathematical concept, such as: two-dimensional figure, geometry, comparing or estimating longer-shorter, smaller-bigger, or more-less.

  9. Osteoblast response to oxygen functionalised plasma polymer surfaces

    International Nuclear Information System (INIS)

    Kelly, Jonathan M.

    2001-01-01

    Thin organic films with oxygen-carbon functionalities were deposited from plasmas containing vapour of the small organic compounds: allyI alcohol, methyl vinyl ketone and acrylic acid with octadiene. Characterisation of the deposits was carried out using X-ray photoelectron spectroscopy, in conjunction with chemical derivatisation, and this showed that plasma polymers retained high levels of original monomer functionality when the plasmas were sustained at low power for a given monomer vapour flow rate. High levels of attachment of rat osteosarcoma (ROS 17/2.8) cells were observed on surfaces that had high concentrations of hydroxyl and carbonyl functionalities and intermediate concentrations of carboxyl functionality. Cells did not attach to the octadiene plasma polymer. Cell attachment to carboxyl and methyl functionalised self-assembled monolayers increased with increasing concentration of surface carboxyl groups. Adsorption of the extracellular matrix protein fibronectin to acrylic acid/octadiene plasma copolymers was studied by enzyme linked immunosorbent assays and by I 125 radiolabelling. Fibronectin adsorbed in largest amounts to surfaces with intermediate concentrations of carboxyl functionality. Spreading of ROS cells and rat bone marrow stromal cells (BMSC) was characterised by computer image analysis. Cell spreading in media containing 10% serum, on a surface deposited from a plasma of 5 O/o acrylic acid was much greater than on the octadiene plasma polymer while most extensive cell spreading was observed on these surfaces when preadsorbed with fibronectin. Growth (proliferation) of BMSC was assessed over nine days and was found to be faster on an 50% acrylic acid plasma polymer than on tissue culture polystyrene or a hydrocarbon plasma polymer, though cell growth was fastest on fibronectin precoated substrates. Expression of cellular alkaline phosphatase, collagen and calcium reached similar levels on the 50% acrylic acid plasma polymer, tissue culture

  10. Osteoblast response to oxygen functionalised plasma polymer surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Kelly, Jonathan M

    2001-07-01

    culture polystyrene and on fibronectin precoated substrates, showing that osteoblast differentiation was promoted by each of these surfaces, but osteoblast differentiation was not demonstrated by cells on the octadiene plasma polymer. (author)

  11. Effects of space-relevant radiation on pre-osteoblasts

    International Nuclear Information System (INIS)

    Hu, Yueyuan

    2014-01-01

    Until now limited research has been conducted to address the mechanisms leading ionizing radiation exposure induced bone loss. This is relevant for cancer radiotherapy and human spaceflight. Exposure to radiation can result in elevated bone fracture risk in patients receiving cancer radiotherapy. In human spaceflight, astronauts are exposed to space radiation which is a very complex mixture consisting primarily of high-energy charged particles. Osteoblasts are of mesenchymal origin and responsible for creating and maintaining skeletal architecture; these cells produce extracellular matrix proteins and regulators of matrix mineralization during initial bone formation and later bone remodeling. The aim of this work was to investigate the effects of ionizing radiation on pre-osteoblasts including cellular survival, cell cycle regulation and differentiation modification. Experiments with the pre-osteoblast cell line OCT-1 and the mesenchymal stem cell line C3H10T1/2 showed that radiation cell killing depends on dose and linear energy transfer (LET) and is most effective at an LET of ∝150 keV/μm. High-LET radiation has a much more pronounced ability to induce cell cycle arrest in the G2/M phase. After both X-rays and heavy ions exposure, expression of the cell cycle regulator CDKN1A was significantly up-regulated in a dose-dependent manner. The findings suggest that cell cycle regulation is more sensitive to high-LET radiation than cell survival, which is not solely regulated through elevated CDKN1A expression. Radiation exposure enhances osteoblastic differentiation and maturation, and mediates Runx2 and TGF-β1 expression during early differentiation of pre-osteoblasts. Osteogenic differentiation did not alter cellular radiosensitivity, DNA repair of radiation-induced damages and the effects of radiation on proliferation. Further experiments are needed to elucidate possible synergistic effects of microgravity and radiation on osteoblast differentiation. This may

  12. Effects of space-relevant radiation on pre-osteoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Yueyuan

    2014-02-12

    Until now limited research has been conducted to address the mechanisms leading ionizing radiation exposure induced bone loss. This is relevant for cancer radiotherapy and human spaceflight. Exposure to radiation can result in elevated bone fracture risk in patients receiving cancer radiotherapy. In human spaceflight, astronauts are exposed to space radiation which is a very complex mixture consisting primarily of high-energy charged particles. Osteoblasts are of mesenchymal origin and responsible for creating and maintaining skeletal architecture; these cells produce extracellular matrix proteins and regulators of matrix mineralization during initial bone formation and later bone remodeling. The aim of this work was to investigate the effects of ionizing radiation on pre-osteoblasts including cellular survival, cell cycle regulation and differentiation modification. Experiments with the pre-osteoblast cell line OCT-1 and the mesenchymal stem cell line C3H10T1/2 showed that radiation cell killing depends on dose and linear energy transfer (LET) and is most effective at an LET of ∝150 keV/μm. High-LET radiation has a much more pronounced ability to induce cell cycle arrest in the G2/M phase. After both X-rays and heavy ions exposure, expression of the cell cycle regulator CDKN1A was significantly up-regulated in a dose-dependent manner. The findings suggest that cell cycle regulation is more sensitive to high-LET radiation than cell survival, which is not solely regulated through elevated CDKN1A expression. Radiation exposure enhances osteoblastic differentiation and maturation, and mediates Runx2 and TGF-β1 expression during early differentiation of pre-osteoblasts. Osteogenic differentiation did not alter cellular radiosensitivity, DNA repair of radiation-induced damages and the effects of radiation on proliferation. Further experiments are needed to elucidate possible synergistic effects of microgravity and radiation on osteoblast differentiation. This may

  13. Establishment of primary cell culture from the temperate symbiotic cnidarian, Anemonia viridis.

    Science.gov (United States)

    Barnay-Verdier, Stéphanie; Dall'osso, Diane; Joli, Nathalie; Olivré, Juliette; Priouzeau, Fabrice; Zamoum, Thamilla; Merle, Pierre-Laurent; Furla, Paola

    2013-10-01

    The temperate symbiotic sea anemone Anemonia viridis, a member of the Cnidaria phylum, is a relevant experimental model to investigate the molecular and cellular events involved in the preservation or in the rupture of the symbiosis between the animal cells and their symbiotic microalgae, commonly named zooxanthellae. In order to increase research tools for this model, we developed a primary culture from A. viridis animal cells. By adapting enzymatic dissociation protocols, we isolated animal host cells from a whole tentacle in regeneration state. Each plating resulted in a heterogeneous primary culture consisted of free zooxanthellae and many regular, small rounded and adherent cells (of 3-5 μm diameter). Molecular analyses conducted on primary cultures, maintained for 2 weeks, confirmed a specific signature of A. viridis cells. Further serial dilutions and micromanipulation allowed us to obtain homogenous primary cultures of the small rounded cells, corresponding to A. viridis "epithelial-like cells". The maintenance and the propagation over a 4 weeks period of primary cells provide, for in vitro cnidarian studies, a preliminary step for further investigations on cnidarian cellular pathways notably in regard to symbiosis interactions.

  14. Adiponectin stimulates human osteoblasts proliferation and differentiation via the MAPK signaling pathway

    International Nuclear Information System (INIS)

    Luo Xianghang; Guo Lijuan; Yuan Lingqing; Xie Hui; Zhou Houde; Wu Xianping; Liao Eryuan

    2005-01-01

    Adipocytes can highly and specifically express adiponectin, and the adiponectin receptor (AdipoR) has been detected in bone-forming cells. The present study was undertaken to investigate the action of adiponectin on osteoblast proliferation and differentiation. AdipoR1 protein was detected in human osteoblasts. Adiponectin promoted osteoblast proliferation and resulted in a dose- and time-dependent increase in alkaline phosphatase (ALP) activity, osteocalcin and type I collagen production, and an increase in mineralized matrix. Suppression of AdipoR1 with small-interfering RNA (siRNA) abolished the adiponectin-induced cell proliferation and ALP expression. Adiponectin induces activation of p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal Kinase (JNK), but not ERK1/2 in osteoblasts, and these effects were blocked by suppression of AdipoR1 with siRNA. Furthermore, pretreatment of osteoblasts with the JNK inhibitor SP600125 abolished the adiponectin-induced cell proliferation. p38 inhibitor SB203580 blocked the adiponectin-induced ALP activity. These data indicate that adiponectin induces human osteoblast proliferation and differentiation, and the proliferation response is mediated by the AdipoR/JNK pathway, while the differentiation response is mediated via the AdipoR/p38 pathway. These findings suggest that osteoblasts are the direct targets of adiponectin

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

    Science.gov (United States)

    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

  16. Pharmacological activation of aldehyde dehydrogenase 2 promotes osteoblast differentiation via bone morphogenetic protein-2 and induces bone anabolic effect

    Energy Technology Data Exchange (ETDEWEB)

    Mittal, Monika; Pal, Subhashis; China, Shyamsundar Pal; Porwal, Konica [Division of Endocrinology and Centre for Research in Anabolic Skeletal Targets in Health and Illness (ASTHI), CSIR-Central Drug Research Institute, Lucknow 226031 (India); Dev, Kapil [Division of Medicinal and Process Chemistry, CSIR-Central Drug Research Institute, Lucknow 226031 (India); Shrivastava, Richa [Division of Toxicology, CSIR-Central Drug Research Institute, Lucknow 226031 (India); Raju, Kanumuri Siva Rama; Rashid, Mamunur [Pharmaceutics Division, CSIR-Central Drug Research Institute, Lucknow 226031 (India); Trivedi, Arun Kumar; Sanyal, Sabyasachi [Biochemistry Division, CSIR-Central Drug Research Institute, Lucknow 226031 (India); Wahajuddin, Muhammad [Pharmaceutics Division, CSIR-Central Drug Research Institute, Lucknow 226031 (India); Bhaduria, Smrati [Division of Toxicology, CSIR-Central Drug Research Institute, Lucknow 226031 (India); Maurya, Rakesh [Division of Medicinal and Process Chemistry, CSIR-Central Drug Research Institute, Lucknow 226031 (India); Chattopadhyay, Naibedya, E-mail: n_chattopadhyay@cdri.res.in [Division of Endocrinology and Centre for Research in Anabolic Skeletal Targets in Health and Illness (ASTHI), CSIR-Central Drug Research Institute, Lucknow 226031 (India)

    2017-02-01

    Aldehyde dehydrogenases (ALDHs) are a family of enzymes involved in detoxifying aldehydes. Previously, we reported that an ALDH inhibitor, disulfiram caused bone loss in rats and among ALDHs, osteoblast expressed only ALDH2. Loss-of-function mutation in ALDH2 gene is reported to cause bone loss in humans which suggested its importance in skeletal homeostasis. We thus studied whether activating ALDH2 by N-(1, 3-benzodioxol-5-ylmethyl)-2, 6-dichlorobenzamide (alda-1) had osteogenic effect. We found that alda-1 increased and acetaldehyde decreased the differentiation of rat primary osteoblasts and expressions of ALDH2 and bone morphogenetic protein-2 (BMP-2). Silencing ALDH2 in osteoblasts abolished the alda-1 effects. Further, alda-1 attenuated the acetaldehyde-induced lipid-peroxidation and oxidative stress. BMP-2 is essential for bone regeneration and alda-1 increased its expression in osteoblasts. We then showed that alda-1 (40 mg/kg dose) augmented bone regeneration at the fracture site with concomitant increase in BMP-2 protein compared with control. The osteogenic dose (40 mg/kg) of alda-1 attained a bone marrow concentration that was stimulatory for osteoblast differentiation, suggesting that the tissue concentration of alda-1 matched its pharmacologic effect. In addition, alda-1 promoted modeling-directed bone growth and peak bone mass achievement, and increased bone mass in adult rats which reiterated its osteogenic effect. In osteopenic ovariectomized (OVX) rats, alda-1 reversed trabecular osteopenia with attendant increase in serum osteogenic marker (procollagen type I N-terminal peptide) and decrease in oxidative stress. Alda-1 has no effect on liver and kidney function. We conclude that activating ALDH2 by alda-1 had an osteoanabolic effect involving increased osteoblastic BMP-2 production and decreased OVX-induced oxidative stress. - Highlights: • Alda-1 induced osteoblast differentiation that involved upregulation of ALDH2 and BMP-2 • Alda-1

  17. Nanostructured magnesium has fewer detrimental effects on osteoblast function

    Directory of Open Access Journals (Sweden)

    Weng L

    2013-05-01

    Full Text Available Lucy Weng, Thomas J Webster School of Engineering and Department of Orthopedics, Brown University, Providence, RI, USA Abstract: 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. Keywords: nanostructured magnesium, degradation, detrimental effects, osteoblasts

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

    International Nuclear Information System (INIS)

    Tasat, D.R.; Orona, N.S.; Mandalunis, P.M.; Cabrini, R.L.; Ubios, A.M.

    2007-01-01

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

  19. Surface Modification of NiTi Alloy via Cathodic Plasma Electrolytic Deposition and its Effect on Ni Ion Release and Osteoblast Behaviors

    International Nuclear Information System (INIS)

    Yan Ying; Cai Kaiyong; Yang Weihu; Liu Peng

    2013-01-01

    To reduce Ni ion release and improve biocompatibility of NiTi alloy, the cathodic plasma electrolytic deposition (CPED) technique was used to fabricate ceramic coating onto a NiTi alloy surface. The formation of a coating with a rough and micro-textured surface was confirmed by X-ray diffraction, scanning electron microscopy, and energy-dispersive X-ray spectroscopy, respectively. An inductively coupled plasma mass spectrometry test showed that the formed coating significantly reduced the release of Ni ions from the NiTi alloy in simulated body fluid. The influence of CPED treated NiTi substrates on the biological behaviors of osteoblasts, including cell adhesion, cell viability, and osteogenic differentiation function (alkaline phosphatase), was investigated in vitro. Immunofluorescence staining of nuclei revealed that the CPED treated NiTi alloy was favorable for cell growth. Osteoblasts on CPED modified NiTi alloy showed greater cell viability than those for the native NiTi substrate after 4 and 7 days cultures. More importantly, osteoblasts cultured onto a modified NiTi sample displayed significantly higher differentiation levels of alkaline phosphatase. The results suggested that surface functionalization of NiTi alloy with ceramic coating via the CPED technique was beneficial for cell proliferation and differentiation. The approach presented here is useful for NiTi implants to enhance bone osteointegration and reduce Ni ion release in vitro

  20. Surface Modification of NiTi Alloy via Cathodic Plasma Electrolytic Deposition and its Effect on Ni Ion Release and Osteoblast Behaviors

    Science.gov (United States)

    Yan, Ying; Cai, Kaiyong; Yang, Weihu; Liu, Peng

    2013-07-01

    To reduce Ni ion release and improve biocompatibility of NiTi alloy, the cathodic plasma electrolytic deposition (CPED) technique was used to fabricate ceramic coating onto a NiTi alloy surface. The formation of a coating with a rough and micro-textured surface was confirmed by X-ray diffraction, scanning electron microscopy, and energy-dispersive X-ray spectroscopy, respectively. An inductively coupled plasma mass spectrometry test showed that the formed coating significantly reduced the release of Ni ions from the NiTi alloy in simulated body fluid. The influence of CPED treated NiTi substrates on the biological behaviors of osteoblasts, including cell adhesion, cell viability, and osteogenic differentiation function (alkaline phosphatase), was investigated in vitro. Immunofluorescence staining of nuclei revealed that the CPED treated NiTi alloy was favorable for cell growth. Osteoblasts on CPED modified NiTi alloy showed greater cell viability than those for the native NiTi substrate after 4 and 7 days cultures. More importantly, osteoblasts cultured onto a modified NiTi sample displayed significantly higher differentiation levels of alkaline phosphatase. The results suggested that surface functionalization of NiTi alloy with ceramic coating via the CPED technique was beneficial for cell proliferation and differentiation. The approach presented here is useful for NiTi implants to enhance bone osseointegration and reduce Ni ion release in vitro.

  1. Primary culture of human Schwann and schwannoma cells: improved and simplified protocol.

    Science.gov (United States)

    Dilwali, Sonam; Patel, Pratik B; Roberts, Daniel S; Basinsky, Gina M; Harris, Gordon J; Emerick, Kevin S; Stankovic, Konstantina M

    2014-09-01

    Primary culture of human Schwann cells (SCs) and vestibular schwannoma (VS) cells are invaluable tools to investigate SC physiology and VS pathobiology, and to devise effective pharmacotherapies against VS, which are sorely needed. However, existing culture protocols, in aiming to create robust, pure cultures, employ methods that can lead to loss of biological characteristics of the original cells, potentially resulting in misleading biological findings. We have developed a minimally manipulative method to culture primary human SC and VS cells, without the use of selective mitogens, toxins, or time-consuming and potentially transformative laboratory techniques. Schwann cell purity was quantified longitudinally using S100 staining in SC cultures derived from the great auricular nerve and VS cultures followed for 7 and 12 weeks, respectively. SC cultures retained approximately ≥85% purity for 2 weeks. VS cultures retained approximately ≥80% purity for the majority of the span of 12 weeks, with maximal purity of 87% at 2 weeks. The VS cultures showed high level of biological similarity (68% on average) to their respective parent tumors, as assessed using a protein array featuring 41 growth factors and receptors. Apoptosis rate in vitro negatively correlated with tumor volume. Our results, obtained using a faster, simplified culturing method than previously utilized, indicate that highly pure, primary human SC and VS cultures can be established with minimal manipulation, reaching maximal purity at 2 weeks of culture. The VS cultures recapitulate the parent tumors' biology to a great degree, making them relevant models to investigate VS pathobiology. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Enhanced osteoblast responses to poly ether ether ketone surface modified by water plasma immersion ion implantation.

    Science.gov (United States)

    Wang, Heying; Lu, Tao; Meng, Fanhao; Zhu, Hongqin; Liu, Xuanyong

    2014-05-01

    Poly ether ether ketone (PEEK) offers a set of characteristics superior for human implants; however, its application is limited by the bio-inert surface property. In this work, PEEK surface was modified using single step plasma immersion ion implantation (PIII) treatment with a gas mixture of water vapor as a plasma resource and argon as an ionization assistant. Field emission scanning electron microscopy, atomic force microscopy and X-ray photoelectron spectroscopy were used to investigate the microstructure and composition of the modified PEEK surface. The water contact angle and zeta-potential of the surfaces were also measured. Osteoblast precursor cells MC3T3-E1 and rat bone mesenchymal stem cells were cultured on the PEEK samples to evaluate their cytocompatibility. The obtained results show that the hydroxyl groups as well as a "ravined structure" are constructed on water PIII modified PEEK. Compared with pristine PEEK, the water PIII treated PEEK is more favorable for osteoblast adhesion, spreading and proliferation, besides, early osteogenic differentiation indicated by the alkaline phosphatase activity is also up-regulated. Our study illustrates enhanced osteoblast responses to the PEEK surface modified by water PIII, which gives positive information in terms of future biomedical applications. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    International Nuclear Information System (INIS)

    Tang Lin; Lin Zhu; Li Yongming

    2006-01-01

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

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

    Science.gov (United States)

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

    2003-01-01

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

  5. Co-Culture with Human Osteoblasts and Exposure to Extremely Low Frequency Pulsed Electromagnetic Fields Improve Osteogenic Differentiation of Human Adipose-Derived Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Sabrina Ehnert

    2018-03-01

    Full Text Available Human adipose-derived mesenchymal stem cells (Ad-MSCs have been proposed as suitable option for cell-based therapies to support bone regeneration. In the bone environment, Ad-MSCs will receive stimuli from resident cells that may favor their osteogenic differentiation. There is recent evidence that this process can be further improved by extremely low frequency pulsed electromagnetic fields (ELF-PEMFs. Thus, the project aimed at (i investigating whether co-culture conditions of human osteoblasts (OBs and Ad-MSCs have an impact on their proliferation and osteogenic differentiation; (ii whether this effect can be further improved by repetitive exposure to two specific ELF-PEMFs (16 and 26 Hz; (iii and the effect of these ELF-PEMFs on human osteoclasts (OCs. Osteogenic differentiation was improved by co-culturing OBs and Ad-MSCs when compared to the individual mono-cultures. An OB to Ad-MSC ratio of 3:1 had best effects on total protein content, alkaline phosphatase (AP activity, and matrix mineralization. Osteogenic differentiation was further improved by both ELF-PEMFs investigated. Interestingly, only repetitive exposure to 26 Hz ELF-PEMF increased Trap5B activity in OCs. Considering this result, a treatment with gradually increasing frequency might be of interest, as the lower frequency (16 Hz could enhance bone formation, while the higher frequency (26 Hz could enhance bone remodeling.

  6. A comparison of the cytotoxicity and proinflammatory cytokine production of EndoSequence root repair material and ProRoot mineral trioxide aggregate in human osteoblast cell culture using reverse-transcriptase polymerase chain reaction.

    Science.gov (United States)

    Ciasca, Maria; Aminoshariae, Anita; Jin, Ge; Montagnese, Thomas; Mickel, Andre

    2012-04-01

    The purpose of this study was to compare the cytotoxicity and cytokine expression profiles of EndoSequence Root Repair Material (ERRM; Brasseler, Savannah, GA) putty, ERRM flowable, and ProRoot mineral trioxide aggregate (MTA; Dentsply Tulsa Dental, Johnson City, TN) using osteoblast cells (MG-63). Four millimeters in diameter of each material was placed in the center of a 6-well culture plate, and a 2-mL suspension (10(5) cells/mL) of human osteoblasts was seeded in each well. Photomicrograph images were used to evaluate cytotoxicity as evidenced by the lack of osteoblast cell growth in relation to the materials with AH-26 (Dentsply Tulsa Dental) as the positive control. In addition, reverse-transcriptase polymerase chain reaction (RT-PCR) was used to evaluate the expression of interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor-α (TNF-α). Cytokine expression of MG-63 cells upon lipopolysaccharide treatment was used as controls. RT-PCR results were normalized by the expression of the housekeeping gene β-actin and were used to measure cytokine expression. Statistical analysis was performed using analysis of variance. Results showed that ERRM putty and MTA exhibited minimal levels of cytotoxicity; however, ERRM was slightly more cytotoxic although not statistically significant. The expression of IL-1β, IL-6, and IL-8 was detected in all samples with minimal TNF-α expression. We concluded that ERRM and MTA showed similar cytotoxicity and cytokine expressions. Copyright © 2012 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  7. Smurf1 Inhibits Osteoblast Differentiation, Bone Formation, and Glucose Homeostasis through Serine 148

    Directory of Open Access Journals (Sweden)

    Junko Shimazu

    2016-04-01

    Full Text Available The E3 ubiquitin ligase Smurf1 targets the master regulator of osteoblast differentiation, Runx2, for degradation, yet the function of Smurf1, if any, during osteoblast differentiation in vivo is ill defined. Here, we show that Smurf1 prevents osteoblast differentiation by decreasing Runx2 accumulation in osteoblasts. Remarkably, mice harboring a substitution mutation at serine 148 (S148 in Smurf1 that prevents its phosphorylation by AMPK (Smurf1ki/ki display a premature osteoblast differentiation phenotype that is equally severe as that of Smurf1−/− mice, as well as a high bone mass, and are also hyperinsulinemic and hypoglycemic. Consistent with the fact that Smurf1 targets the insulin receptor for degradation, there is, in Smurf1ki/ki mice, an increase in insulin signaling in osteoblasts that triggers a rise in the circulating levels of osteocalcin, a hormone that favors insulin secretion. These results identify Smurf1 as a determinant of osteoblast differentiation during the development of bone formation and glucose homeostasis post-natally and demonstrate the necessity of S148 for these functions.

  8. Notch Signaling in Prostate Cancer Cells Promotes Osteoblastic Metastasis

    Science.gov (United States)

    2017-06-01

    information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this...function and number while inducing osteoblast proliferation. Our results suggest that Notch signaling from cancer cells promotes osteoblastic...Participants and other collaborating organizations: I initiated collaboration with Dr. Evan Keller at University of Michigan to interrogate PCa bone

  9. Osteoblastic response to pectin nanocoating on titanium surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Gurzawska, Katarzyna, E-mail: kagu@sund.ku.dk [Research Center for Ageing and Osteoporosis, Departments of Medicine and Diagnostics, Copenhagen University Hospital Glostrup, Ndr. Ringvej 57, 2600 Glostrup (Denmark); Institute of Odontology, Faculty of Health and Medical Sciences, University of Copenhagen, Nørre Allé 20, 2200 Copenhagen N (Denmark); Svava, Rikke [Department of Plant Environment Sciences, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C (Denmark); Copenhagen Center for Glycomics, Institute for Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen N (Denmark); Yihua, Yu; Haugshøj, Kenneth Brian [Microtechnology and Surface Analysis, Danish Technological Institute, Gregersensvej 8, 2630 Taastrup (Denmark); Dirscherl, Kai [Dansk Fundamental Metrologi A/S, Matematiktorvet 307, 2800 Lyngby (Denmark); Levery, Steven B. [Copenhagen Center for Glycomics, Institute for Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen N (Denmark); Byg, Inge [Department of Plant Environment Sciences, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C (Denmark); Damager, Iben [Novozymes A/S, Krogshoejvej 36, 2880 Bagsvaerd (Denmark); Nielsen, Martin W. [Department of Systems Biology, Technical University of Denmark, Matematiktorvet, Building 301, Kgs. Lyngby DK-2800 (Denmark); Jørgensen, Bodil [Department of Plant Environment Sciences, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C (Denmark); Jørgensen, Niklas Rye [Research Center for Ageing and Osteoporosis, Departments of Medicine and Diagnostics, Copenhagen University Hospital Glostrup, Ndr. Ringvej 57, 2600 Glostrup (Denmark); and others

    2014-10-01

    Osseointegration of titanium implants can be improved by organic and inorganic nanocoating of the surface. The aim of our study was to evaluate the effect of organic nanocoating of titanium surface with unmodified and modified pectin Rhamnogalacturonan-Is (RG-Is) isolated from potato and apple with respect to surface properties and osteogenic response in osteoblastic cells. Nanocoatings on titanium surfaces were evaluated by scanning electron microscopy, contact angle measurements, atomic force microscopy, and X-ray photoelectron spectroscopy. The effect of coated RG-Is on cell adhesion, cell viability, bone matrix formation and mineralization was tested using SaOS-2 cells. Nanocoating with pectin RG-Is affected surface properties and in consequence changed the environment for cellular response. The cells cultured on surfaces coated with RG-Is from potato with high content of linear 1.4-linked galactose produced higher level of mineralized matrix compared with control surfaces and surfaces coated with RG-I with low content of linear 1.4-linked galactose. The study showed that the pectin RG-Is nanocoating not only changed chemical and physical titanium surface properties, but also specific coating with RG-Is containing high amount of galactan increased mineralized matrix formation of osteoblastic cells in vitro. - Highlights: • Surface nanocoating with plant-derived Rhamnogalacturonan-I (RG-I) is proposed. • Titanium surface became more hydrophilic after RG-Is nanocoating. • RG-Is with high galactose content resulted in high level of mineralized matrix. • RG-I is a new candidate for improvement of bone healing and osseointegration.

  10. Intrinsically superparamagnetic Fe-hydroxyapatite nanoparticles positively influence osteoblast-like cell behaviour

    Science.gov (United States)

    2012-01-01

    Background Superparamagnetic nanoparticles (MNPs) have been progressively explored for their potential in biomedical applications and in particular as a contrast agent for diagnostic imaging, for magnetic drug delivery and more recently for tissue engineering applications. Considering the importance of having safe MNPs for such applications, and the essential role of iron in bone remodelling, this study developed and analysed novel biocompatible and bioreabsorbable superparamagnetic nanoparticles, that avoid the use of poorly tolerated magnetite based nanoparticles, for bone tissue engineering applications. Results MNPs were obtained by doping hydroxyapatite (HA) with Fe ions, by directly substituting Fe2+ and Fe3+ into the HA structure yielding superparamagnetic bioactive phase. In the current study, we have investigated the effects of increasing concentrations (2000 μg/ml; 1000 μg/ml; 500 μg/ml; 200 μg/ml) of FeHA MNPs in vitro using Saos-2 human osteoblast-like cells cultured for 1, 3 and 7 days with and without the exposure to a static magnetic field of 320 mT. Results demonstrated not only a comparable osteoblast viability and morphology, but increased in cell proliferation, when compared to a commercially available Ha nanoparticles, even with the highest dose used. Furthermore, FeHA MNPs exposure to the static magnetic field resulted in a significant increase in cell proliferation throughout the experimental period, and higher osteoblast activity. In vivo preliminary results demonstrated good biocompatibility of FeHA superparamagnetic material four weeks after implantation into a critical size lesion of the rabbit condyle. Conclusions The results of the current study suggest that these novel FeHA MNPs may be particularly relevant for strategies of bone tissue regeneration and open new perspectives for the application of a static magnetic field in a clinical setting of bone replacement, either for diagnostic imaging or magnetic drug delivery

  11. Identification of differentiation-stage specific molecular markers for the osteoblastic phenotype

    DEFF Research Database (Denmark)

    Twine, Natalie; Chen, Li; Wilkins, Marc

    to age-matched control (n=4). Using RNA-seq and cluster analysis, we identified a set of stage-specific molecular markers that define the progression of OB phenotype during ex vivo culture of hMSC, predict in vivo bone formation capacity of hMSC and can be employed to study the mechanisms of impaired......The phenotype of osteoblastic (OB) cells in culture is currently defined using a limited number of markers of low sensitivity and specificity which belong mostly to extracellular matrix proteins. Also, for clinical use of human skeletal (mesenchymal) stem cells (hMSC) in bone regeneration......, there is a need to identify predictive markers for in vivo bone forming capacity. Thus, we employed Illumina RNA sequencing (RNASeq) to examine changes in gene expression across 8 time points between 0-12 days of ex vivo OB differentiation of hMSC. We identified a subset of expressed genes as potentially...

  12. Accumulation of pyrethroid compounds in primary cultures of rat cortical neurons

    Science.gov (United States)

    Recent studies have demonstrated that lipophilic compounds (e.g. methylmercury, polychlorinated biphenyls (PCBs) and polybrominated diphenylethers (PBDEs)) rapidly accumulate in cells in culture to concentrations much higher than in the surrounding media. Primary cultures of neur...

  13. Reprogramming of Mouse Calvarial Osteoblasts into Induced Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Yinxiang Wang

    2018-01-01

    Full Text Available Previous studies have demonstrated the ability of reprogramming endochondral bone into induced pluripotent stem (iPS cells, but whether similar phenomenon occurs in intramembranous bone remains to be determined. Here we adopted fluorescence-activated cell sorting-based strategy to isolate homogenous population of intramembranous calvarial osteoblasts from newborn transgenic mice carrying both Osx1-GFP::Cre and Oct4-EGFP transgenes. Following retroviral transduction of Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc, enriched population of osteoblasts underwent silencing of Osx1-GFP::Cre expression at early stage of reprogramming followed by late activation of Oct4-EGFP expression in the resulting iPS cells. These osteoblast-derived iPS cells exhibited gene expression profiles akin to embryonic stem cells and were pluripotent as demonstrated by their ability to form teratomas comprising tissues from all germ layers and also contribute to tail tissue in chimera embryos. These data demonstrate that iPS cells can be generated from intramembranous osteoblasts.

  14. Renal cell carcinoma primary cultures maintain genomic and phenotypic profile of parental tumor tissues

    International Nuclear Information System (INIS)

    Cifola, Ingrid; Magni, Fulvio; Signorini, Stefano; Battaglia, Cristina; Perego, Roberto A; Bianchi, Cristina; Mangano, Eleonora; Bombelli, Silvia; Frascati, Fabio; Fasoli, Ester; Ferrero, Stefano; Di Stefano, Vitalba; Zipeto, Maria A

    2011-01-01

    Clear cell renal cell carcinoma (ccRCC) is characterized by recurrent copy number alterations (CNAs) and loss of heterozygosity (LOH), which may have potential diagnostic and prognostic applications. Here, we explored whether ccRCC primary cultures, established from surgical tumor specimens, maintain the DNA profile of parental tumor tissues allowing a more confident CNAs and LOH discrimination with respect to the original tissues. We established a collection of 9 phenotypically well-characterized ccRCC primary cell cultures. Using the Affymetrix SNP array technology, we performed the genome-wide copy number (CN) profiling of both cultures and corresponding tumor tissues. Global concordance for each culture/tissue pair was assayed evaluating the correlations between whole-genome CN profiles and SNP allelic calls. CN analysis was performed using the two CNAG v3.0 and Partek software, and comparing results returned by two different algorithms (Hidden Markov Model and Genomic Segmentation). A very good overlap between the CNAs of each culture and corresponding tissue was observed. The finding, reinforced by high whole-genome CN correlations and SNP call concordances, provided evidence that each culture was derived from its corresponding tissue and maintained the genomic alterations of parental tumor. In addition, primary culture DNA profile remained stable for at least 3 weeks, till to third passage. These cultures showed a greater cell homogeneity and enrichment in tumor component than original tissues, thus enabling a better discrimination of CNAs and LOH. Especially for hemizygous deletions, primary cultures presented more evident CN losses, typically accompanied by LOH; differently, in original tissues the intensity of these deletions was weaken by normal cell contamination and LOH calls were missed. ccRCC primary cultures are a reliable in vitro model, well-reproducing original tumor genetics and phenotype, potentially useful for future functional approaches

  15. Sr-containing hydroxyapatite: morphologies of HA crystals and bioactivity on osteoblast cells

    Energy Technology Data Exchange (ETDEWEB)

    Aina, Valentina [Department of Chemistry, Università degli Studi di Torino, Via P. Giuria 7, 10125 Torino (Italy); Centre of Excellence NIS (Nanostructured Interfaces and Surface) Università degli Studi di Torino (Italy); INSTM (Italian National Consortium for Materials Science and Technology), UdR Università di Torino (Italy); Bergandi, Loredana, E-mail: loredana.bergandi@unito.it [Department of Oncology, Università degli Studi di Torino, Via Santena 5/bis, 10126 Torino (Italy); Lusvardi, Gigliola; Malavasi, Gianluca [Department of Chemical and Geological Sciences, Università di Modena and Reggio Emilia, Via Campi 183, 41125 Modena (Italy); Imrie, Flora E.; Gibson, Iain R. [School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD (United Kingdom); Cerrato, Giuseppina [Department of Chemistry, Università degli Studi di Torino, Via P. Giuria 7, 10125 Torino (Italy); Centre of Excellence NIS (Nanostructured Interfaces and Surface) Università degli Studi di Torino (Italy); INSTM (Italian National Consortium for Materials Science and Technology), UdR Università di Torino (Italy); Ghigo, Dario [Department of Oncology, Università degli Studi di Torino, Via Santena 5/bis, 10126 Torino (Italy)

    2013-04-01

    A series of Sr-substituted hydroxyapatites (HA), of general formula Ca{sub (10−x)}Sr{sub x}(PO{sub 4}){sub 6}(OH){sub 2}, where x = 2 and 4, were synthesized by solid state methods and characterized extensively. The reactivity of these materials in cell culture medium was evaluated, and the behavior towards MG-63 osteoblast cells (in terms of cytotoxicity and proliferation assays) was studied. Future in vivo studies will give further insights into the behavior of the materials. A paper by Lagergren et al. (1975), concerning Sr-substituted HA prepared by a solid state method, reports that the presence of Sr in the apatite composition strongly influences the apatite diffraction patterns. Zeglinsky et al. (2012) investigated Sr-substituted HA by ab initio methods and Rietveld analyses and reported changes in the HA unit cell volume and shape due to the Sr addition. To further clarify the role played by the addition of Sr on the physico-chemical properties of these materials we prepared Sr-substituted HA compositions by a solid state method, using different reagents, thermal treatments and a multi-technique approach. Our results indicated that the introduction of Sr at the levels considered here does influence the structure of HA. There is also evidence of a decrease in the crystallinity degree of the materials upon Sr addition. The introduction of increasing amounts of Sr into the HA composition causes a decrease in the specific surface area and an enrichment of Sr-apatite phase at the surface of the samples. Bioactivity tests show that the presence of Sr causes changes in particle size and/or morphology during soaking in MEM solution; on the contrary the morphology of pure HA does not change after 14 days of reaction. The presence of Sr, as Sr-substituted HA and SrCl{sub 2,} in cultures of human MG-63 osteoblasts did not produce any cytotoxic effect. In fact, Sr-substituted HA increased the proliferation of osteoblast cells and enhanced cell differentiation: Sr in

  16. Sr-containing hydroxyapatite: morphologies of HA crystals and bioactivity on osteoblast cells

    International Nuclear Information System (INIS)

    Aina, Valentina; Bergandi, Loredana; Lusvardi, Gigliola; Malavasi, Gianluca; Imrie, Flora E.; Gibson, Iain R.; Cerrato, Giuseppina; Ghigo, Dario

    2013-01-01

    A series of Sr-substituted hydroxyapatites (HA), of general formula Ca (10−x) Sr x (PO 4 ) 6 (OH) 2 , where x = 2 and 4, were synthesized by solid state methods and characterized extensively. The reactivity of these materials in cell culture medium was evaluated, and the behavior towards MG-63 osteoblast cells (in terms of cytotoxicity and proliferation assays) was studied. Future in vivo studies will give further insights into the behavior of the materials. A paper by Lagergren et al. (1975), concerning Sr-substituted HA prepared by a solid state method, reports that the presence of Sr in the apatite composition strongly influences the apatite diffraction patterns. Zeglinsky et al. (2012) investigated Sr-substituted HA by ab initio methods and Rietveld analyses and reported changes in the HA unit cell volume and shape due to the Sr addition. To further clarify the role played by the addition of Sr on the physico-chemical properties of these materials we prepared Sr-substituted HA compositions by a solid state method, using different reagents, thermal treatments and a multi-technique approach. Our results indicated that the introduction of Sr at the levels considered here does influence the structure of HA. There is also evidence of a decrease in the crystallinity degree of the materials upon Sr addition. The introduction of increasing amounts of Sr into the HA composition causes a decrease in the specific surface area and an enrichment of Sr-apatite phase at the surface of the samples. Bioactivity tests show that the presence of Sr causes changes in particle size and/or morphology during soaking in MEM solution; on the contrary the morphology of pure HA does not change after 14 days of reaction. The presence of Sr, as Sr-substituted HA and SrCl 2, in cultures of human MG-63 osteoblasts did not produce any cytotoxic effect. In fact, Sr-substituted HA increased the proliferation of osteoblast cells and enhanced cell differentiation: Sr in HA has a positive effect

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

    International Nuclear Information System (INIS)

    Torricelli, Paola; Fini, Milena; Giavaresi, Gianluca; Borsari, Veronica; Rimondini, Lia; Rimondini, Roberto; Carrassi, Antonio; Giardino, Roberto

    2007-01-01

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

  18. Temporal Profiling and Pulsed SILAC Labeling Identify Novel Secreted Proteins During Ex Vivo Osteoblast Differentiation of Human Stromal Stem Cells*

    Science.gov (United States)

    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

  19. Anti-inflammatory drugs suppress proliferation and induce apoptosis through altering expressions of cell cycle regulators and pro-apoptotic factors in cultured human osteoblasts

    International Nuclear Information System (INIS)

    Chang, J.-K.; Li, C.-J.; Liao, H.-J.; Wang, C.-K.; Wang, G.-J.; Ho, M.-L.

    2009-01-01

    It has been reported that anti-inflammatory drugs (AIDs) inhibited bone repair in animal studies, and suppressed proliferation and induced cell death in rat osteoblast cultures. In this study, we further investigated the molecular mechanisms of AID effects on proliferation and cell death in human osteoblasts (hOBs). We examined the effects of dexamethasone (10 -7 and 10 -6 M), non-selective non-steroidal anti-inflammatory drugs (NSAIDs): indomethacin, ketorolac, piroxicam and diclofenac (10 -5 and 10 -4 M), and COX-2 inhibitor: celecoxib (10 -6 and 10 -5 M) on proliferation, cytotoxicity, cell death, and mRNA and protein levels of cell cycle and apoptosis-related regulators in hOBs. All the tested AIDs significantly inhibited proliferation and arrested cell cycle at G0/G1 phase in hOBs. Celecoxib and dexamethasone, but not non-selective NSAIDs, were found to have cytotoxic effects on hOB, and further demonstrated to induce apoptosis and necrosis (at higher concentration) in hOBs. We further found that indomethacin, celecoxib and dexamethasone increased the mRNA and protein expressions of p27 kip1 and decreased those of cyclin D2 and p-cdk2 in hOBs. Bak expression was increased by celecoxib and dexamethasone, while Bcl-XL level was declined only by dexamethasone. Furthermore, the replenishment of PGE1, PGE2 or PGF2α did not reverse the effects of AIDs on proliferation and expressions of p27 kip1 and cyclin D2 in hOBs. We conclude that the changes in expressions of regulators of cell cycle (p27 kip1 and cyclin D2) and/or apoptosis (Bak and Bcl-XL) by AIDs may contribute to AIDs caused proliferation suppression and apoptosis in hOBs. This effect might not relate to the blockage of prostaglandin synthesis by AIDs

  20. The role of nanocrystalline titania coating on nanostructured austenitic stainless steel in enhancing osteoblasts functions for regeneration of tissue

    Energy Technology Data Exchange (ETDEWEB)

    Shah, J.S.; Venkatsurya, P.K.C.; Thein-Han, W.W. [Biomaterials and Biomedical Engineering Research Laboratory, Center for Structural and Functional Materials, University of Louisiana at Lafayette, P.O. Box 44130, Lafayette, LA 70504 (United States); Misra, R.D.K., E-mail: dmisra@louisiana.edu [Biomaterials and Biomedical Engineering Research Laboratory, Center for Structural and Functional Materials, University of Louisiana at Lafayette, P.O. Box 44130, Lafayette, LA 70504 (United States); Pesacreta, T.C. [Department of Biology, University of Louisiana at Lafayette, P.O. Box 42451, Lafayette, LA 70504 (United States); Somani, M.C.; Karjalainen, L.P. [Department of Mechanical Engineering, University of Oulu, P.O. Box 4200, 90014 Oulu (Finland)

    2011-03-12

    In the context of osseointegration of metallic implants, while nanostructuring the surface favorably modulates cellular response, the disinfective attributes required during the healing process are not available. Thus, in the present study, we demonstrate that nanocrystalline titania provides cumulative benefit of enhancing osteoblasts functions to promote the efficacy of metal implants together with the disinfective attributes. To this end, the primary objective here is to examine the select functions of bone forming cells (osteoblasts) on electrocrystallized nanonodular titania-coated nanograined/ultrafine grained (NG/UFG) austenitic stainless steel. The accompanying objective is to study the disinfective/antimicrobial activity. To the best of our understanding this is the first study of nanophase titania on a non-titanium substrate. The osteoblasts functions were investigated in terms of cell attachment, proliferation, and quantitative analysis of proteins, actin and vinculin. In comparison to the bare NG/UFG substrate, the nanophase titania-coated substrate exhibited higher degree of cell attachment and proliferation which are regulated via cellular and molecular interactions with proteins, actin and vinculin. The enhanced functions of osteoblasts suggest that nanophase titania adsorbs extracellular matrix proteins, fibronectin and vitronectin from serum enhancing protein, with subsequent binding of integrins and osteoblasts precursor to titania. The antimicrobial attributes assessed in terms of degradation of methyl orange and effectiveness in killing E. coli supports the viewpoint that large surface area of titania would be instrumental in reducing the detrimental effect of biologically reactive oxygen species produced by macrophages in the vicinity of the metal bone/implant interface. In summary, the study provides some new insights concerning nanostructuring of metallic substrates with specific physical and surface properties for medical devices with

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

    Science.gov (United States)

    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.

  2. Disinfection of titanium dioxide nanotubes using super-oxidized water decrease bacterial viability without disrupting osteoblast behavior

    Energy Technology Data Exchange (ETDEWEB)

    Beltrán-Partida, Ernesto [Department of Biomaterials, Dental Materials and Tissue Engineering, Faculty of Dentistry Mexicali, Autonomous University of Baja California, Av. Zotoluca and Chinampas St., 21040 Mexicali, Baja California (Mexico); Department of Corrosion and Materials, Engineering Institute, Autonomous University of Baja California, Blvd. Benito Juarez and Normal St., 21280 Mexicali, Baja California (Mexico); Valdez-Salas, Benjamín, E-mail: benval@uabc.edu.mx [Department of Corrosion and Materials, Engineering Institute, Autonomous University of Baja California, Blvd. Benito Juarez and Normal St., 21280 Mexicali, Baja California (Mexico); Escamilla, Alan; Curiel, Mario [Department of Corrosion and Materials, Engineering Institute, Autonomous University of Baja California, Blvd. Benito Juarez and Normal St., 21280 Mexicali, Baja California (Mexico); Valdez-Salas, Ernesto [Ixchel Medical Centre, Av. Bravo y Obregón, 21000 Mexicali, Baja California (Mexico); Nedev, Nicola [Department of Corrosion and Materials, Engineering Institute, Autonomous University of Baja California, Blvd. Benito Juarez and Normal St., 21280 Mexicali, Baja California (Mexico); Bastidas, Jose M. [National Centre for Metallurgical Research, CSIC, Av. Gregorio del Amo 8, 28040 Madrid (Spain)

    2016-03-01

    Amorphous titanium dioxide (TiO{sub 2}) nanotubes (NTs) on Ti6Al4V alloy were synthesized by anodization using a commercially available super-oxidized water (SOW). The NT surfaces were sterilized by ultraviolet (UV) irradiation and disinfected using SOW. The adhesion and cellular morphology of pig periosteal osteoblast (PPO) cells and the behavior of Staphylococcus aureus (S. aureus) cultured on the sterilized and disinfected surfaces were investigated. A non-anodized Ti6Al4V disc sterilized by UV irradiation (without SOW) was used as control. The results of this study reveal that the adhesion, morphology and filopodia development of PPO cells in NTs are dramatically improved, suggesting that SOW cleaning may not disrupt the benefits obtained by NTs. Significantly decreased bacterial viability in NTs after cleaning with SOW and comparing with non-cleaned NTs was seen. The results suggest that UV and SOW could be a recommendable method for implant sterilization and disinfection without altering osteoblast behavior while decreasing bacterial viability. - Highlights: • The effect of super-oxidized water cleaning was studied on Ti6Al4V nanotubes. • Super oxidized-water cleaning caused a decline in S. aureus viability. • Osteoblast behavior was not disrupted after super-oxidized water disinfection. • Super-oxidized water is suggested as a cleaning protocol for TiO{sub 2} nanotubes.

  3. Disinfection of titanium dioxide nanotubes using super-oxidized water decrease bacterial viability without disrupting osteoblast behavior

    International Nuclear Information System (INIS)

    Beltrán-Partida, Ernesto; Valdez-Salas, Benjamín; Escamilla, Alan; Curiel, Mario; Valdez-Salas, Ernesto; Nedev, Nicola; Bastidas, Jose M.

    2016-01-01

    Amorphous titanium dioxide (TiO_2) nanotubes (NTs) on Ti6Al4V alloy were synthesized by anodization using a commercially available super-oxidized water (SOW). The NT surfaces were sterilized by ultraviolet (UV) irradiation and disinfected using SOW. The adhesion and cellular morphology of pig periosteal osteoblast (PPO) cells and the behavior of Staphylococcus aureus (S. aureus) cultured on the sterilized and disinfected surfaces were investigated. A non-anodized Ti6Al4V disc sterilized by UV irradiation (without SOW) was used as control. The results of this study reveal that the adhesion, morphology and filopodia development of PPO cells in NTs are dramatically improved, suggesting that SOW cleaning may not disrupt the benefits obtained by NTs. Significantly decreased bacterial viability in NTs after cleaning with SOW and comparing with non-cleaned NTs was seen. The results suggest that UV and SOW could be a recommendable method for implant sterilization and disinfection without altering osteoblast behavior while decreasing bacterial viability. - Highlights: • The effect of super-oxidized water cleaning was studied on Ti6Al4V nanotubes. • Super oxidized-water cleaning caused a decline in S. aureus viability. • Osteoblast behavior was not disrupted after super-oxidized water disinfection. • Super-oxidized water is suggested as a cleaning protocol for TiO_2 nanotubes.

  4. Osteoblast interaction with DLC-coated Si substrates.

    Science.gov (United States)

    Chai, Feng; Mathis, Nicolas; Blanchemain, Nicolas; Meunier, Cathy; Hildebrand, Hartmut F

    2008-09-01

    Diamond-like carbon (DLC) coating is a convenient means of modifying material surfaces that are sensitive to wear, such as titanium and silica substrates. This work aims to evaluate the osteoblast-like cells' response to DLC-coated Si (Si-DLC), which was treated under different conditions. DLC and deuterated DLC films were deposited by plasma-enhanced chemical vapor deposition to obtain a 200-nm-thick layer on all the samples. Three types of precursor gas were applied for deposition: pure methane (CH(4)), pure deuterated methane (CD(4)) and their half/half mixture. All surface treatments were performed under two different self-bias voltages (V(sb)): -400 and -600V. The modified surfaces were characterized by X-ray photoelectron spectroscopy, Raman spectroscopy, Rutherford backscattering spectroscopy, elastic recoil detection analysis, X-ray reflectometry and the sessile-drop method. MC3T3-E1 osteoblasts were cultured on the Si-DLC wafers for 3 and 6 days. Biological tests to measure cell proliferation, cell vitality, cell morphology and cell adhesion were performed. All DLC coatings produced a slightly more hydrophobic state than non-treated Si. Certain types of amorphous DLC coating, such as the surface treated under the V(sb) of -600V in pure methane (600CH(4)) or in pure deuterated methane (600CD(4)), offered a significantly higher cell proliferation rate to Si substrate. Scanning electron microscopy observations confirmed that the optimal cell adhesion behavior, among all the treated surfaces, occurred on the surface of the 600CH(4) and 600CD(4) groups, which showed increased amounts of filopodia and microvilli to enhance cell-environment exchange. In conclusion, DLC coating on Si could produce better surface stability and improved cellular responses.

  5. Cultural democracy: the way forward for primary care of hard to reach New Zealanders.

    Science.gov (United States)

    Finau, Sitaleki A; Finau, Eseta

    2007-09-01

    The use of cultural democracy, the freedom to practice one's culture without fear, as a framework for primary care service provision is essential for improved health service in a multi cultural society like New Zealand. It is an effective approach to attaining health equity for all. Many successful health ventures are ethnic specific and have gone past cultural competency to the practice of cultural democracy. That is, the services are freely taking on the realities of clients without and malice from those of other ethnicities. In New Zealand the scientific health service to improve the health of a multi cultural society are available but there is a need to improve access and utilization by hard to reach New Zealanders. This paper discusses cultural democracy and provide example of how successful health ventures that had embraced cultural democracy were implemented. It suggests that cultural democracy will provide the intellectual impetus and robust philosophy for moving from equality to equity in health service access and utilization. This paper would provide a way forward to improved primary care utilization, efficiency, effectiveness and equitable access especially for the hard to reach populations. use the realities of Pacificans in New Zealand illustrate the use of cultural democracy, and thus equity to address the "inverse care law" of New Zealand. The desire is for primary care providers to take cognizance and use cultural democracy and equity as the basis for the design and practice of primary health care for the hard to reach New Zealanders.

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

    Directory of Open Access Journals (Sweden)

    Annussek Tobias

    2012-09-01

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

  7. Zirconia coated titanium for implants and their interactions with osteoblast cells

    International Nuclear Information System (INIS)

    Kaluđerović, Milena R.; Schreckenbach, Joachim P.; Graf, Hans-Ludwig

    2014-01-01

    The anodic plasma-electrochemical oxidation in aqueous electrolytes of Zr(SO 4 ) 2 was used to prepare new zirconia/titania-based surfaces M1 (Ti, Zr and O: 7–10, 22–27 and 65–69 at.%) and M2 (Ti, Zr and O: 11–13, 20–23 and 64–69 at.%). The chemical composition and the microstructure of these coatings were characterized by surface and solid state techniques such as scanning electron microscopy, electron probe microanalysis, Raman spectroscopy and X-ray diffraction. These mixed oxides of ZrO 2 /TiO 2 surfaces consist up to 84% (m/m) of ZrO 2 and 16% (m/m) of TiO 2 . Monoclinic zirconia was detected as the dominant microcrystalline phase. In vitro studies were conducted on primary human osteoblast cells. MTT and DAPI assays were used for assessment on cell proliferation. Immunohistochemical analyses of morphology, cell cluster formation and expression of bone sialoprotein (BSP) and osteocalcin (OC) were performed. Novel surfaces M1 and M2 induced proliferation and expression of OC and BSP similarly to Ticer, used in clinical practice. Furthermore, the presence of zirconia on titanium surface has a higher beneficial effect on the osteoblast morphological changes and cell cluster formation. - Highlights: • Surfaces M1 and M2 (up to 84% (m/m) ZrO 2 and 16% (m/m) TiO 2 ) were prepared. • Novel materials promote proliferation of human osteoblasts similarly to Ticer. • Morphological changes and cell cluster formation are induced faster on M1 and M2. • Higher expression of OC and BSP is caused by M1 and M2. • M1 and M2 may influence the rate of bone formation

  8. Myostatin inhibits osteoblastic differentiation by suppressing osteocyte-derived exosomal microRNA-218: A novel mechanism in muscle-bone communication.

    Science.gov (United States)

    Qin, Yiwen; Peng, Yuanzhen; Zhao, Wei; Pan, Jianping; Ksiezak-Reding, Hanna; Cardozo, Christopher; Wu, Yingjie; Divieti Pajevic, Paola; Bonewald, Lynda F; Bauman, William A; Qin, Weiping

    2017-06-30

    Muscle and bone are closely associated in both anatomy and function, but the mechanisms that coordinate their synergistic action remain poorly defined. Myostatin, a myokine secreted by muscles, has been shown to inhibit muscle growth, and the disruption of the myostatin gene has been reported to cause muscle hypertrophy and increase bone mass. Extracellular vesicle-exosomes that carry microRNA (miRNA), mRNA, and proteins are known to perform an important role in cell-cell communication. We hypothesized that myostatin may play a crucial role in muscle-bone interactions and may promote direct effects on osteocytes and on osteocyte-derived exosomal miRNAs, thereby indirectly influencing the function of other bone cells. We report herein that myostatin promotes expression of several bone regulators such as sclerostin (SOST), DKK1, and RANKL in cultured osteocytic (Ocy454) cells, concomitant with the suppression of miR-218 in both parent Ocy454 cells and derived exosomes. Exosomes produced by Ocy454 cells that had been pretreated with myostatin could be taken up by osteoblastic MC3T3 cells, resulting in a marked reduction of Runx2, a key regulator of osteoblastic differentiation, and in decreased osteoblastic differentiation via the down-regulation of the Wnt signaling pathway. Importantly, the inhibitory effect of myostatin-modified osteocytic exosomes on osteoblast differentiation is completely reversed by expression of exogenous miR-218, through a mechanism involving miR-218-mediated inhibition of SOST. Together, our findings indicate that myostatin directly influences osteocyte function and thereby inhibits osteoblastic differentiation, at least in part, through the suppression of osteocyte-derived exosomal miR-218, suggesting a novel mechanism in muscle-bone communication. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Cultural activities in primary school students' spare time

    Directory of Open Access Journals (Sweden)

    Mikanović Brane

    2014-01-01

    Full Text Available Culture is a form of creative expression of a human being through which he reshapes the world, acts on it adding it value and creating new, cultural values. A human being is able to create a product of culture only when he is free and able to express himself. A contemporary man can incorporate various cultural activities into his spare time. They are especially important when they concern children and young people: regardless of whether they are used in institutional settings or in spare time. The authors conducted an empirical research of students' assumptions and beliefs concerning cultural activities in their free time. The sample comprised 233 fifth grade students. The findings show that in their spare time fifth graders: engage in various cultural activities; that students who live in urban areas attend more cultural events; that students have the opportunity to engage in extra-curricular activities in the area of culture - join cultural and artistic groups and associations and engage in various creative pursuits at different levels of participation (as consumers, full participants; and that students' attitudes concerning the influence of parents and teachers on the selection of cultural activities to be pursued do not vary greatly by gender, location or school achievement. Cultural activities do play a significant part in the free time of primary school students. This is why it is important that guidance provided in school and in spare time should be brought in greaer harmony.

  10. Seawater Polluted with Highly Concentrated Polycyclic Aromatic Hydrocarbons Suppresses Osteoblastic Activity in the Scales of Goldfish, Carassius auratus.

    Science.gov (United States)

    Suzuki, Nobuo; Sato, Masayuki; Nassar, Hossam F; Abdel-Gawad, Fagr Kh; Bassem, Samah M; Yachiguchi, Koji; Tabuchi, Yoshiaki; Endo, Masato; Sekiguchi, Toshio; Urata, Makoto; Hattori, Atsuhiko; Mishima, Hiroyuki; Shimasaki, Youhei; Oshima, Yuji; Hong, Chun-Sang; Makino, Fumiya; Tang, Ning; Toriba, Akira; Hayakawa, Kazuichi

    2016-08-01

    We have developed an original in vitro bioassay using teleost scale, that has osteoclasts, osteoblasts, and bone matrix as each marker: alkaline phosphatase (ALP) for osteoblasts and tartrate-resistant acid phosphatase (TRAP) for osteoclasts. Using this scale in vitro bioassay, we examined the effects of seawater polluted with highly concentrated polycyclic aromatic hydrocarbons (PAHs) and nitro-polycyclic aromatic hydrocarbons (NPAHs) on osteoblastic and osteoclastic activities in the present study. Polluted seawater was collected from two sites (the Alexandria site on the Mediterranean Sea and the Suez Canal site on the Red Sea). Total levels of PAHs in the seawater from the Alexandria and Suez Canal sites were 1364.59 and 992.56 ng/l, respectively. We were able to detect NPAHs in both seawater samples. Total levels of NPAHs were detected in the seawater of the Alexandria site (12.749 ng/l) and the Suez Canal site (3.914 ng/l). Each sample of polluted seawater was added to culture medium at dilution rates of 50, 100, and 500, and incubated with the goldfish scales for 6 hrs. Thereafter, ALP and TRAP activities were measured. ALP activity was significantly suppressed by both polluted seawater samples diluted at least 500 times, but TRAP activity did not change. In addition, mRNA expressions of osteoblastic markers (ALP, osteocalcin, and the receptor activator of the NF-κB ligand) decreased significantly, as did the ALP enzyme activity. In fact, ALP activity decreased on treatment with PAHs and NPAHs. We conclude that seawater polluted with highly concentrated PAHs and NPAHs influences bone metabolism in teleosts.

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

    International Nuclear Information System (INIS)

    Tsukasaki, Masayuki; Yamada, Atsushi; Suzuki, Dai; Aizawa, Ryo; Miyazono, Agasa; Miyamoto, Yoichi; Suzawa, Tetsuo; Takami, Masamichi; Yoshimura, Kentaro; Morimura, Naoko; Yamamoto, Matsuo; Kamijo, Ryutaro

    2011-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-07-15

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

  13. MSM enhances GH signaling via the Jak2/STAT5b pathway in osteoblast-like cells and osteoblast differentiation through the activation of STAT5b in MSCs.

    Directory of Open Access Journals (Sweden)

    Youn Hee Joung

    Full Text Available Methylsulfonylmethane (MSM is a naturally occurring sulfur compound with well-known anti-oxidant properties and anti-inflammatory activities. But, its effects on bone are unknown. Growth hormone (GH is regulator of bone growth and bone metabolism. GH activates several signaling pathways such as the Janus kinase (Jak/signal transducers and activators of transcription (STAT pathway, thereby regulating expression of genes including insulin-like growth factor (IGF-1. GH exerts effects both directly and via IGF-1, which signals by activating the IGF-1 receptor (IGF-1R. In this study, we investigated the effects of MSM on the GH signaling via the Jak/STAT pathway in osteoblasts and the differentiation of primary bone marrow mesenchymal stem cells (MSCs. MSM was not toxic to osteoblastic cells and MSCs. MSM increased the expression of GH-related proteins including IGF-1R, p-IGF-1R, STAT5b, p-STAT5b, and Jak2 in osteoblastic cells and MSCs. MSM increased IGF-1R and GHR mRNA expression in osteoblastic cells. The expression of MSM-induced IGF-1R and GHR was inhibited by AG490, a Jak2 kinase inhibitor. MSM induced binding of STAT5 to the IGF-1R and increased IGF-1 and IGF-1R promoter activities. Analysis of cell extracts by immunoprecipitation and Western blot showed that MSM enhanced GH-induced activation of Jak2/STAT5b. We found that MSM and GH, separately or in combination, activated GH signaling via the Jak2/STAT5b pathway in UMR-106 cells. Using siRNA analysis, we found that STAT5b plays an essential role in GH signaling activation in C3H10T1/2 cells. Osteogenic marker genes (ALP, ON, OCN, BSP, OSX, and Runx2 were activated by MSM, and siRNA-mediated STAT5b knockdown inhibited MSM-induced expression of osteogenic markers. Furthermore, MSM increased ALP activity and the mineralization of MSCs. Taken together, these results indicated that MSM can promote osteogenic differentiation of MSCs through activation of STAT5b.

  14. Biocompatibility of chitosan-coated iron oxide nanoparticles with osteoblast cells

    Directory of Open Access Journals (Sweden)

    Shi S

    2012-10-01

    Full Text Available Si-Feng Shi,1 Jing-Fu Jia,2 Xiao-Kui Guo,3 Ya-Ping Zhao,2 De-Sheng Chen,1 Yong-Yuan Guo,1 Tao Cheng,1 Xian-Long Zhang11Department of Orthopedic Surgery, Shanghai Sixth People’s Hospital, School of Medicine, 2School of Chemistry and Chemical Technology, 3Department of Medical Microbiology and Parasitology, School of Medicine, Shanghai Jiao Tong University Shanghai, ChinaBackground: Bone disorders (including osteoporosis, loosening of a prosthesis, and bone infections are of great concern to the medical community and are difficult to cure. Therapies are available to treat such diseases, but all have drawbacks and are not specifically targeted to the site of disease. Chitosan is widely used in the biomedical community, including for orthopedic applications. The aim of the present study was to coat chitosan onto iron oxide nanoparticles and to determine its effect on the proliferation and differentiation of osteoblasts.Methods: Nanoparticles were characterized using transmission electron microscopy, dynamic light scattering, x-ray diffraction, zeta potential, and vibrating sample magnetometry. Uptake of nanoparticles by osteoblasts was studied by transmission electron microscopy and Prussian blue staining. Viability and proliferation of osteoblasts were measured in the presence of uncoated iron oxide magnetic nanoparticles or those coated with chitosan. Lactate dehydrogenase, alkaline phosphatase, total protein synthesis, and extracellular calcium deposition was studied in the presence of the nanoparticles.Results: Chitosan-coated iron oxide nanoparticles enhanced osteoblast proliferation, decreased cell membrane damage, and promoted cell differentiation, as indicated by an increase in alkaline phosphatase and extracellular calcium deposition. Chitosan-coated iron oxide nanoparticles showed good compatibility with osteoblasts.Conclusion: Further research is necessary to optimize magnetic nanoparticles for the treatment of bone disease

  15. Renal cell carcinoma primary cultures maintain genomic and phenotypic profile of parental tumor tissues.

    Science.gov (United States)

    Cifola, Ingrid; Bianchi, Cristina; Mangano, Eleonora; Bombelli, Silvia; Frascati, Fabio; Fasoli, Ester; Ferrero, Stefano; Di Stefano, Vitalba; Zipeto, Maria A; Magni, Fulvio; Signorini, Stefano; Battaglia, Cristina; Perego, Roberto A

    2011-06-13

    Clear cell renal cell carcinoma (ccRCC) is characterized by recurrent copy number alterations (CNAs) and loss of heterozygosity (LOH), which may have potential diagnostic and prognostic applications. Here, we explored whether ccRCC primary cultures, established from surgical tumor specimens, maintain the DNA profile of parental tumor tissues allowing a more confident CNAs and LOH discrimination with respect to the original tissues. We established a collection of 9 phenotypically well-characterized ccRCC primary cell cultures. Using the Affymetrix SNP array technology, we performed the genome-wide copy number (CN) profiling of both cultures and corresponding tumor tissues. Global concordance for each culture/tissue pair was assayed evaluating the correlations between whole-genome CN profiles and SNP allelic calls. CN analysis was performed using the two CNAG v3.0 and Partek software, and comparing results returned by two different algorithms (Hidden Markov Model and Genomic Segmentation). A very good overlap between the CNAs of each culture and corresponding tissue was observed. The finding, reinforced by high whole-genome CN correlations and SNP call concordances, provided evidence that each culture was derived from its corresponding tissue and maintained the genomic alterations of parental tumor. In addition, primary culture DNA profile remained stable for at least 3 weeks, till to third passage. These cultures showed a greater cell homogeneity and enrichment in tumor component than original tissues, thus enabling a better discrimination of CNAs and LOH. Especially for hemizygous deletions, primary cultures presented more evident CN losses, typically accompanied by LOH; differently, in original tissues the intensity of these deletions was weaken by normal cell contamination and LOH calls were missed. ccRCC primary cultures are a reliable in vitro model, well-reproducing original tumor genetics and phenotype, potentially useful for future functional approaches

  16. Notch Inhibits Osteoblast Differentiation and Causes Osteopenia

    Science.gov (United States)

    Zanotti, Stefano; Smerdel-Ramoya, Anna; Stadmeyer, Lisa; Durant, Deena; Radtke, Freddy; Canalis, Ernesto

    2008-01-01

    Notch receptors are determinants of cell fate decisions. To define the role of Notch in the adult skeleton, we created transgenic mice overexpressing the Notch intracellular domain (NICD) under the control of the type I collagen promoter. First-generation transgenics were small and osteopenic. Bone histomorphometry revealed that NICD caused a decrease in bone volume, secondary to a reduction in trabecular number; osteoblast and osteoclast number were decreased. Low fertility of founder mice and lethality of young pups did not allow the complete establishment of transgenic lines. To characterize the effect of Notch overexpression in vitro, NICD was induced in osteoblasts and stromal cells from Rosanotch mice, in which a STOP cassette flanked by loxP sites is upstream of NICD, by transduction with an adenoviral vector expressing Cre recombinase (Cre) under the control of the cytomegalovirus (CMV) promoter (Ad-CMV-Cre). NICD impaired osteoblastogenesis and inhibited Wnt/β-catenin signaling. To determine the effects of notch1 deletion in vivo, mice in which notch1 was flanked by loxP sequences (notch1loxP/loxP) were mated with mice expressing Cre recombinase under the control of the osteocalcin promoter. Conditional null notch1 mice had no obvious skeletal phenotype, possibly because of rescue by notch2; however, 1-month-old females exhibited a modest increase in osteoclast surface and eroded surface. Osteoblasts from notch1loxP/loxP mice, transduced with Ad-CMV-Cre and transfected with Notch2 small interfering RNA, displayed increased alkaline phosphatase activity. In conclusion, Notch signaling in osteoblasts causes osteopenia and impairs osteo-blastogenesis by inhibiting the Wnt/β-catenin pathway. PMID:18420737

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

    Directory of Open Access Journals (Sweden)

    Yun-Yun Ma

    Full Text Available 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.

  18. Measurement tools and process indicators of patient safety culture in primary care. A mixed methods study by the LINNEAUS collaboration on patient safety in primary care

    Science.gov (United States)

    Parker, Dianne; Wensing, Michel; Esmail, Aneez; Valderas, Jose M

    2015-01-01

    ABSTRACT Background: There is little guidance available to healthcare practitioners about what tools they might use to assess the patient safety culture. Objective: To identify useful tools for assessing patient safety culture in primary care organizations in Europe; to identify those aspects of performance that should be assessed when investigating the relationship between safety culture and performance in primary care. Methods: Two consensus-based studies were carried out, in which subject matter experts and primary healthcare professionals from several EU states rated (a) the applicability to their healthcare system of several existing safety culture assessment tools and (b) the appropriateness and usefulness of a range of potential indicators of a positive patient safety culture to primary care settings. The safety culture tools were field-tested in four countries to ascertain any challenges and issues arising when used in primary care. Results: The two existing tools that received the most favourable ratings were the Manchester patient safety framework (MaPsAF primary care version) and the Agency for healthcare research and quality survey (medical office version). Several potential safety culture process indicators were identified. The one that emerged as offering the best combination of appropriateness and usefulness related to the collection of data on adverse patient events. Conclusion: Two tools, one quantitative and one qualitative, were identified as applicable and useful in assessing patient safety culture in primary care settings in Europe. Safety culture indicators in primary care should focus on the processes rather than the outcomes of care. PMID:26339832

  19. Celecoxib inhibits osteoblast maturation by suppressing the expression of Wnt target genes

    Directory of Open Access Journals (Sweden)

    Akihiro Nagano

    2017-01-01

    Full Text Available Non-steroidal anti-inflammatory drugs (NSAIDs have been shown to impair bone healing. We previously reported that in colon cancer cells, celecoxib, a COX-2-selective NSAID, inhibited the canonical Wnt/β-catenin signaling pathway. Since this pathway also plays an important role in osteoblast growth and differentiation, we examined the effect of celecoxib on maturation of osteoblast-like cell line MC3T3-E1. Celecoxib induced degradation of transcription factor 7-like 2, a key transcription factor of the canonical Wnt pathway. Subsequently, we analyzed the effect of celecoxib on two osteoblast differentiation markers; runt-related transcription factor 2 (RUNX2 and alkaline phosphatase (ALP, both of which are the products of the canonical Wnt pathway target genes. Celecoxib inhibited the expression of both RUNX2 and ALP by suppressing their promoter activity. Consistent with these observations, celecoxib also strongly inhibited osteoblast-mediated mineralization. These results suggest that celecoxib inhibits osteoblast maturation by suppressing Wnt target genes, and this could be the mechanism that NSAIDs inhibit bone formation and fracture healing.

  20. RNA synthesis in primary cultures of adult rat hepatocytes

    International Nuclear Information System (INIS)

    Fugassa, E.; Gallo, G.; Voci, A.; Cordone, A.

    1983-01-01

    The ability of hepatocyte monolayers to synthesize RNA was investigated by measuring [3H]orotic acid incorporation into RNA and the total nuclear RNA polymerase activity as a function of the time in culture. The results demonstrate that primary cultures of hepatocytes maintained in a chemically defined serum- and hormone-free medium are able to synthesize RNA actively. This ability increases within the first 2 d of culture, despite the concomitant decrease in [3H]orotic acid uptake, and decreases only after 3 d. Factors such as serum, insulin, and dexamethasone, known to improve maintenance of functional hepatocytes, markedly stimulate the uptake of labeled precursor without apparently affecting the rate of RNA synthesis by cultured cells. It is suggested that the culture of adult rat hepatocytes provides a useful experimental model for the studies of hormonal regulation of transcription in liver

  1. Exploring Culture in Locally Published English Textbooks for Primary Education in Turkey

    Science.gov (United States)

    Kirkgöz, Yasemin; Agçam, Reyhan

    2011-01-01

    Since language and culture are closely interwoven, the integration of culture into textbooks used for teaching English as a second/foreign language has become a widely accepted phenomenon. This study investigates the cultural elements in locally published English textbooks used for Turkish primary schools following two major curriculum innovations…

  2. Establishment of primary bovine intestinal epithelial cell culture and clone method.

    Science.gov (United States)

    Zhan, Kang; Lin, Miao; Liu, Ming-Mei; Sui, Yang-Nan; Zhao, Guo-Qi

    2017-01-01

    The aim of this study was to establish bovine intestinal epithelial cell (BIEC) line and provide a novel clone cell method. Although various strategies of bovine cell culture and clone techniques have been reported, these methods remain not established. Here, we culture successfully primary BIECs and establish a novel clone cell method. Our result showed that BIECs could be successfully cultured and passaged about generation 5. These cellular aggregates and clusters were adherent loosely at day 2 of culture. Cell aggregates and clusters start to proliferate after approximately 4 d. The BIECs showed positive reaction against cytokeratin 18, E-cadherin, and characteristics of epithelial-like morphology. In addition, the fatty acid-binding proteins (FABPs), villin, and intestinal peptidase (IP) band were positive in BIECs. Our results suggest that the establishment of culturing and clone BIEC methods will apply to isolate and clone other primary cells. These BIECs could therefore contribute to the study of bovine intestinal nutrient absorption and regulation, immune regulation, and the pathogenesis of the bovine intestinal disease, which will provide intestinal cell model in vitro.

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

    Science.gov (United States)

    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

  4. Osteoblast growth behavior on porous-structure titanium surface

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-11-15

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

  5. Osteoblast growth behavior on porous-structure titanium surface

    International Nuclear Information System (INIS)

    Tian Yuan; Ding Siyang; Peng Hui; Lu Shanming; Wang Guoping; Xia Lu; Wang Peizhi

    2012-01-01

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

  6. Nanostructured magnesium has fewer detrimental effects on osteoblast function

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  8. Identification of differentiation-stage specific markers that define the ex vivo osteoblastic phenotype

    DEFF Research Database (Denmark)

    Twine, Natalie A; Chen, Li; Pang, Chi N

    2014-01-01

    The phenotype of osteoblastic (OB) cells in culture is currently defined using a limited number of markers of low sensitivity and specificity. For the clinical use of human skeletal (stromal, mesenchymal) stem cells (hMSC) in therapy, there is also a need to identify a set of gene markers...... clustering and Pearson's correlation generated 4 groups of genes: early stage differentiation genes (peak expression: 0-24h, n=28) which were enriched for extracellular matrix organisation, e.g. COL1A1, LOX, and SERPINH1; middle stage differentiating genes (peak expression days: 3 and 6, n=20) which were...

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

    Science.gov (United States)

    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.

  10. Mechanically induced intracellular calcium waves in osteoblasts demonstrate calcium fingerprints in bone cell mechanotransduction.

    Science.gov (United States)

    Godin, Lindsay M; Suzuki, Sakiko; Jacobs, Christopher R; Donahue, Henry J; Donahue, Seth W

    2007-11-01

    An early response to mechanical stimulation of bone cells in vitro is an increase in intracellular calcium concentration ([Ca (2+)](i)). This study analyzed the [Ca (2+)](i) wave area, magnitude, duration, rise time, fall time, and time to onset in individual osteoblasts for two identical bouts of mechanical stimulation separated by a 30-min rest period. The area under the [Ca (2+)](i) wave increased in the second loading bout compared to the first. This suggests that rest periods may potentiate mechanically induced intracellular calcium signals. Furthermore, many of the [Ca (2+)](i) wave parameters were strongly, positively correlated between the two bouts of mechanical stimulation. For example, in individual primary osteoblasts, if a cell had a large [Ca (2+)](i) wave area in the first bout it was likely to have a large [Ca (2+)](i) wave area in the second bout (r (2) = 0.933). These findings support the idea that individual bone cells have "calcium fingerprints" (i.e., a unique [Ca (2+)](i) wave profile that is reproducible for repeated exposure to a given stimulus).

  11. Chitosan-based hydrogel tissue scaffolds made by 3D plotting promotes osteoblast proliferation and mineralization.

    Science.gov (United States)

    Liu, I-Hsin; Chang, Shih-Hsin; Lin, Hsin-Yi

    2015-05-13

    A 3D plotting system was used to make chitosan-based tissue scaffolds with interconnected pores using pure chitosan (C) and chitosan cross-linked with pectin (CP) and genipin (CG). A freeze-dried chitosan scaffold (CF/D) was made to compare with C, to observe the effects of structural differences. The fiber size, pore size, porosity, compression strength, swelling ratio, drug release efficacy, and cumulative weight loss of the scaffolds were measured. Osteoblasts were cultured on the scaffolds and their proliferation, type I collagen production, alkaline phosphatase activity, calcium deposition, and morphology were observed. C had a lower swelling ratio, degradation, porosity and drug release efficacy and a higher compressional stiffness and cell proliferation compared to CF/D (p 3D-plotted samples, cells on CP exhibited the highest degree of mineralization after 21 d (p 3D-plotted scaffolds were stronger, less likely to degrade and better promoted osteoblast cell proliferation in vitro compared to the freeze-dried scaffolds. C, CP and CG were structurally similar, and the different crosslinking caused significant changes in their physical and biological performances.

  12. Nanocrystallinity effects on osteoblast and osteoclast response to silicon substituted hydroxyapatite.

    Science.gov (United States)

    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.

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

    International Nuclear Information System (INIS)

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

    2015-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-03-15

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

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

    Science.gov (United States)

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

    2016-06-01

    Insulin-like growth factor-1 (IGF-1) is anabolic for muscle by enhancing the rate of mRNA translation via activation of AKT and subsequent activation of the mammalian target of rapamycin complex 1 (mTOR), thereby increasing cellular protein production. IGF-1 is also anabolic for bone, but whether the mTOR pathway plays a role in the rate of bone matrix protein production by osteoblasts is unknown. We hypothesized that anabolic stimuli such as mechanical loading and IGF-1 stimulate protein synthesis in osteoblasts via activation of the AKT-mTOR pathway. MC3T3-E1 osteoblasts were either or not subjected for 1 h to mechanical loading by pulsating fluid flow (PFF) or treated with or without human recombinant IGF-1 (1-100 ng/ml) for 0.5-6 h, to determine phosphorylation of AKT and p70S6K (downstream of mTOR) by Western blot. After 4 days of culture with or without the mTOR inhibitor rapamycin, total protein, DNA, and gene expression were quantified. IGF-1 (100 ng/ml) reduced IGF-1 gene expression, although PFF enhanced IGF-1 expression. IGF-1 did not affect collagen-I gene expression. IGF-1 dose-dependently enhanced AKT and p70S6K phosphorylation at 2 and 6 h. PFF enhanced phosphorylation of AKT and p70S6K already within 1 h. Both IGF-1 and PFF enhanced total protein per cell by ∼30%, but not in the presence of rapamycin. Our results show that IGF-1 and PFF activate mTOR, thereby stimulating the rate of mRNA translation in osteoblasts. The known anabolic effect of mechanical loading and IGF-1 on bone may thus be partly explained by mTOR-mediated enhanced protein synthesis in osteoblasts. © 2015 Wiley Periodicals, Inc.

  16. Phenolic Modified Ceramic Coating on Biodegradable Mg Alloy: The Improved Corrosion Resistance and Osteoblast-Like Cell Activity

    Directory of Open Access Journals (Sweden)

    Hung-Pang Lee

    2017-06-01

    Full Text Available Magnesium alloys have great potential for developing orthopedic implants due to their biodegradability and mechanical properties, but the rapid corrosion rate of the currently-available alloys limits their clinical applications. To increase the corrosion resistance of the substrate, a protective ceramic coating is constructed by a micro-arc oxidation (MAO process on ZK60 magnesium alloy. The porous ceramic coating is mainly composed of magnesium oxide and magnesium silicate, and the results from cell cultures show it can stimulate osteoblastic cell growth and proliferation. Moreover, gallic acid, a phenolic compound, was successfully introduced onto the MAO coating by grafting on hydrated oxide and chelating with magnesium ions. The gallic acid and rough surface of MAO altered the cell attachment behavior, making it difficult for fibroblasts to adhere to the MAO coating. The viability tests showed that gallic acid could suppress fibroblast growth and stimulate osteoblastic cell proliferation. Overall, the porous MAO coating combined with gallic acid offered a novel strategy for increasing osteocompatibility.

  17. Repression of osteoblast maturation by ERRα accounts for bone loss induced by estrogen deficiency.

    Directory of Open Access Journals (Sweden)

    Marlène Gallet

    Full Text Available 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.

  18. Repression of osteoblast maturation by ERRα accounts for bone loss induced by estrogen deficiency.

    Science.gov (United States)

    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.

  19. Nurses' perceptions of workplace culture in primary health care in Finland.

    Science.gov (United States)

    Hahtela, N; Paavilainen, E; McCormack, B; Helminen, M; Slater, P; Suominen, T

    2015-12-01

    This study aimed to describe nurses' perceptions of workplace culture, especially in regard to stress levels, job satisfaction and the practice environment in primary health care. Health care is facing many challenges related to its attractiveness as a place of employment and the maintenance of a sufficient workforce supply. Previous studies report increasing rates of nurse job dissatisfaction and intentions to leave their current positions both in Finland and also globally. Improving workplace culture is thus vital in meeting the challenges related to recruitment and retention. A cross-sectional descriptive design was used to describe nurses' perceptions of workplace culture. Data were collected by questionnaire from 22 units in nine primary healthcare organizations in Finland, and analysed using descriptive and inferential statistics. Most of the respondents indicated that they were not certain whether their workplace culture was either positive or negative. Profession, age and work shift characteristics had an effect on the respondents' perceptions of workplace culture. Younger licensed practical and registered nurses assessed their workplace culture more positively, whereas older registered nurses and those working rotating rosters viewed workplace culture more negatively. The findings suggest that both unit and demographic characteristics affect workplace culture. This survey highlights that a positive workplace culture is one of the key factors in retaining and recruiting nurses, and provides an essential evidence that may be considered by other healthcare organizations. Nurse managers and healthcare leaders need to address workload management and take into account the related variables that affect a unit's workplace culture. © 2015 International Council of Nurses.

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

    Directory of Open Access Journals (Sweden)

    Neda Shams

    2015-11-01

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

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

    Science.gov (United States)

    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

  2. Relevant principal factors affecting the reproducibility of insect primary culture.

    Science.gov (United States)

    Ogata, Norichika; Iwabuchi, Kikuo

    2017-06-01

    The primary culture of insect cells often suffers from problems with poor reproducibility in the quality of the final cell preparations. The cellular composition of the explants (cell number and cell types), surgical methods (surgical duration and surgical isolation), and physiological and genetic differences between donors may be critical factors affecting the reproducibility of culture. However, little is known about where biological variation (interindividual differences between donors) ends and technical variation (variance in replication of culture conditions) begins. In this study, we cultured larval fat bodies from the Japanese rhinoceros beetle, Allomyrina dichotoma, and evaluated, using linear mixed models, the effect of interindividual variation between donors on the reproducibility of the culture. We also performed transcriptome analysis of the hemocyte-like cells mainly seen in the cultures using RNA sequencing and ultrastructural analyses of hemocytes using a transmission electron microscope, revealing that the cultured cells have many characteristics of insect hemocytes.

  3. Problems of culture of written expression in primary school

    Directory of Open Access Journals (Sweden)

    Zlatić Marina V.

    2014-01-01

    Full Text Available This paper investigates the issue of the culture of written expression in primary school students. Starting from the fact that teaching practices increasingly points to the fact that knowledge of rules of writing in primary school students presents the weakest link in teaching Serbian language, we sought to describe the problem, point to the possible causes, propose measures and illustrate all this on concrete examples of students' essays. Our microinvestigation showed that primary school students display considerably poorer mastery of rules of writing than previously thought, to the extent that it presents a serious obstacle in language teaching as well as in other areas of educational process.

  4. Eyewitness Culture and History: Primary Written Sources. The Iconoclast.

    Science.gov (United States)

    McMurtry, John

    1995-01-01

    Asserts that contemporary history and historiography is "official" history that ignores the daily struggles of people for their continued survival. Argues that, while public illiteracy has nearly disappeared, individuals are ignorant of the wealth of primary-source materials of other cultures' histories. (CFR)

  5. Hydroxyapatite and Calcified Elastin Induce Osteoblast-like Differentiation in Rat Aortic Smooth Muscle Cells

    Science.gov (United States)

    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

  6. Osteoblast cell membrane chromatography coupled with liquid chromatography and time-of-flight mass spectrometry for screening specific active components from traditional Chinese medicines.

    Science.gov (United States)

    Wang, Nani; Zhang, Qiaoyan; Xin, Hailiang; Shou, Dan; Qin, Luping

    2017-11-01

    A method using osteoblast membrane chromatography coupled with liquid chromatography and time-of-flight mass spectrometry was developed to recognize and identify the specific active components from traditional Chinese medicines. Primary rat osteoblasts were used for the preparation of the stationary phase in the cell chromatography method. Retention components from the cell chromatography were collected and analyzed by liquid chromatography with time-of-flight mass spectrometry. This method was applied in screening active components from extracts of four traditional Chinese medicines. In total, 24 potentially active components with different structures were retained by osteoblast cell chromatography. There were five phenolic glucosides and one triterpenoid saponin from Curculigo orchioides Gaertn, two organic acids and ten flavonoids from Epimedium sagittatum Maxim, one phthalide compound and one organic acid from Angelica sinensis Diels, and two flavonoids and two saponins from Anemarrhena asphodeloides Bunge. Among those, four components (icariin, curculigoside, ferulaic acid, and timosaponin BII) were used for in vitro pharmacodynamics validation. They significantly increased the osteoblast proliferation, alkaline phosphatase activity, levels of bone gla protein and collagen type 1, and promoted mineralized nodule formation. The developed method was an effective screening method for finding active components from complex medicines that act on bone diseases. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. In vitro biological evaluation of beta-TCP/HDPE--A novel orthopedic composite: a survey using human osteoblast and fibroblast bone cells.

    Science.gov (United States)

    Homaeigohar, S Sh; Shokrgozar, M A; Khavandi, A; Sadi, A Yari

    2008-02-01

    Beta-tricalcium phosphate reinforced high density polyethylene (beta-TCP/HDPE) was prepared to simulate bone composition and to study its capacity to act as bone tissue. This material was produced by replacing the mineral component and collagen soft tissue of the bone with beta-TCP and HDPE, respectively. The biocompatibility of the composite samples with different volume fractions of TCP (20, 30 and 40 vol %) was examined in vitro using two osteoblast cell lines G-292 and Saos-2, and also a type of fibroblast cell isolated from bone tissue, namely human bone fibroblast (HBF) by proliferation, and cell adhesion assays. Cell-material interaction with the surface of the composite samples was examined by scanning electron microscopy (SEM). The effect of beta-TCP/HDPE on the behavior of osteoblast and fibroblast cells was compared with those of composite and negative control samples; polyethylene (PE) and tissue culture polystyrene (TPS), respectively. In general, the results showed that the composite samples containing beta-TCP as reinforcement supported a higher rate of proliferation by various bone cells after 3, 7, and 14 days of incubation compared to the composite control sample. Furthermore, more osteoblast cells were attached to the surface of the composite samples when compared to the composite control samples after the above incubation periods (p HDPE composites are biocompatible, nontoxic, and act to stimulate proliferation and adhesion of the cells, whether osteoblast or fibroblast. (c) 2007 Wiley Periodicals, Inc. J Biomed Mater Res, 2008.

  8. In vitro study of vancomycin release and osteoblast-like cell growth on structured calcium phosphate-collagen

    International Nuclear Information System (INIS)

    Pon-On, Weeraphat; Charoenphandhu, Narattaphol; Teerapornpuntakit, Jarinthorn; Thongbunchoo, Jirawan; Krishnamra, Nateetip; Tang, I-Ming

    2013-01-01

    A drug delivery vehicle consisting of spherical calcium phosphate-collagen particles covered by flower-like (SFCaPCol) blossoms composed of nanorod building blocks and their cellular response is studied. The spherical structure was achieved by a combination of sonication and freeze-drying. The SFCaPCol blossoms have a high surface area of approximately 280 m 2 g −1 . The blossom-like formation having a high surface area allows a drug loading efficiency of 77.82%. The release profile for one drug, vancomycin (VCM), shows long term sustained release in simulated body fluid (SBF), in a phosphate buffer saline (PBS, pH 7.4) solution and in culture media over 2 weeks with a cumulative release ∼ 53%, 75% and 50%, respectively, over the first 7 days. The biocompatibility of the VCM-loaded SFCaPCol scaffold was determined by in vitro cell adhesion and proliferation tests of rat osteoblast-like UMR-106 cells. MTT tests indicated that UMR-106 cells were viable after exposure to the VCM loaded SFCaPCol, meaning that the scaffold (the flower-like blossoms) did not impair the cell's viability. The density of cells on the substrate was seen to increase with increasing cultured time. - Graphical abstract: A spherical calcium phosphate-collagen with flower-like blossoms consisting of nanorod building blocks (SFCaPCol) particles was achieved by a combination of sonication and freeze-drying. In vitro drug release profile and the biocompatibility of the VCM-loaded SFCaPCol composite cell adhesion and proliferation in rat osteoblast-like UMR-106 cells were determined for biomaterial applications. Highlights: ► SFCaPCol and VCM-loaded SFCaPCol composite were synthesized by a combination of ultra sonication and freeze-drying. ► VCM drug-loaded SFCaPCol composite was used as substrate for the growth of rat osteoblast-like UMR-106 cells. ► Controlled release of VCM from the composite is critically medium dependent. ► The VCM-loaded SFCaPCol composite is also bioactive by in

  9. Proteomic Characterization of Primary Mouse Hepatocytes in Collagen Monolayer and Sandwich Culture.

    Science.gov (United States)

    Orsini, Malina; Sperber, Saskia; Noor, Fozia; Hoffmann, Esther; Weber, Susanne N; Hall, Rabea A; Lammert, Frank; Heinzle, Elmar

    2018-01-01

    Dedifferentiation of primary hepatocytes in vitro makes their application in long-term studies difficult. Embedding hepatocytes in a sandwich of extracellular matrix is reported to delay the dedifferentiation process to some extent. In this study, we compared the intracellular proteome of primary mouse hepatocytes (PMH) in conventional monolayer cultures (ML) to collagen sandwich culture (SW) after 1 day and 5 days of cultivation. Quantitative proteome analysis of PMH showed no differences between collagen SW and ML cultures after 1 day. Glycolysis and gluconeogenesis were strongly affected by long-term cultivation in both ML and SW cultures. Interestingly, culture conditions had no effect on cellular lipid metabolism. After 5 days, PMH in collagen SW and ML cultures exhibit characteristic indications of oxidative stress. However, in the SW culture the defense system against oxidative stress is significantly up-regulated to deal with this, whereas in the ML culture a down-regulation of these important enzymes takes place. Regarding the multiple effects of ROS and oxidative stress in cells, we conclude that the down-regulation of these enzymes seem to play a role in the loss of hepatic function observed in the ML cultivation. In addition, enzymes of the urea cycle were clearly down-regulated in ML culture. Proteomics confirms lack in oxidative stress defense mechanisms as the major characteristic of hepatocytes in monolayer cultures compared to sandwich cultures. J. Cell. Biochem. 119: 447-454, 2018. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  10. Subcellular imaging of freeze-fractured cell cultures by TOF-SIMS and Laser-SNMS

    International Nuclear Information System (INIS)

    Fartmann, M.; Dambach, S.; Kriegeskotte, C.; Lipinsky, D.; Wiesmann, H.P.; Wittig, A.; Sauerwein, W.; Arlinghaus, H.F.

    2003-01-01

    We have examined atomic and molecular distributions in freeze-fractured freeze-dried primary osteoblasts and cancer cells using time-of-flight secondary ion mass spectrometry (TOF-SIMS) and non-resonant laser secondary neutral mass spectrometry (NR-Laser-SNMS). A pulsed Ga primary ion beam with a diameter of approximately 200 nm was employed to bombard the sample. Ion-induced electron-images were used to identify individual cells. High resolution elemental and molecular images were obtained from cell cultures. From these data the K/Na ratio was determined. It shows a higher K-concentration inside individual cells demonstrating that the chemical and structural integrity of living cells were preserved by the applied preparation technique. Consecutive presputtering of the sample with different primary ion dose densities was used to move the analysis plane toward the inside of the cell. It can be concluded that TOF-SIMS and Laser-SNMS are well suited for imaging trace element and molecule concentrations in biological samples

  11. On the importances of cultivating cross-cultural awareness of pre-service English teachers of primary schools

    Institute of Scientific and Technical Information of China (English)

    聂冬霞

    2016-01-01

    Cross-cultural communication becomes more important in English education in primary schools and one of the main tasks of English learning in primary schools is to improve students' cross-cultural awareness. For pre-service English teachers they should pay more attention on the importances of cultivating their cross-cultural awareness in English learning.

  12. Effects of silica and calcium levels in nanobioglass ceramic particles on osteoblast proliferation

    International Nuclear Information System (INIS)

    Moorthi, A.; Parihar, P.R.; Saravanan, S.; Vairamani, M.; Selvamurugan, N.

    2014-01-01

    At nanoscale, bioglass ceramic (nBGC) particles containing calcium oxide (lime), silica and phosphorus pentoxide promote osteoblast proliferation. However, the role of varied amounts of calcium and silica present in nBGC particles on osteoblast proliferation is not yet completely known. Hence, the current work was aimed at synthesizing two different nBGC particles with varied amounts of calcium oxide and silica, nBGC-1: SiO 2 :CaO:P 2 O 5 ; mol% ∼ 70:25:5 and nBGC-2: SiO 2 :CaO:P 2 O 5 ; mol% ∼ 64:31:5, and investigating their role on osteoblast proliferation. The synthesized nBGC particles were characterized by transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDS), Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction (XRD) studies. They exhibited their size at nanoscale and were non-toxic to human osteoblastic cells (MG-63). The nBGC-2 particles were found to have more effect on stimulation of osteoblast proliferation and promoted entering of more cells into G2/M cell cycle phase compared to nBGC-1 particles. There was a differential expression of cyclin proteins in MG-63 cells by nBGC-1 and nBGC-2 treatments, and the expression of cyclin B1 and E proteins was found to be more by nBGC-2 treatment. Thus, these results provide us a new insight in understanding the design of various nBGC particles by altering their ionic constituents with desirable biological properties thereby supporting bone augmentation. - Highlights: • nBGC particles with varied amounts of calcium and silica were synthesized. • They were non-toxic to human osteoblastic cells. • nBGC-2 particles had more effect on stimulation of osteoblast proliferation. • nBGC-2 particles promoted entering of osteoblasts into G2/M cell cycle phase. • Expression of cyclin B1 and E proteins was found to be more by nBGC-2 treatment

  13. Effects of silica and calcium levels in nanobioglass ceramic particles on osteoblast proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Moorthi, A.; Parihar, P.R.; Saravanan, S.; Vairamani, M.; Selvamurugan, N., E-mail: selvamurugan.n@ktr.srmuniv.ac.in

    2014-10-01

    At nanoscale, bioglass ceramic (nBGC) particles containing calcium oxide (lime), silica and phosphorus pentoxide promote osteoblast proliferation. However, the role of varied amounts of calcium and silica present in nBGC particles on osteoblast proliferation is not yet completely known. Hence, the current work was aimed at synthesizing two different nBGC particles with varied amounts of calcium oxide and silica, nBGC-1: SiO{sub 2}:CaO:P{sub 2}O{sub 5}; mol% ∼ 70:25:5 and nBGC-2: SiO{sub 2}:CaO:P{sub 2}O{sub 5}; mol% ∼ 64:31:5, and investigating their role on osteoblast proliferation. The synthesized nBGC particles were characterized by transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDS), Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction (XRD) studies. They exhibited their size at nanoscale and were non-toxic to human osteoblastic cells (MG-63). The nBGC-2 particles were found to have more effect on stimulation of osteoblast proliferation and promoted entering of more cells into G2/M cell cycle phase compared to nBGC-1 particles. There was a differential expression of cyclin proteins in MG-63 cells by nBGC-1 and nBGC-2 treatments, and the expression of cyclin B1 and E proteins was found to be more by nBGC-2 treatment. Thus, these results provide us a new insight in understanding the design of various nBGC particles by altering their ionic constituents with desirable biological properties thereby supporting bone augmentation. - Highlights: • nBGC particles with varied amounts of calcium and silica were synthesized. • They were non-toxic to human osteoblastic cells. • nBGC-2 particles had more effect on stimulation of osteoblast proliferation. • nBGC-2 particles promoted entering of osteoblasts into G2/M cell cycle phase. • Expression of cyclin B1 and E proteins was found to be more by nBGC-2 treatment.

  14. Collagen films with stabilized liquid crystalline phases and concerns on osteoblast behaviors

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Minjian; Ding, Shan; Min, Xiang; Jiao, Yanpeng, E-mail: tjiaoyp@jnu.edu.cn; Li, Lihua; Li, Hong; Zhou, Changren, E-mail: tcrz9@jnu.edu.cn

    2016-01-01

    To duplicate collagen's in vivo liquid crystalline (LC) phase and investigate the relationship between the morphology of LC collagen and osteoblast behavior, a self-assembly method was introduced for preparing collagen films with a stabilized LC phase. The LC texture and topological structure of the films before and after stabilization were observed with polarizing optical microscopy, scanning electron microscopy (SEM), and atomic force microscopy (AFM). The relationship between the collagen films and osteoblast behavior was studied with the 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide method, proliferation index detection, alkaline phosphatase measurements, osteocalcin assay, inverted microscopy, SEM observation, AFM observation, and cytoskeleton fluorescence staining. The results showed that the LC collagen film had continuously twisting orientations in the cholesteric phase with a typical series of arced patterns. The collagen fibers assembled in a well-organized orientation in the LC film. Compared to the non-LC film, the LC collagen film can promote cell proliferation, and increase ALP and osteocalcin expression, revealing a contact guide effect on osteoblasts. - Highlights: • Collagen film with liquid crystalline (LC) phase was observed by POM, SEM and AFM. • The effect of LC collagen film on osteoblasts behaviors was studied in detail. • LC collagen film promoted osteoblast proliferation and osteogenesis activity.

  15. The controlled release of simvastatin from TiO{sub 2} nanotubes to promote osteoblast differentiation and inhibit osteoclast resorption

    Energy Technology Data Exchange (ETDEWEB)

    Lai, Min, E-mail: minlai@jsnu.edu.cn [School of Life Science, Jiangsu Normal University, Xuzhou, Jiangsu 221116 (China); Jin, Ziyang; Yang, Xinyi; Wang, Huaying [School of Life Science, Jiangsu Normal University, Xuzhou, Jiangsu 221116 (China); Xu, Kui [Biomedical Engineering Center, School of Medicine, Ningbo University, Ningbo, Zhejiang 315211 (China)

    2017-02-28

    Highlights: • The TiO{sub 2} nanotube substrates filled with simvastatin were successfully coated using chitosan/gelatin multilayers. • The bio-functionalized substrates display controlled release of simvastatin in a sustained manner. • The bio-functionalized substrates have great potential for improving osteoblast differentiation. • The bio-functionalized substrates effectively inhibit osteoclast differentiation. - Abstract: The aim of this study was to fabricate a novel drug-releasing bioactive platform that has excellent potential for improving osteoblast differentiation and inhibiting osteoclast resorption. TiO{sub 2} nanotubes (TNTs) with an outer diameter of around 70 nm were prepared by an anodization method. TNTs were filled with simvastatin (SV) and then coated using chitosan/gelatin multilayers (TNT-SV-LBL). The successful fabrication of TNT-SV-LBL substrates was confirmed by field emission scanning electron microscopy (FE-SEM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and contact angle measurement, respectively. The in vitro release behavior of simvastatin from TNT-SV-LBL substrates showed a sustained release as compared to the uncoated group. Osteoblasts adhering to TNT-SV-LBL substrates attached well and displayed significantly higher (p < 0.01) cell viability compared with the other substrates. More importantly, osteoblasts grown on TNT-SV-LBL substrates displayed a statistically significant (p < 0.01 or p < 0.05) increase in protein production levels of alkaline phosphatase (ALP), osteocalcin (OC) and mRNA expression of runt related transcription factor 2 (Runx2), ALP, collagen type I (Col I), osteopontin (OPN), OC and osteoprotegerin (OPG) compared to the other groups after 4, 7 and 14 days of culture, respectively. Additionally, multinuclear osteoclastic differentiation of RAW264.7 cells grown on TNT-SV-LBL substrates was inhibited as confirmed by tartrate-resistant acid phosphatase (TRAP) analysis. These

  16. Water soluble bioactives of nacre mediate antioxidant activity and osteoblast differentiation.

    Directory of Open Access Journals (Sweden)

    Ratna Chaturvedi

    Full Text Available The water soluble matrix of nacre is a proven osteoinductive material. In spite of the differences in the biomolecular compositions of nacre obtained from multiple species of oysters, the common biochemical properties of those principles substantiate their biological activity. However, the mechanism by which nacre stimulates bone differentiation remains largely unknown. Since the positive impact of antioxidants on bone metabolism is well acknowledged, in this study we investigated the antioxidant potential of a water soluble matrix (WSM obtained from the nacre of the marine oyster Pinctada fucata, which could regulate its osteoblast differentiation activity. Enhanced levels of ALP activity observed in pre-osteoblast cells upon treatment with WSM, suggested the induction of bone differentiation events. Furthermore, bone nodule formation and up-regulation of bone differentiation marker transcripts, i.e. collagen type-1 and osteocalcin by WSM confirmed its ability to induce differentiation of the pre-osteoblasts into mature osteoblasts. Remarkably, same WSM fraction upon pre-treatment lowered the H2O2 and UV-B induced oxidative damages in keratinocytes, thus indicating the antioxidant potential of WSM. This was further confirmed from the in vitro scavenging of ABTS and DPPH free radicals and inhibition of lipid peroxidation by WSM. Together, these results indicate that WSM poses both antioxidant potential and osteoblast differentiation property. Thus, bioactivities associated with nacre holds potential in the development of therapeutics for bone regeneration and against oxidative stress induced damages in cells.

  17. Basic reactions of osteoblasts on structured material surfaces

    Directory of Open Access Journals (Sweden)

    U. Meyer

    2005-04-01

    Full Text Available In order to assess how bone substitute materials determine bone formation in vivo it is useful to understand the mechanisms of the material surface/tissue interaction on a cellular level. Artificial materials are used in two applications, as biomaterials alone or as a scaffold for osteoblasts in a tissue engineering approach. Recently, many efforts have been undertaken to improve bone regeneration by the use of structured material surfaces. In vitro studies of bone cell responses to artificial materials are the basic tool to determine these interactions. Surface properties of materials surfaces as well as biophysical constraints at the biomaterial surface are of major importance since these features will direct the cell responses. Studies on osteoblast-like cell reactivity towards materials will have to focus on the different steps of protein and cell reactions towards defined surface properties. The introduction of new techniques allows nowadays the fabrication of materials with ordered surface structures. This paper gives a review of present knowledge on the various stages of osteoblast reactions on material surfaces, focused on basic cell events under in vitro conditions. Special emphasis is given to cellular reactions towards ordered nano-sized topographies.

  18. Cytotoxic and phenotypic effects of uranium and lead on osteoblastic cellular models

    International Nuclear Information System (INIS)

    Milgram, S.

    2008-04-01

    This study is involved in the evaluation of bio-hazard associated with the use of uranium in nuclear activities and industrial research. The uranium, known in the literature as potentially carcinogenic or toxic for reproduction, can become a public health problem with the views of the various possibilities of human infections (military of the Gulf War, Finnish populations exposed to drinking water contaminated by example). The skeleton represents the organ of long-term storage of uranium and can be a target of its toxicity. Lead sharing this way of fixing in the bone matrix and have the same adverse effects on bone formation. The osteoblasts, cells responsible in bone formation, are specific targets of these two metals. The aim of this study was to evaluate the effects of acute toxicity of speciation controlled uranium and lead on osteoblasts culture. The intracellular accumulation, distribution and speciation were then studied to explain the observed toxicity. A cell death and phenotypic disorder were highlighted. The speciation is seen as crucial in biological effects of these metals. The most toxic species of both metals have been identified. The accumulation or cell distribution could not alone explain the impact of speciation on the toxicity observed. However, a phenomenon of intracellular precipitation of uranium and lead has been stressed and could be involved in a detoxification mechanism. (author)

  19. Gelatin for purification and proliferation of primary keratinocyte culture for use in chronic wounds and burns.

    Science.gov (United States)

    Rahsaz, Marjan; Geramizadeh, Bita; Kaviani, Maryam; Marzban, Saeed

    2015-04-01

    Human epidermal keratinocytes are currently established as a treatment for burns and wounds and have laboratory applications. Keratinocyte culture contamination by unwanted cells and inhibition of cell proliferation are barriers in primary keratinocyte culture. According to the recent literature, these cells are hard to culture. The present study was conducted to evaluate the efficacy of gelatin-coated surfaces in keratinocyte cultures. After enzymatic isolation of keratinocytes from normal epidermis by trypsin, the cells were cultured on gelatin-coated flasks in serum-free medium. Another group of cells were cultured as a control group without gelatin coating. We showed positive effects of surface coating with gelatin on the primary culture of keratinocytes. Culture of these cells on a gelatincoated surface showed better proliferation with suitable morphology. By using gelatin, adhesion of these cells to the surface was more efficient and without contamination by small round cells. Successful primary culture of keratinocytes on a gelatin-coated surface may provide better yield and optimal number of cells for research and clinical applications.

  20. Gene expression analysis in human osteoblasts exposed to dexamethasone identifies altered developmental pathways as putative drivers of osteoporosis

    Directory of Open Access Journals (Sweden)

    Sadlier Denise M

    2007-02-01

    Full Text Available Abstract Background Osteoporosis, a disease of decreased bone mineral density represents a significant and growing burden in the western world. Aging population structure and therapeutic use of glucocorticoids have contributed in no small way to the increase in the incidence of this disease. Despite substantial investigative efforts over the last number of years the exact molecular mechanism underpinning the initiation and progression of osteoporosis remain to be elucidated. This has meant that no significant advances in therapeutic strategies have emerged, with joint replacement surgery being the mainstay of treatment. Methods In this study we have used an integrated genomics profiling and computational biology based strategy to identify the key osteoblast genes and gene clusters whose expression is altered in response to dexamethasone exposure. Primary human osteoblasts were exposed to dexamethasone in vitro and microarray based transcriptome profiling completed. Results These studies identified approximately 500 osteoblast genes whose expression was altered. Functional characterization of the transcriptome identified developmental networks as being reactivated with 106 development associated genes found to be differentially regulated. Pathway reconstruction revealed coordinate alteration of members of the WNT signaling pathway, including frizzled-2, frizzled-7, DKK1 and WNT5B, whose differential expression in this setting was confirmed by real time PCR. Conclusion The WNT pathway is a key regulator of skeletogenesis as well as differentiation of bone cells. Reactivation of this pathway may lead to altered osteoblast activity resulting in decreased bone mineral density, the pathological hallmark of osteoporosis. The data herein lend weight to the hypothesis that alterations in developmental pathways drive the initiation and progression of osteoporosis.

  1. Impaired cholesterol esterification in primary brain cultures of the lysosomal cholesterol storage disorder (LCSD) mouse mutant

    International Nuclear Information System (INIS)

    Patel, S.C.; Suresh, S.; Weintroub, H.; Brady, R.O.; Pentchev, P.G.

    1987-01-01

    Esterification of cholesterol was investigated in primary neuroglial cultures obtained from newborn lysosomal cholesterol storage disorder (LCSD) mouse mutants. An impairment in 3 H-oleic acid incorporation into cholesteryl esters was demonstrated in cultures of homozygous LCSD brain. Primary cultures derived from other phenotypically normal pups of the carrier breeders esterified cholesterol at normal levels or at levels which were intermediary between normal and deficient indicating a phenotypic expression of the LCSD heterozygote genotype. These observations on LCSD mutant brain cells indicate that the defect in cholesterol esterification is closely related to the primary genetic defect and is expressed in neuroglial cells in culture

  2. BMP-2 Induced Expression of Alx3 That Is a Positive Regulator of Osteoblast Differentiation.

    Directory of Open Access Journals (Sweden)

    Takashi Matsumoto

    Full Text Available Bone morphogenetic proteins (BMPs regulate many aspects of skeletal development, including osteoblast and chondrocyte differentiation, cartilage and bone formation, and cranial and limb development. Among them, BMP-2, one of the most potent osteogenic signaling molecules, stimulates osteoblast differentiation, while it inhibits myogenic differentiation in C2C12 cells. To evaluate genes involved in BMP-2-induced osteoblast differentiation, we performed cDNA microarray analyses to compare BMP-2-treated and -untreated C2C12 cells. We focused on Alx3 (aristaless-like homeobox 3 which was clearly induced during osteoblast differentiation. Alx3, a homeobox gene related to the Drosophilaaristaless gene, has been linked to developmental functions in craniofacial structures and limb development. However, little is known about its direct relationship with bone formation. In the present study, we focused on the mechanisms of Alx3 gene expression and function during osteoblast differentiation induced by BMP-2. In C2C12 cells, BMP-2 induced increase of Alx3 gene expression in both time- and dose-dependent manners through the BMP receptors-mediated SMAD signaling pathway. In addition, silencing of Alx3 by siRNA inhibited osteoblast differentiation induced by BMP-2, as showed by the expressions of alkaline phosphatase (Alp, Osteocalcin, and Osterix, while over-expression of Alx3 enhanced osteoblast differentiation induced by BMP-2. These results indicate that Alx3 expression is enhanced by BMP-2 via the BMP receptors mediated-Smad signaling and that Alx3 is a positive regulator of osteoblast differentiation induced by BMP-2.

  3. Comparison of the Osteogenic Potential of Mineral Trioxide Aggregate and Endosequence Root Repair Material in a 3-dimensional Culture System.

    Science.gov (United States)

    Rifaey, Hisham S; Villa, Max; Zhu, Qiang; Wang, Yu-Hsiung; Safavi, Kamran; Chen, I-Ping

    2016-05-01

    The ability to promote osteoblast differentiation is a desirable property of root-end filling materials. Several in vitro studies compare the cytotoxicity and physical properties between mineral trioxide aggregate (MTA) and Endosequence root repair material (ERRM), but not their osteogenic potential. Three-dimensional cultures allow cells to better maintain their physiological morphology and better resemble in vivo cellular response than 2-dimensional cultures. Here we examined the osteogenic potential of MTA and ERRM by using a commercially available 3-dimensional Alvetex scaffold. Mandibular osteoblasts were derived from 3-week-old male transgenic reporter mice where mature osteoblasts express green fluorescent protein (GFP) driven by a 2.3-kilobase type I collagen promoter (Col(I)-2.3). Mandibular osteoblasts were grown on Alvetex in direct contact with MTA, ERRM, or no material (negative control) for 14 days. Osteoblast differentiation was evaluated by expression levels of osteogenic genes by using quantitative polymerase chain reaction and by the spatial dynamics of Col(I)-2.3 GFP-positive mature osteoblasts within the Alvetex scaffolds by using 2-photon microscopy. ERRM significantly increased alkaline phosphatase (Alp) and bone sialoprotein (Bsp) expression compared with MTA and negative control groups. Both MTA and ERRM increased osterix (Osx) mRNA significantly compared with the negative control group. The percentage of Col(I)-2.3 GFP-positive cells over total cells within Alvetex was the highest in the ERRM group, followed by MTA and by negative controls. ERRM promotes osteoblast differentiation better than MTA and controls with no material in a 3-dimensional culture system. Alvetex scaffolds can be used to test endodontic materials. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    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.

  5. Measuring safety culture in Dutch primary care: psychometric characteristics of the SCOPE-PC questionnaire.

    Science.gov (United States)

    Verbakel, Natasha J; Zwart, Dorien L M; Langelaan, Maaike; Verheij, Theo J M; Wagner, Cordula

    2013-09-17

    Patient safety has been a priority in primary healthcare in the last years. The prevailing culture is seen as an important condition for patient safety in practice and several tools to measure patient safety culture have therefore been developed. Although Dutch primary care consists of different professions, such as general practice, dental care, dietetics, physiotherapy and midwifery, a safety culture questionnaire was only available for general practices. The purpose of this study was to modify and validate this existing questionnaire to a generic questionnaire for all professions in Dutch primary care. A validated Dutch questionnaire for general practices was modified to make it usable for all Dutch primary care professions. Subsequently, this questionnaire was administered to a random sample of 2400 practices from eleven primary care professions. The instrument's factor structure, reliability and validity were examined using confirmatory and explorative factor analyses. 921 questionnaires were returned. Of these, 615 were eligible for factor analysis. The resulting SCOPE-PC questionnaire consisted of seven dimensions: 'open communication and learning from errors', 'handover and teamwork', 'adequate procedures and working conditions', 'patient safety management', 'support and fellowship', 'intention to report events' and 'organisational learning' with a total of 41 items. All dimensions had good reliability with Cronbach's alphas ranging from 0.70-0.90, and the questionnaire had a good construct validity. The SCOPE-PC questionnaire has sound psychometric characteristics for use by the different professions in Dutch primary care to gain insight in their safety culture.

  6. Creative Partnerships? Cultural Policy and Inclusive Arts Practice in One Primary School

    Science.gov (United States)

    Hall, Christine; Thomson, Pat

    2007-01-01

    This article traces the "cultural turn" in UK educational policy through an analysis of the Creative Partnerships policy (New Labour's "flagship programme in the cultural education field") and a consideration of an arts project funded under this initiative in one primary school. It argues that current educational policy…

  7. Improved osteoblasts growth on osteomimetic hydroxyapatite/BaTiO_3 composites with aligned lamellar porous structure

    International Nuclear Information System (INIS)

    Liu, Beilei; Chen, Liangjian; Shao, Chunsheng; Zhang, Fuqiang; Zhou, Kechao; Cao, Jun; Zhang, Dou

    2016-01-01

    Osteoblasts growing into bone substitute is an important step of bone regeneration. This study prepared porous hydroxyapatite (HA)/BaTiO_3 piezoelectric composites with porosity of 40%, 50% and 60% by ice-templating method. Effects of HA/BaTiO_3 composites with different porosities, with and without polarizing treatment on adhesion, proliferation and differentiation of osteoblasts were investigated in vitro. Results revealed that cell densities of the porous groups were significantly higher than those of the dense group (p 0.05). The absence of mechanical loading on the polarized samples may account for this. The results indicated that hierarchically porous HA/BaTiO_3 played a favorable part in osteoblasts proliferation, differentiation and adhesion process and is a promising bone substitute material. - Graphical abstract: Aligned porous structure of HA/BaTiO_3 piezoelectric composites prepared by ice-templating method was similar to the lamellar Haversian system in bone tissue. When co-cultured with human osteosarcoma cells (MG63), porous HA/BaTiO_3 composites exhibited remarkable biological activity in promoting proliferation, differentiation and adhesion of MG63 cells. - Highlights: • The aligned porous structure of HA/BaTiO_3 composite was similar to the lamellar Haversian system in bone tissue. • The piezoelectric d_3_3 coefficient of HA/BaTiO_3 with porosity of 50% was 5.0 pC/N, much higher than that of natural bone. • HA/BaTiO_3 with porosity of 50% promoted proliferation, differentiation and adhesion of MG63 cells remarkably.

  8. Design of a social constructivism-based curriculum for primary science education in Confucian heritage culture

    NARCIS (Netherlands)

    Vu Thu Hang, N.

    2014-01-01

    This study is about the application of social constructivism in primary science curriculum in Confucian heritage culture. It was found that the implementation of social constructivism in Confucian heritage culture was low and influenced by cultural divergences between Confucian cultural philosophy

  9. Effect of 670-nm Light-Emitting Diode Light On Neuronal Cultures

    Science.gov (United States)

    Wong-Riley, Margaret T. T.; Whelan, Harry T.

    2002-01-01

    Light close to and within the near infrared range has documented benefits for promoting wound healing in human and animal studies. Our preliminary results using light-emitting diodes (LEDs) in this range have also demonstrated two-to five-fold increases in growth-phase-specific DNA synthesis in normal fibroblasts, muscle cells, osteoblasts, and mucosal epithelial cells in tissue cultures. However, the mechanisms of action of such light on cells are poorly understood. We hypothesized that the therapeutic effects of such light result from the stimulation of cellular events associated with increases in cytochrome oxidase activity. As a first step in testing our hypothesis, we subjected primary neuronal cultures to impulse blockade by tetrodotoxin (TTX), a voltage-dependent sodium channel blocker, and applied LED light at 670 nm to determine if it could partially or fully reverse the reduction of cytochrome oxidase activity by TTX. The wavelength and parameters were previously tested to be beneficial for wound healing.

  10. Comparative genomic and proteomic analysis of high grade glioma primary cultures and matched tumor in situ.

    LENUS (Irish Health Repository)

    Howley, R

    2012-10-15

    Developing targeted therapies for high grade gliomas (HGG), the most common primary brain tumor in adults, relies largely on glioma cultures. However, it is unclear if HGG tumorigenic signaling pathways are retained under in-vitro conditions. Using array comparative genomic hybridization and immunohistochemical profiling, we contrasted the epidermal and platelet-derived growth factor receptor (EGFR\\/PDGFR) in-vitro pathway status of twenty-six primary HGG cultures with the pathway status of their original HGG biopsies. Genomic gains or amplifications were lost during culturing while genomic losses were more likely to be retained. Loss of EGFR amplification was further verified immunohistochemically when EGFR over expression was decreased in the majority of cultures. Conversely, PDGFRα and PDGFRβ were more abundantly expressed in primary cultures than in the original tumor (p<0.05). Despite these genomic and proteomic differences, primary HGG cultures retained key aspects of dysregulated tumorigenic signaling. Both in-vivo and in-vitro the presence of EGFR resulted in downstream activation of P70s6K while reduced downstream activation was associated with the presence of PDGFR and the tumor suppressor, PTEN. The preserved pathway dysregulation make this glioma model suitable for further studies of glioma tumorigenesis, however individual culture related differences must be taken into consideration when testing responsiveness to chemotherapeutic agents.

  11. Isolation and culture of primary adult skin fibroblasts from the Asian elephant (Elephas maximus

    Directory of Open Access Journals (Sweden)

    Puntita Siengdee

    2018-01-01

    Full Text Available Background Primary cultures from Asian elephants (Elephas maximus allow scientists to obtain representative cells that have conserved most of their original characteristics, function, physiology and biochemistry. This technique has thus gained significant importance as a foundation for further cellular, cell biology and molecular research. Therefore, the aim of this study was to describe conditions for the successful establishment of primary adult fibroblasts from Asian elephant carcasses. Methods Ear tissue sample collection from Asian elephant carcasses and our recommendations are given. We describe here a simple modified protocol for successful isolation and maintenance of primary adult fibroblasts from elephant ear skin. Ear samples from each individual (five 3 × 3 cm2 pieces were brought to the laboratory within 3 h after collection, kept in transportation medium at 0–4 °C. The ear tissues were prepared by a combination of 10% collagenase type II digestion procedure together with a simple explant procedure. Primary fibroblasts were cultured at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM with 20% fetal calf serum (FCS in a humidified atmosphere containing 5% CO2. After the third passage, fibroblasts were routinely trypsinized with 0.25% trypsin/EDTA and cultured in DMEM with 10% FCS at 37 °C and 5% CO2. Traditional cell counting method was used to measure cell viability and growth curve. Long-term storage of cells used freezing medium consisting of 40% FCS (v/v. Results We explored the most suitable conditions during sample collection (post-mortem storage time and sample storage temperature, which is the most important step in determining primary outgrowth. Our study successfully established and cultured primary adult skin fibroblasts obtained from post-mortem E. maximus ear skin tissues from six carcasses, with a success rate of around 83.3%. Outgrowth could be seen 4–12 days after explantation, and epithelial

  12. NELL-1 increases pre-osteoblast mineralization using both phosphate transporter Pit1 and Pit2

    Energy Technology Data Exchange (ETDEWEB)

    Cowan, Catherine M. [Department of Bioengineering, University of California, Los Angeles, 420 Westwood Plaza,7523 Boelter Hall, Los Angeles, CA 90095 (United States); Dental and Craniofacial Research Institute and Section of Orthodontics, School of Dentistry, University of California, Los Angeles, 40833 Le Conte Ave, Los Angeles, CA 90095 (United States); Zhang, Xinli; James, Aaron W.; Mari Kim, T.; Sun, Nichole [Dental and Craniofacial Research Institute and Section of Orthodontics, School of Dentistry, University of California, Los Angeles, 40833 Le Conte Ave, Los Angeles, CA 90095 (United States); Wu, Benjamin [Department of Bioengineering, University of California, Los Angeles, 420 Westwood Plaza,7523 Boelter Hall, Los Angeles, CA 90095 (United States); Dental and Craniofacial Research Institute and Section of Orthodontics, School of Dentistry, University of California, Los Angeles, 40833 Le Conte Ave, Los Angeles, CA 90095 (United States); Ting, Kang [Dental and Craniofacial Research Institute and Section of Orthodontics, School of Dentistry, University of California, Los Angeles, 40833 Le Conte Ave, Los Angeles, CA 90095 (United States); Soo, Chia, E-mail: bsoo@ucla.edu [UCLA and Orthopaedic Hospital Department of Orthopaedic Surgery and the Orthopaedic, Hospital Research Center, University of California, Los Angeles, 2641 Charles E. Young Dr. South, Los Angeles, CA 90095 (United States)

    2012-06-08

    Highlights: Black-Right-Pointing-Pointer NELL-1 accelerates extracellular matrix mineralization in MC3T3-E1 pre-osteoblasts. Black-Right-Pointing-Pointer NELL-1 significantly increases intracellular inorganic phosphate levels. Black-Right-Pointing-Pointer NELL-1 positively regulates osteogenesis but not proliferation in MC3T3-E1 cells. Black-Right-Pointing-Pointer NELL-1 regulates inorganic phosphate transporter activity. -- Abstract: NELL-1 is a potent osteoinductive molecule that enhances bone formation in multiple animal models through currently unidentified pathways. In the present manuscript, we hypothesized that NELL-1 may regulate osteogenic differentiation accompanied by alteration of inorganic phosphate (Pi) entry into the osteoblast via sodium dependent phosphate (NaPi) transporters. To determine this, MC3T3-E1 pre-osteoblasts were cultured in the presence of recombinant human (rh)NELL-1 or rhBMP-2. Analysis was performed for intracellular Pi levels through malachite green staining, Pit-1 and Pit-2 expression, and forced upregulation of Pit-1 and Pit-2. Results showed rhNELL-1 to increase MC3T3-E1 matrix mineralization and Pi influx associated with activation of both Pit-1 and Pit-2 channels, with significantly increased Pit-2 production. In contrast, Pi transport elicited by rhBMP-2 showed to be associated with increased Pit-1 production only. Next, neutralizing antibodies against Pit-1 and Pit-2 completely abrogated the Pi influx effect of rhNELL-1, suggesting rhNELL-1 is dependent on both transporters. These results identify one potential mechanism of action for rhNELL-1 induced osteogenesis and highlight a fundamental difference between NELL-1 and BMP-2 signaling.

  13. NELL-1 increases pre-osteoblast mineralization using both phosphate transporter Pit1 and Pit2

    International Nuclear Information System (INIS)

    Cowan, Catherine M.; Zhang, Xinli; James, Aaron W.; Mari Kim, T.; Sun, Nichole; Wu, Benjamin; Ting, Kang; Soo, Chia

    2012-01-01

    Highlights: ► NELL-1 accelerates extracellular matrix mineralization in MC3T3-E1 pre-osteoblasts. ► NELL-1 significantly increases intracellular inorganic phosphate levels. ► NELL-1 positively regulates osteogenesis but not proliferation in MC3T3-E1 cells. ► NELL-1 regulates inorganic phosphate transporter activity. -- Abstract: NELL-1 is a potent osteoinductive molecule that enhances bone formation in multiple animal models through currently unidentified pathways. In the present manuscript, we hypothesized that NELL-1 may regulate osteogenic differentiation accompanied by alteration of inorganic phosphate (Pi) entry into the osteoblast via sodium dependent phosphate (NaPi) transporters. To determine this, MC3T3-E1 pre-osteoblasts were cultured in the presence of recombinant human (rh)NELL-1 or rhBMP-2. Analysis was performed for intracellular Pi levels through malachite green staining, Pit-1 and Pit-2 expression, and forced upregulation of Pit-1 and Pit-2. Results showed rhNELL-1 to increase MC3T3-E1 matrix mineralization and Pi influx associated with activation of both Pit-1 and Pit-2 channels, with significantly increased Pit-2 production. In contrast, Pi transport elicited by rhBMP-2 showed to be associated with increased Pit-1 production only. Next, neutralizing antibodies against Pit-1 and Pit-2 completely abrogated the Pi influx effect of rhNELL-1, suggesting rhNELL-1 is dependent on both transporters. These results identify one potential mechanism of action for rhNELL-1 induced osteogenesis and highlight a fundamental difference between NELL-1 and BMP-2 signaling.

  14. Effects of Ranolazine on Astrocytes and Neurons in Primary Culture.

    Directory of Open Access Journals (Sweden)

    Martin Aldasoro

    Full Text Available Ranolazine (Rn is an antianginal agent used for the treatment of chronic angina pectoris when angina is not adequately controlled by other drugs. Rn also acts in the central nervous system and it has been proposed for the treatment of pain and epileptic disorders. Under the hypothesis that ranolazine could act as a neuroprotective drug, we studied its effects on astrocytes and neurons in primary culture. We incubated rat astrocytes and neurons in primary cultures for 24 hours with Rn (10-7, 10-6 and 10-5 M. Cell viability and proliferation were measured using trypan blue exclusion assay, MTT conversion assay and LDH release assay. Apoptosis was determined by Caspase 3 activity assay. The effects of Rn on pro-inflammatory mediators IL-β and TNF-α was determined by ELISA technique, and protein expression levels of Smac/Diablo, PPAR-γ, Mn-SOD and Cu/Zn-SOD by western blot technique. In cultured astrocytes, Rn significantly increased cell viability and proliferation at any concentration tested, and decreased LDH leakage, Smac/Diablo expression and Caspase 3 activity indicating less cell death. Rn also increased anti-inflammatory PPAR-γ protein expression and reduced pro-inflammatory proteins IL-1 β and TNFα levels. Furthermore, antioxidant proteins Cu/Zn-SOD and Mn-SOD significantly increased after Rn addition in cultured astrocytes. Conversely, Rn did not exert any effect on cultured neurons. In conclusion, Rn could act as a neuroprotective drug in the central nervous system by promoting astrocyte viability, preventing necrosis and apoptosis, inhibiting inflammatory phenomena and inducing anti-inflammatory and antioxidant agents.

  15. Blockade of Drp1 rescues oxidative stress-induced osteoblast dysfunction

    Energy Technology Data Exchange (ETDEWEB)

    Gan, Xueqi; Huang, Shengbin; Yu, Qing [Department of Pharmacology and Toxicology and Higuchi Bioscience Center, University of Kansas, Lawrence, KS, 66047 (United States); State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, 610041 (China); Yu, Haiyang [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, 610041 (China); Yan, Shirley ShiDu, E-mail: shidu@ku.edu [Department of Pharmacology and Toxicology and Higuchi Bioscience Center, University of Kansas, Lawrence, KS, 66047 (United States)

    2015-12-25

    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) activity 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.

  16. Sodium nitroprusside induces autophagic cell death in glutathione-depleted osteoblasts.

    Science.gov (United States)

    Son, Min Jeong; Lee, Seong-Beom; Byun, Yu Jeong; Lee, Hwa Ok; Kim, Ho-Shik; Kwon, Oh-Joo; Jeong, Seong-Whan

    2010-01-01

    Previous studies reported that high levels of nitric oxide (NO) induce apoptotic cell death in osteoblasts. We examined molecular mechanisms of cytotoxic injury induced by sodium nitroprusside (SNP), a NO donor, in both glutathione (GSH)-depleted and control U2-OS osteoblasts. Cell viability was reduced by much lower effective concentrations of SNP in GSH-depleted cells compared to normal cells. The data suggest that the level of intracellular GSH is critical in SNP-induced cell death processes of osteoblasts. The level of oxidative stress due to SNP treatments doubled in GSH-depleted cells when measured with fluorochrome H2DCFDA. Pretreatment with the NO scavenger PTIO preserved the viability of cells treated with SNP. Viability of cells treated with SNP was recovered by pretreatment with Wortmannin, an autophagy inhibitor, but not by pretreatment with zVAD-fmk, a pan-specific caspase inhibitor. Large increases of LC3-II were shown by immunoblot analysis of the SNP-treated cells, and the increase was blocked by pretreatment with PTIO or Wortmannin; this implies that under GSH-depleted conditions SNP induces different molecular signaling that lead to autophagic cell death. The ultrastructural morphology of SNP-treated cells in transmission electron microscopy showed numerous autophagic vacuoles. These data suggest NO produces oxidative stress and cellular damage that culminate in autophagic cell death of GSH-depleted osteoblasts. Copyright 2010 Wiley Periodicals, Inc.

  17. Osteogenic gene expression of murine osteoblastic (MC3T3-E1) cells under cyclic tension

    International Nuclear Information System (INIS)

    Kao, C T; Chen, C C; Cheong, U-I; Liu, S L; Huang, T H

    2014-01-01

    Low-level laser therapy (LLLT) can promote cell proliferation. The remodeling ability of the tension side of orthodontic teeth affects post-orthodontic stability. The purpose of the present study was to investigate the osteogenic effects of LLLT on osteoblast-like cells treated with a simulated tension system that provides a mechanical tension regimen. Murine osteoblastic (MC3T3-E1) cells were cultured in a Flexcell strain unit with programmed loads of 12% elongation at a frequency of 0.5 Hz for 24 and 48 h. The cultured cells were treated with a low-level diode laser using powers of 5 J and 10 J. The proliferation of MC3T3-E1 cells was determined using the Alamar Blue assay. The expression of osteogenic genes (type I collagen (Col-1), osteopontin (OPN), osteocalcin (OC), osteoprotegerin (OPG), receptor activator of nuclear factor kappa B ligand (RANKL), bone morphologic protein (BMP-2), and bone morphologic protein (BMP-4)) in MC3T3-E1 cells was analyzed using reverse transcription polymerase chain reaction (RT-PCR). The data were analyzed using one-way analysis of variance. The proliferation rate of tension-cultured MC3T3-E1 cells under 5 J and 10 J LLLT increased compared with that of the control group (p < 0.05). Prominent mineralization of the MC3T3-E1 cells was visible using a von Kossa stain in the 5 J LLLT group. Osteogenic genes (Col-1, OC, OPG and BMP-2) were significantly expressed in the MC3T3-E1 cells treated with 5 J and 10 J LLLT (p < 0.05). LLLT in tension-cultured MC3T3-E1 cells showed synergistic osteogenic effects, including increases in cell proliferation and Col-1, OPN, OC, OPG and BMP-2 gene expression. LLLT might be beneficial for bone remodeling on the tension side of orthodontics. (paper)

  18. Evaluation of the effect of three monazite constituents on the radiosensitivity of human osteoblasts

    International Nuclear Information System (INIS)

    Iwahara, Lucas Kiyoshi da Fonseca; Oliveira, Monica Stuck de; Alencar, Marcus Alexandre Vallim de

    2017-01-01

    Thorium has gained notoriety in recent years, as a potential source of nuclear energy, substituting uranium in power plants. Monazite is an important font of thorium, as well of uranium and rare earths elements. Professionals involved in the extraction and manipulation of this mineral are occupationally exposed to aerosols containing metals and to ionizing radiation. This paper analyzed the effects of thorium, cerium and lanthanum on cell radiosensitivity. As an osteotropic substance, thorium is mostly deposited in bone tissue and may interfere in cellular radiosensitivity. A human osteoblast cell line was used to evaluate the effects of thorium, cerium and lanthanum on cell radiosensitivity, using proliferation as indicator. Assays were performed using cell cultures exposed to metals and to ionizing radiation. As a result, metals in combination with ionizing radiation induced changes on cell proliferation, in a concentration-dependent manner, in comparison with the exposure to metals alone. That suggests the possibility of combination interfering with radiosensitivity of osteoblasts, indicating an enhancement in occupational risk for workers that manipulate monazite byproducts and are subject to radiation in the environment. Thus, the development of risk assessment models that include the evaluation of metal-radiation mixtures and their cytotoxic and radiotoxic effects on tissues and organs must be highlighted. (author)

  19. Lysophosphatidic acid induces chemotaxis in MC3T3-E1 osteoblastic cells

    Energy Technology Data Exchange (ETDEWEB)

    Masiello, Lisa M.; Fotos, Joseph S.; Galileo, Deni S.; Karin, Norm J.

    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 fewer 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.

  20. Sugammadex, a Neuromuscular Blockade Reversal Agent, Causes Neuronal Apoptosis in Primary Cultures

    Science.gov (United States)

    Palanca, José M.; Aguirre-Rueda, Diana; Granell, Manuel V.; Aldasoro, Martin; Garcia, Alma; Iradi, Antonio; Obrador, Elena; Mauricio, Maria Dolores; Vila, Jose; Gil-Bisquert, Anna; Valles, Soraya L.

    2013-01-01

    Sugammadex, a γ-cyclodextrin that encapsulates selectively steroidal neuromuscular blocking agents, such as rocuronium or vecuronium, has changed the face of clinical neuromuscular pharmacology. Sugammadex allows a rapid reversal of muscle paralysis. Sugammadex appears to be safe and well tolerated. Its blood-brain barrier penetration is poor (Sugammadex in neurons in primary culture. Here we show that clinically relevant sugammadex concentrations cause apoptotic/necrosis neuron death in primary cultures. Studies on the underlying mechanism revealed that sugammadex-induced activation of mitochondria-dependent apoptosis associates with depletion of neuronal cholesterol levels. Furthermore SUG increase CytC, AIF, Smac/Diablo and CASP-3 protein expression in cells in culture. Potential association of SUG-induced alteration in cholesterol homeostasis with oxidative stress and apoptosis activation occurs. Furthermore, resistance/sensitivity to oxidative stress differs between neuronal cell types. PMID:23983586

  1. Low bone mass and changes in the osteocyte network in mice lacking autophagy in the osteoblast lineage.

    Science.gov (United States)

    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.

  2. Estrogen/ERα signaling axis participates in osteoblast maturation via upregulating chromosomal and mitochondrial complex gene expressions

    Science.gov (United States)

    Lin, Pei-I; Tai, Yu-Ting; Chan, Wing P.; Lin, Yi-Ling; Liao, Mei-Hsiu; Chen, Ruei-Ming

    2018-01-01

    Estrogen deficiency usually leads to bone loss and osteoporosis in postmenopausal women. Osteoblasts play crucial roles in bone formation. However, osteoblast functions are influenced by mitochondrial bioenergetic conditions. In this study, we investigated the roles of the estrogen and estrogen receptor alpha (ERα) axis in mitochondrial energy metabolism and subsequent osteoblast mineralization. Exposure of rat calvarial osteoblasts to estradiol caused substantial improvements in alkaline phosphatase activities and cell calcification. In parallel, treatment of human osteoblast-like U2OS cells, derived from a female osteosarcoma patient, with estradiol specifically augmented ERα levels. Sequentially, estradiol stimulated translocation of ERα to nuclei in human osteoblasts and induced expressions of genomic respiratory chain complex NDUFA10, UQCRC1, cytochrome c oxidase (COX)8A, COX6A2, COX8C, COX6C, COX6B2, COX412, and ATP12A genes. Concurrently, estradiol stimulated translocation of ERα to mitochondria from the cytoplasm. A bioinformatic search found the existence of four estrogen response elements in the 5’-promoter region of the mitochondrial cox i gene. Interestingly, estradiol induced COX I mRNA and protein expressions in human osteoblasts or rat calvarial osteoblasts. Knocking-down ERα translation concurrently downregulated estradiol-induced COX I mRNA expression. Consequently, exposure to estradiol led to successive increases in the mitochondrial membrane potential, the mitochondrial enzyme activity, and cellular adenosine triphosphate levels. Taken together, this study showed the roles of the estradiol/ERα signaling axis in improving osteoblast maturation through upregulating the mitochondrial bioenergetic system due to induction of definite chromosomal and mitochondrial complex gene expressions. Our results provide novel insights elucidating the roles of the estrogen/ERα alliance in regulating bone formation. PMID:29416685

  3. Generation of primary cultures of bovine brain endothelial cells and setup of cocultures with rat astrocytes

    DEFF Research Database (Denmark)

    Helms, Hans C; Brodin, Birger

    2014-01-01

    -brain barrier. The present protocol describes the setup of an in vitro coculture model based on primary cultures of endothelial cells from bovine brain microvessels and primary cultures of rat astrocytes. The model displays a high electrical tightness and expresses blood-brain barrier marker proteins....

  4. Biocompatibility evaluation in vitro. Part I: Morphology expression and proliferation of human and rat osteoblasts on the biomaterials

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The biocompatibility evaluation of calcium phosphate based biomaterials is performed by tissue culture in vitro model. Three kinds of bioceramic materials which are potential to deal with bone trauma and/or conduct tissue growth are recommodated. The biological research results show that human and animal osteoblast cells anchor the materials surface in two hours in culture. Confocal laser scanning microscopy (CLSM) demonstrated the normal cell distribution and proliferation on both of dense and porous biomaterials. Hydroxyapatite and tricalcium phosphate stimulate cell proliferation. However, DNA and protein synthesis were considerably limited and the apoptosis phenomenon would be present on the hydroxyapatite (HA) materials by adding Al, Mg elements. Several important methods of biocompatibility evaluation of implant materials are described and the related biological molecular techniques such as tissue culture, cell transfection, cellular DNA stain, and Lowry assay are involved in the present research.

  5. New titanium and titanium/hydroxyapatite coatings on ultra-high-molecular-weight polyethylene-in vitro osteoblastic performance

    International Nuclear Information System (INIS)

    Silva, M A; Lopes, M A; Santos, J D; Fernandes, M H; Gomes, P S; Vila, M; Silva, R F

    2010-01-01

    The development of optimized hip joint materials is one of the most challenging opportunities in prosthetic technologies. In current approaches, ultra-high-molecular-weight polyethylene (UHMWPE) has been a favorite material for the acetabular component and, regarding the cementless technique, several coating options may be considered to contain and stabilize bearing surfaces and establish an improved interface with bone. In this work, newly developed constructs of UHMWPE coated with either commercially pure titanium (cpTi-UHMWPE), by DC magnetron sputtering, or with commercially pure titanium and hydroxyapatite (cpTi/HA-UHMWPE), by DC/RF magnetron co-sputtering, have been prepared and biologically characterized with human bone marrow-derived osteoblastic cultures. The cpTi-UHMWPE samples allowed a high cell growth and the expression of the complete osteoblastic phenotype, with high alkaline phosphatase activity, expression of osteogenic-associated genes and evident cell-mediated mineralization of the extracellular matrix. In comparison, the cpTi/HA-UHMWPE samples reported lower cell proliferation but earlier cell-mediated matrix mineralization. Accordingly, these newly developed systems may be suitable candidates to improve the osteointegration process in arthroplastic devices; nevertheless, further biological evaluation should be conducted.

  6. Multi-lipofection efficiently transfected genes into astrocytes in primary culture.

    Science.gov (United States)

    Wu, B Y; Liu, R Y; So, K L; Yu, A C

    2000-10-30

    This study demonstrated that liposome-mediated transfection - lipofection - is suitable for delivering genes into astrocytes. By repeatedly lipofecting the same astrocyte cultures, a process we call multi-lipofection, the transfection efficiency of the beta-galactosidase (beta-gal) gene was improved from 2.6+/-0.6 to 17. 4+/-1.1%. This is the highest efficiency ever reported in gene-transfer with Lipofectin(R) in a primary culture of mouse cerebral cortical astrocytes. Furthermore, multi-lipofection did not cause observable disturbance to astrocytes as indicated by insignificant changes in the glial fibrillary acidic protein content in the cultures. In order to demonstrate that the transfected gene achieved a physiologically relevant expression level, a plasmid containing the pEF-hsp70 protein gene was lipofected into astrocytes. This produced colonies of astrocytes showing an increased resistance to heat-induced cell death. A similar experiment was performed with the glial-derived neurotrophic factor (GDNF) gene. Control astrocytes had no detectable GDNF. In the transfected astrocytes, the GDNF protein could be identified intracellularly by immunocytochemistry. Western blot analysis revealed, as compared to astrocytes with one lipofection, a 2.9-fold increase of GDNF with four lipofections. GDNF remained detectable in astrocytes 2 weeks after four lipofections. Thus, multi-lipofection provides a mild and efficient means of delivering foreign genes into astrocytes in a primary culture, making astrocytes good candidate vehicle cells for gene/cell therapy in the CNS.

  7. Cellular Microenvironment Dictates Androgen Production by Murine Fetal Leydig Cells in Primary Culture1

    Science.gov (United States)

    Carney, Colleen M.; Muszynski, Jessica L.; Strotman, Lindsay N.; Lewis, Samantha R.; O'Connell, Rachel L.; Beebe, David J.; Theberge, Ashleigh B.; Jorgensen, Joan S.

    2014-01-01

    ABSTRACT Despite the fact that fetal Leydig cells are recognized as the primary source of androgens in male embryos, the mechanisms by which steroidogenesis occurs within the developing testis remain unclear. A genetic approach was used to visualize and isolate fetal Leydig cells from remaining cells within developing mouse testes. Cyp11a1-Cre mice were bred to mT/mG dual reporter mice to target membrane-tagged enhanced green fluorescent protein (GFP) within steroidogenic cells, whereas other cells expressed membrane-tagged tandem-dimer tomato red. Fetal Leydig cell identity was validated using double-labeled immunohistochemistry against GFP and the steroidogenic enzyme 3beta-HSD, and cells were successfully isolated as indicated by qPCR results from sorted cell populations. Because fetal Leydig cells must collaborate with neighboring cells to synthesize testosterone, we hypothesized that the fetal Leydig cell microenvironment defined their capacity for androgen production. Microfluidic culture devices were used to measure androstenedione and testosterone production of fetal Leydig cells that were cultured in cell-cell contact within a mixed population, were isolated but remained in medium contact via compartmentalized co-culture with other testicular cells, or were isolated and cultured alone. Results showed that fetal Leydig cells maintained their identity and steroidogenic activity for 3–5 days in primary culture. Microenvironment dictated proficiency of testosterone production. As expected, fetal Leydig cells produced androstenedione but not testosterone when cultured in isolation. More testosterone accumulated in medium from mixed cultures than from compartmentalized co-cultures initially; however, co-cultures maintained testosterone synthesis for a longer time. These data suggest that a combination of cell-cell contact and soluble factors constitute the ideal microenvironment for fetal Leydig cell activity in primary culture. PMID:25143354

  8. Osteoblast and osteoclast behaviors in the turnover of attachment bones during medaka tooth replacement.

    Science.gov (United States)

    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.

  9. rhEPO Enhances Cellular Anti-oxidant Capacity to Protect Long-Term Cultured Aging Primary Nerve Cells.

    Science.gov (United States)

    Wang, Huqing; Fan, Jiaxin; Chen, Mengyi; Yao, Qingling; Gao, Zhen; Zhang, Guilian; Wu, Haiqin; Yu, Xiaorui

    2017-08-01

    Erythropoietin (EPO) may protect the nervous system of animals against aging damage, making it a potential anti-aging drug for the nervous system. However, experimental evidence from natural aging nerve cell models is lacking, and the efficacy of EPO and underlying mechanism of this effect warrant further study. Thus, the present study used long-term cultured primary nerve cells to successfully mimic the natural aging process of nerve cells. Starting on the 11th day of culture, cells were treated with different concentrations of recombinant human erythropoietin (rhEPO). Using double immunofluorescence labeling, we found that rhEPO significantly improved the morphology of long-term cultured primary nerve cells and increased the total number of long-term cultured primary cells. However, rhEPO did not improve the ratio of nerve cells. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to measure nerve cell activity and showed that rhEPO significantly improved the activity of long-term cultured primary nerve cells. Moreover, Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double immunofluorescence labeling flow cytometry revealed that rhEPO reduced the apoptotic rate of long-term cultured primary nerve cells. Senescence-associated β-galactosidase (SA-β-gal) immunohistochemistry staining showed that rhEPO significantly reduced the aging rate of long-term cultured primary nerve cells. Immunochemistry revealed that rhEPO enhanced intracellular superoxide dismutase (SOD) activity and glutathione (GSH) abundance and reduced the intracellular malondialdehyde (MDA) level. In addition, this effect depended on the dose, was maximized at a dose of 100 U/ml and was more pronounced than that of vitamin E. In summary, this study finds that rhEPO protects long-term cultured primary nerve cells from aging in a dose-dependent manner. The mechanism of this effect may be associated with the enhancement of the intracellular anti

  10. Internal structures of scaffold-free 3D cell cultures visualized by synchrotron radiation-based micro-computed tomography

    Science.gov (United States)

    Saldamli, Belma; Herzen, Julia; Beckmann, Felix; Tübel, Jutta; Schauwecker, Johannes; Burgkart, Rainer; Jürgens, Philipp; Zeilhofer, Hans-Florian; Sader, Robert; Müller, Bert

    2008-08-01

    Recently the importance of the third dimension in cell biology has been better understood, resulting in a re-orientation towards three-dimensional (3D) cultivation. Yet adequate tools for their morphological characterization have to be established. Synchrotron radiation-based micro computed tomography (SRμCT) allows visualizing such biological systems with almost isotropic micrometer resolution, non-destructively. We have applied SRμCT for studying the internal morphology of human osteoblast-derived, scaffold-free 3D cultures, termed histoids. Primary human osteoblasts, isolated from femoral neck spongy bone, were grown as 2D culture in non-mineralizing osteogenic medium until a rather thick, multi-cellular membrane was formed. This delicate system was intentionally released to randomly fold itself. The folded cell cultures were grown to histoids of cubic milli- or centimeter size in various combinations of mineralizing and non-mineralizing osteogenic medium for a total period of minimum 56 weeks. The SRμCT-measurements were performed in the absorption contrast mode at the beamlines BW 2 and W 2 (HASYLAB at DESY, Hamburg, Germany), operated by the GKSS-Research Center. To investigate the entire volume of interest several scans were performed under identical conditions and registered to obtain one single dataset of each sample. The histoids grown under different conditions exhibit similar external morphology of globular or ovoid shape. The SRμCT-examination revealed the distinctly different morphological structures inside the histoids. One obtains details of the histoids that permit to identify and select the most promising slices for subsequent histological characterization.

  11. Role of a new member of IGFBP superfamily, IGFBP-rP10, in proliferation and differentiation of osteoblastic cells

    International Nuclear Information System (INIS)

    Shibata, Yasuaki; Tsukazaki, Tomoo; Hirata, Kazunari; Xin Cheng; Yamaguchi, Akira

    2004-01-01

    Bone regeneration is critically regulated by various molecules. To identify the new genes involved in bone regeneration, we performed microarray-based gene expression analysis using a mouse bone regeneration model. We identified a new member of the IGFBP superfamily, designated IGFBP-rP10, whose expression is up-regulated at the early phase of bone regeneration. IGFBP-rP10 consists of an IGFBP homologous domain followed by a Kazal-type protein inhibitor domain and an immunoglobulin G-like domain. A real-time-based RT-PCR analysis demonstrated that various tissues including bone expressed IGFBP-rP10 mRNA in various degrees, and confirmed an up-regulation at the early phase of bone regeneration. In situ hybridization revealed that osteoblastic cells expressed IGFPB-rP10 mRNA during bone regeneration. Bone morphogenetic protein-2 increased the expression level of IGFBP-rP10 mRNA in various cells including C3H10T1/2, MC3T3-E1, C2C12, and primary murine osteoblastic cells. The addition of recombinant mouse IGFBP-rP10 promoted the proliferation of these cells but failed to stimulate alkaline phosphatase activity. These results suggest that IGFBP-rP10 is involved in the proliferation of osteoblasts during bone formation and bone regeneration

  12. Primary care resident perceived preparedness to deliver cross-cultural care: an examination of training and specialty differences.

    Science.gov (United States)

    Greer, Joseph A; Park, Elyse R; Green, Alexander R; Betancourt, Joseph R; Weissman, Joel S

    2007-08-01

    Previous research has shown that resident physicians report differences in training across primary care specialties, although limited data exist on education in delivering cross-cultural care. The goals of this study were to identify factors that relate to primary care residents' perceived preparedness to provide cross-cultural care and to explore the extent to which these perceptions vary across primary care specialties. Cross-sectional, national mail survey of resident physicians in their last year of training. Eleven hundred fifty primary care residents specializing in family medicine (27%), internal medicine (23%), pediatrics (26%), and obstetrics/gynecology (OB/GYN) (24%). Male residents as well as those who reported having graduated from U.S. medical schools, access to role models, and a greater cross-cultural case mix during residency felt more prepared to deliver cross-cultural care. Adjusting for these demographic and clinical factors, family practice residents were significantly more likely to feel prepared to deliver cross-cultural care compared to internal medicine, pediatric, and OB/GYN residents. Yet, when the quantity of instruction residents reported receiving to deliver cross-cultural care was added as a predictor, specialty differences became nonsignificant, suggesting that training opportunities better account for the variability in perceived preparedness than specialty. Across primary care specialties, residents reported different perceptions of preparedness to deliver cross-cultural care. However, this variation was more strongly related to training factors, such as the amount of instruction physicians received to deliver such care, rather than specialty affiliation. These findings underscore the importance of formal education to enhance residents' preparedness to provide cross-cultural care.

  13. Intracortical osteoblastic osteosarcoma with oncogenic rickets

    International Nuclear Information System (INIS)

    Hasegawa, T.; Hirohashi, Setsuo; Shimoda, Tadakazu; Yokoyama, Ryohei; Beppu, Yasuo; Maeda, Shotaro

    1999-01-01

    Intracortical osteosarcoma is the rarest variant of osteosarcoma, occurring within, and usually confined to, the cortical bone. Oncogenic osteomalacia, or rickets, is an unusual clinicopathologic entity in which vitamin D-resistant osteomalacia, or rickets, occurs in association with some tumors of soft tissue or bone. We present a case of oncogenic rickets associated with intracortical osteosarcoma of the tibia in a 9-year-old boy, whose roentgenographic abnormalities of rickets disappeared and pertinent laboratory data except for serum alkaline phosphatase became normal after surgical resection of the tumor. Histologically, the tumor was an osteosarcoma with a prominent osteoblastic pattern. An unusual microscopic feature was the presence of matrix mineralization showing rounded calcified structures (calcified spherules). Benign osteoblastic tumors, such as osteoid osteoma and osteoblastoma, must be considered in the differential diagnosis because of the relatively low cellular atypia and mitotic activity of this tumor. The infiltrating pattern with destruction or engulfment of normal bone is a major clue to the correct diagnosis of intracortical osteosarcoma. The co-existing radiographic changes of rickets were due to the intracortical osteosarcoma. (orig.)

  14. The role of kaempferol-induced autophagy on differentiation and mineralization of osteoblastic MC3T3-E1 cells.

    Science.gov (United States)

    Kim, In-Ryoung; Kim, Seong-Eon; Baek, Hyun-Su; Kim, Bok-Joo; Kim, Chul-Hoon; Chung, In-Kyo; Park, Bong-Soo; Shin, Sang-Hun

    2016-08-31

    beclin-1, SQSTM1/p62 and the conversion of LC3-II from LC3-I. The application of 3-MA decreased the activity of ALP and the autophagy induced by kaempferol. In the RT-PCR analysis, the expression of RUNX-2, osterix, BMP-2 and collagen I was decreased. The present study showed that kaempferol stimulated the osteogenic differentiation of cultured osteoblasts by inducing autophagy.

  15. The blood-brain barrier in vitro using primary culture

    DEFF Research Database (Denmark)

    Larsen, Annette Burkhart

    The brain is protected from the entry of unwanted substances by means of the blood-brain barrier (BBB) formed by the brain microvasculature. This BBB is composed of non-fenestrated brain capillary endothelial cells (BCECs) with their intermingling tight junctions. The presence of the BBB is a huge...... obstacle for the treatment of central nervous system (CNS) diseases, as many potentially CNS active drugs are unable to reach their site of action within the brain. In vitro BBB models are, therefore, being developed to investigate the BBB permeability of a drug early in its development. The first part...... of the thesis involves the establishment and characterization of an in vitro BBB models based on primary cells isolated from the rat brain. Co-culture and triple culture models with astrocytes and pericytes were found to be the superior to mono cultured BCECs with respect to many important BBB characteristics...

  16. Lysophosphatidic acid-functionalised titanium as a superior surface for supporting human osteoblast (MG63 maturation

    Directory of Open Access Journals (Sweden)

    JP Mansell

    2012-05-01

    Full Text Available Covalent modifications of titanium with small molecules known to promote human osteoblast maturation are especially attractive in developing superior biomaterials. An important step in securing competent bone formation at implant sites is promoting the formation of mature osteoblasts, either from committed pre-osteoblasts or from their mesenchymal progenitors. To this end our research has focussed on identifying molecules that enhance human osteoblast formation and maturation and to develop ways of covalently attaching these molecules to implant surfaces so that they are more likely to withstand the rigors of the implantation process whilst still retaining their bioactivity. Herein we report the novel production of lipid-functionalised titanium using lysophosphatidic acid or a related compound, (3S 1-fluoro-3-hydroxy-4-butyl-1-phosphonate. Both lipids were especially effective at co-operating with calcitriol to promote human osteoblast maturation at these modified Ti surfaces in vitro. The novel findings presented offer enticing new developments towards the fabrication of next-generation implant devices with the potential to significantly enhance the osseointegration process and with it improvements in future prosthesis performance and longevity.

  17. Designing a primary science curriculum in a globalizing world: How do social constructivism and Vietnamese culture meet?

    Science.gov (United States)

    Hằng, Ngô Vũ Thu; Meijer, Marijn Roland; Bulte, Astrid M. W.; Pilot, Albert

    2017-09-01

    The implementation of social constructivist approaches to learning science in primary education in Vietnamese culture as an example of Confucian heritage culture remains challenging and problematic. This theoretical paper focuses on the initial phase of a design-based research approach; that is, the description of the design of a formal, written curriculum for primary science education in which features of social constructivist approaches to learning are synthesized with essential aspects of Vietnamese culture. The written design comprises learning aims, a framework that is the synthesis of learning functions, learning settings and educational expectations for learning phases, and exemplary curriculum units. Learning aims are formulated to comprehensively develop scientific knowledge, skills, and attitudes toward science for primary students. Derived from these learning aims, the designed framework consists of four learning phases respectively labeled as Engagement, Experience, Exchange, and Follow-up. The designed framework refers to knowledge of the "nature of science" education and characteristics of Vietnamese culture as an example of Confucian heritage culture. The curriculum design aims to serve as an educational product that addresses previously analyzed problems of primary science education in the Vietnamese culture in a globalizing world.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-10-24

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

  19. Where Cultural Games Count: The Voices of Primary Classroom Teachers

    Science.gov (United States)

    Nabie, Michael Johnson

    2015-01-01

    This study explored Ghanaian primary school teachers' values and challenges of integrating cultural games in teaching mathematics. Using an In-depth conversational interview, ten (10) certificated teachers' voices on the values and challenges of integrating games were examined. Thematic data analysis was applied to the qualitative data from the…

  20. Improving Patient Safety Culture in Primary Care: A Systematic Review

    NARCIS (Netherlands)

    Verbakel, Natasha J.; Langelaan, Maaike; Verheij, Theo J. M.; Wagner, Cordula; Zwart, Dorien L. M.

    Background: Patient safety culture, described as shared values, attitudes and behavior of staff in a health-care organization, gained attention as a subject of study as it is believed to be related to the impact of patient safety improvements. However, in primary care, it is yet unknown, which

  1. Preventive effect of piracetam and vinpocetine on hypoxia-reoxygenation induced injury in primary hippocampal culture.

    Science.gov (United States)

    Solanki, P; Prasad, D; Muthuraju, S; Sharma, A K; Singh, S B; Ilavzhagan, G

    2011-04-01

    The present study investigates the potential of Piracetam and Vinpocetine (nootropic drugs, known to possess neuroprotective properties) in preventing hypoxia-reoxygenation induced oxidative stress in primary hippocampal cell culture. The hippocampal culture was exposed to hypoxia (95% N(2), 5% CO(2)) for 3h and followed by 1h of reoxygenation (21% O(2) and 5% CO(2)) at 37 °C. The primary hippocampal cultures were supplemented with the optimum dose of Piracetam and Vinpocetine, independently, and the cultures were divided into six groups, viz. Control/Normoxia, Hypoxia, Hypoxia+Piracetam, Hypoxia+Vinpocetine, Normoxia + Piracetam and Normoxia+Vinpocetine. The cell-viability assays and biochemical oxidative stress parameters were evaluated for each of the six groups. Administration of 1mM Piracetam or 500 nM Vinpocetine significantly prevents the culture from hypoxia-reoxygenation injury when determined by Neutral Red assay, LDH release and Acetylcholine esterase activity. Results showed that Piracetam and Vinpocetine supplementation significantly prevented the fall of mitochondrial membrane potential, rise in ROS generation and reduction in antioxidant levels associated with the hypoxia-reoxygenation injury. In conclusion, the present study establishes that both Piracetam and Vinpocetine give neuroprotection against hypoxia-reoxygenation injury in primary hippocampal cell culture. Copyright © 2010 Elsevier Ltd. All rights reserved.

  2. Cultural aspects of primary healthcare in india: A case- based analysis

    Directory of Open Access Journals (Sweden)

    Worthington Roger P

    2011-06-01

    Full Text Available Abstract Delivering quality primary care to large populations is always challenging, and that is certainly the case in India. While the sheer magnitude of patients can create difficulties, not all challenges are about logistics. Sometimes patient health-seeking behaviour leads to delays in obtaining medical help for reasons that have more to do with culture, social practice and religious belief. When primary care is accessed via busy state-run outpatient departments there is often little time for the physician to investigate causes behind a patient's condition, and these factors can adversely affect patient outcomes. We consider the case of a woman with somatic symptoms seemingly triggered by psychological stresses associated with social norms and familial cultural expectations. These expectations conflict with her personal and professional aspirations, and although she eventually receives psychiatric help and her problems are addressed, initially, psycho-social factors underlying her condition posed a hurdle in terms of accessing appropriate medical care. While for many people culture, belief and social norms exert a stabilising, positive influence, in situations where someone's personal expectations differ significantly from accepted social norms, individual autonomy can be directly challenged, and in which case, something has to give. The result of such challenges can negatively impact on health and well-being, and for patients with immature defence mechanisms for dealing with inner conflict, such an experience can be damaging and ensuing somatic disturbances are often difficult to treat. Patients with culture-bound symptoms are not uncommon within primary care in India or in other Asian countries and communities. We argue that such cases need to be properly understood if satisfactory patient outcomes are to be achieved. While some causes are structural, having to do with how healthcare is accessed and delivered, others are about cultural

  3. Cultural aspects of primary healthcare in india: A case- based analysis.

    Science.gov (United States)

    Worthington, Roger P; Gogne, Anupriya

    2011-06-16

    Delivering quality primary care to large populations is always challenging, and that is certainly the case in India. While the sheer magnitude of patients can create difficulties, not all challenges are about logistics. Sometimes patient health-seeking behaviour leads to delays in obtaining medical help for reasons that have more to do with culture, social practice and religious belief. When primary care is accessed via busy state-run outpatient departments there is often little time for the physician to investigate causes behind a patient's condition, and these factors can adversely affect patient outcomes. We consider the case of a woman with somatic symptoms seemingly triggered by psychological stresses associated with social norms and familial cultural expectations. These expectations conflict with her personal and professional aspirations, and although she eventually receives psychiatric help and her problems are addressed, initially, psycho-social factors underlying her condition posed a hurdle in terms of accessing appropriate medical care. While for many people culture, belief and social norms exert a stabilising, positive influence, in situations where someone's personal expectations differ significantly from accepted social norms, individual autonomy can be directly challenged, and in which case, something has to give. The result of such challenges can negatively impact on health and well-being, and for patients with immature defence mechanisms for dealing with inner conflict, such an experience can be damaging and ensuing somatic disturbances are often difficult to treat. Patients with culture-bound symptoms are not uncommon within primary care in India or in other Asian countries and communities. We argue that such cases need to be properly understood if satisfactory patient outcomes are to be achieved. While some causes are structural, having to do with how healthcare is accessed and delivered, others are about cultural values, social practices and

  4. Bioactive treatment promotes osteoblast differentiation on titanium materials fabricated by selective laser melting technology.

    Science.gov (United States)

    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.

  5. The effect of variation in physical properties of porous bioactive glass on the expression and maintenance of the osteoblastic phenotype

    Science.gov (United States)

    Effah Kaufmann, Elsie Akosua Biraa

    pore sizes and porosity and determined the effect of substrate properties on the expression and maintenance of the osteoblastic phenotype, using an in vitro culture of osteoblast-like cells. Our data showed that porous bioactive glass substrates support the proliferation and maturation of osteoblast-like cells. Within the conditions of the experiment, we also found that at a given porosity of 44% the pore size of bioactive glass neither directs nor modulates the in vitro expression of the osteoblastic phenotype. On the other hand, at an average pore size of 92 mum, when cultures are maintained for 14 days, cell activity is greatly affected by the substrate porosity. As the porosity increases from 35% to 59%, osteoblast activity is adversely affected. (Abstract shortened by UMI.)

  6. Cloning and regulation of rat tissue inhibitor of metalloproteinases-2 in osteoblastic cells

    Science.gov (United States)

    Cook, T. F.; Burke, J. S.; Bergman, K. D.; Quinn, C. O.; Jeffrey, J. J.; Partridge, N. C.

    1994-01-01

    Rat tissue inhibitor of metalloproteinases-2 (TIMP-2) was cloned from a UMR 106-01 rat osteoblastic osteosarcoma cDNA library. The 969-bp full-length clone demonstrates 98 and 86% sequence identity to human TIMP-2 at the amino acid and nucleic acid levels, respectively. Parathyroid hormone (PTH), at 10(-8) M, stimulates an approximately twofold increase in both the 4.2- and 1.0-kb transcripts over basal levels in UMR cells after 24 h of exposure. The PTH stimulation of TIMP-2 transcripts was not affected by the inhibitor of protein synthesis, cycloheximide (10(-5) M), suggesting a primary effect of the hormone. This is in contradistinction to regulation of interstitial collagenase (matrix metalloproteinase-1) by PTH in these same cells. Nuclear run-on assays demonstrate that PTH causes an increase in TIMP-2 transcription that parallels the increase in message levels. Parathyroid hormone, in its stimulation of TIMP-2 mRNA, appears to act through a signal transduction pathway involving protein kinase A (PKA) since the increase in TIMP-2 mRNA is reproduced by treatment with the cAMP analogue, 8-bromo-cAMP (5 x 10(-3) M). The protein kinase C and calcium pathways do not appear to be involved due to the lack of effect of phorbol 12-myristate 13-acetate (2.6 x 10(-6) M) and the calcium ionophore, ionomycin (10(-7) M), on TIMP-2 transcript abundance. In this respect, regulation of TIMP-2 and collagenase in osteoblastic cells by PTH are similar. However, we conclude that since stimulation of TIMP-2 transcription is a primary event, the PKA pathway must be responsible for a direct increase in transcription of this gene.

  7. Accumulation of type VI collagen in the primary osteon of the rat femur during postnatal development.

    Science.gov (United States)

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

    2015-05-01

    In rodents, the long bone diaphysis is expanded by forming primary osteons at the periosteal surface of the cortical bone. This ossification process is thought to be regulated by the microenvironment in the periosteum. Type VI collagen (Col VI), a component of the extracellular matrix (ECM) in the periosteum, is involved in osteoblast differentiation at early stages. In several cell types, Col VI interacts with NG2 on the cytoplasmic membrane to promote cell proliferation, spreading and motility. However, the detailed functions of Col VI and NG2 in the ossification process in the periosteum are still under investigation. In this study, to clarify the relationship between localization of Col VI and formation of the primary osteon, we examined the distribution of Col VI and osteoblast lineages expressing NG2 in the periosteum of rat femoral diaphysis during postnatal growing periods by immunohistochemistry. Primary osteons enclosing the osteonal cavity were clearly identified in the cortical bone from 2 weeks old. The size of the osteonal cavities decreased from the outer to the inner region of the cortical bone. In addition, the osteonal cavities of newly formed primary osteons at the outermost region started to decrease in size after rats reached the age of 4 weeks. Immunohistochemistry revealed concentrated localization of Col VI in the ECM in the osteonal cavity. Col VI-immunoreactive areas were reduced and they disappeared as the osteonal cavities became smaller from the outer to the inner region. In the osteonal cavities of the outer cortical regions, Runx2-immunoreactive spindle-shaped cells and mature osteoblasts were detected in Col VI-immunoreactive areas. The numbers of Runx2-immunoreactive cells were significantly higher in the osteonal cavities than in the osteogenic layers from 2 to 4 weeks. Most of these Runx2-immunoreactive cells showed NG2-immunoreactivity. Furthermore, PCNA-immunoreactivity was detected in the Runx2-immunoreactive spindle

  8. Aluminum Templates of Different Sizes with Micro-, Nano- and Micro/Nano-Structures for Cell Culture

    Directory of Open Access Journals (Sweden)

    Ming-Liang Yen

    2017-10-01

    Full Text Available This study investigates the results of cell cultures on aluminum (Al templates with flat-structures, micro-structures, nano-structures and micro/nano-structures. An Al template with flat-structure was obtained by electrolytic polishing; an Al template with micro-structure was obtained by micro-powder blasting; an Al template with nano-structure was obtained by aluminum anodization; and an Al template with micro/nano-structure was obtained by micro-powder blasting and then anodization. Osteoblast-like cells were cultured on aluminum templates with various structures. The microculture tetrazolium test assay was utilized to assess the adhesion, elongation, and proliferation behaviors of cultured osteoblast-like cells on aluminum templates with flat-structures, micro-structures, nano-structures, and micro/nano-structures. The results showed that the surface characterization of micro/nano-structure of aluminum templates had superhydrophilic property, and these also revealed that an aluminum template with micro/nano-structure could provide the most suitable growth situation for cell culture.

  9. Does collagen trigger the recruitment of osteoblasts into vacated bone resorption lacunae during bone remodeling?

    DEFF Research Database (Denmark)

    Abdelgawad, Mohamed Essameldin; Søe, Kent; Andersen, Thomas Levin

    2014-01-01

    matrix molecules, collagen's potency was superior and only equaled by fibronectin. Next, the majority of the newly recruited osteoblast lineage cells positioned immediately next to the osteoclasts exhibit uPARAP/Endo180, an endocytic collagen receptor reported to be involved in collagen internalization......Osteoblast recruitment during bone remodeling is obligatory to re-construct the bone resorbed by the osteoclast. This recruitment is believed to be triggered by osteoclast products and is therefore likely to start early during the remodeling cycle. Several osteoclast products with osteoblast...... recruitment potential are already known. Here we draw the attention on the osteoblast recruitment potential of the collagen that is freshly demineralized by the osteoclast. Our evidence is based on observations on adult human cancellous bone, combined with in vitro assays. First, freshly eroded surfaces where...

  10. Primary Care Resident Perceived Preparedness to Deliver Cross-cultural Care: An Examination of Training and Specialty Differences

    Science.gov (United States)

    Park, Elyse R.; Green, Alexander R.; Betancourt, Joseph R.; Weissman, Joel S.

    2007-01-01

    Objective Previous research has shown that resident physicians report differences in training across primary care specialties, although limited data exist on education in delivering cross-cultural care. The goals of this study were to identify factors that relate to primary care residents’ perceived preparedness to provide cross-cultural care and to explore the extent to which these perceptions vary across primary care specialties. Design Cross-sectional, national mail survey of resident physicians in their last year of training. Participants Eleven hundred fifty primary care residents specializing in family medicine (27%), internal medicine (23%), pediatrics (26%), and obstetrics/gynecology (OB/GYN) (24%). Results Male residents as well as those who reported having graduated from U.S. medical schools, access to role models, and a greater cross-cultural case mix during residency felt more prepared to deliver cross-cultural care. Adjusting for these demographic and clinical factors, family practice residents were significantly more likely to feel prepared to deliver cross-cultural care compared to internal medicine, pediatric, and OB/GYN residents. Yet, when the quantity of instruction residents reported receiving to deliver cross-cultural care was added as a predictor, specialty differences became nonsignificant, suggesting that training opportunities better account for the variability in perceived preparedness than specialty. Conclusions Across primary care specialties, residents reported different perceptions of preparedness to deliver cross-cultural care. However, this variation was more strongly related to training factors, such as the amount of instruction physicians received to deliver such care, rather than specialty affiliation. These findings underscore the importance of formal education to enhance residents’ preparedness to provide cross-cultural care. PMID:17516107

  11. Surface modifications of dental ceramic implants with different glass solder matrices: in vitro analyses with human primary osteoblasts and epithelial cells.

    Science.gov (United States)

    Markhoff, Jana; Mick, Enrico; Mitrovic, Aurica; Pasold, Juliane; Wegner, Katharina; Bader, Rainer

    2014-01-01

    Ceramic materials show excellent esthetic behavior, along with an absence of hypersensitivity, making them a possible alternative implant material in dental surgery. However, their surface properties enable only limited osseointegration compared to titanium implants. Within this study, a novel surface coating technique for enhanced osseointegration was investigated biologically and mechanically. Specimens of tetragonal zirconia polycrystal (TZP) and aluminum toughened zirconia (ATZ) were modified with glass solder matrices in two configurations which mainly consisted of SiO2, Al2O3, K2O, and Na2O. The influence on human osteoblastic and epithelial cell viability was examined by means of a WST-1 assay as well as live/dead staining. A C1CP-ELISA was carried out to verify procollagen type I production. Uncoated/sandblasted ceramic specimens and sandblasted titanium surfaces were investigated as a reference. Furthermore, mechanical investigations of bilaterally coated pellets were conducted with respect to surface roughness and adhesive strength of the different coatings. These tests could demonstrate a mechanically stable implant coating with glass solder matrices. The coated ceramic specimens show enhanced osteoblastic and partly epithelial viability and matrix production compared to the titanium control. Hence, the new glass solder matrix coating could improve bone cell growth as a prerequisite for enhanced osseointegration of ceramic implants.

  12. Surface Modifications of Dental Ceramic Implants with Different Glass Solder Matrices: In Vitro Analyses with Human Primary Osteoblasts and Epithelial Cells

    Science.gov (United States)

    Mick, Enrico

    2014-01-01

    Ceramic materials show excellent esthetic behavior, along with an absence of hypersensitivity, making them a possible alternative implant material in dental surgery. However, their surface properties enable only limited osseointegration compared to titanium implants. Within this study, a novel surface coating technique for enhanced osseointegration was investigated biologically and mechanically. Specimens of tetragonal zirconia polycrystal (TZP) and aluminum toughened zirconia (ATZ) were modified with glass solder matrices in two configurations which mainly consisted of SiO2, Al2O3, K2O, and Na2O. The influence on human osteoblastic and epithelial cell viability was examined by means of a WST-1 assay as well as live/dead staining. A C1CP-ELISA was carried out to verify procollagen type I production. Uncoated/sandblasted ceramic specimens and sandblasted titanium surfaces were investigated as a reference. Furthermore, mechanical investigations of bilaterally coated pellets were conducted with respect to surface roughness and adhesive strength of the different coatings. These tests could demonstrate a mechanically stable implant coating with glass solder matrices. The coated ceramic specimens show enhanced osteoblastic and partly epithelial viability and matrix production compared to the titanium control. Hence, the new glass solder matrix coating could improve bone cell growth as a prerequisite for enhanced osseointegration of ceramic implants. PMID:25295270

  13. Fabrication of dopamine-modified hyaluronic acid/chitosan multilayers on titanium alloy by layer-by-layer self-assembly for promoting osteoblast growth

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Xinming, E-mail: xmzhang@tju.edu.cn [School of Materials Science and Engineering, Tianjin University, Tianjin 300072 (China); Li, Zhaoyang, E-mail: zyli@tju.edu.cn [School of Materials Science and Engineering, Tianjin University, Tianjin 300072 (China); Tianjin Key Laboratory of Composite and Functional Materials, Tianjin 300072 (China); Yuan, Xubo [School of Materials Science and Engineering, Tianjin University, Tianjin 300072 (China); Cui, Zhenduo; Yang, Xianjin [School of Materials Science and Engineering, Tianjin University, Tianjin 300072 (China); Tianjin Key Laboratory of Composite and Functional Materials, Tianjin 300072 (China)

    2013-11-01

    The bare inert surface of titanium (Ti) alloy typically causes early failures in implants. Layer-by-layer self-assembly is one of the simple methods for fabricating bioactive multilayer coatings on titanium implants. In this study, a dopamine-modified hyaluronic acid/chitosan (DHA/CHI) bioactive multilayer was built on the surface of Ti–24Nb–2Zr (TNZ) alloy. Zeta potential oscillated between −2 and 17 mV for DHA- and CHI-ending layers during the assembly process, respectively. The DHA/CHI multilayer considerably decreased the contact angle and dramatically improved the wettability of TNZ alloy. Atomic force microscopy results revealed a rough surface on the original TNZ alloy, while the surface became smoother and more homogeneous after the deposition of approximately 5 bilayers (TNZ/(DHA/CHI){sub 5}). X-ray photoelectron spectroscopy analysis indicated that the TNZ/(DHA/CHI){sub 5} sample was completely covered by polyelectrolytes. Pre-osteoblast MC3T3-E1 cells were cultured on the original TNZ alloy and TNZ/(DHA/CHI){sub 5} to evaluate the effects of DHA/CHI multilayer on osteoblast proliferation in vitro. The proliferation of osteoblasts on TNZ/(DHA/CHI){sub 5} was significantly higher than that on the original TNZ alloy. The results of this study indicate that the proposed technique improves the biocompatibility of TNZ alloy and can serve as a potential modification method in orthopedic applications.

  14. Fabrication of dopamine-modified hyaluronic acid/chitosan multilayers on titanium alloy by layer-by-layer self-assembly for promoting osteoblast growth

    International Nuclear Information System (INIS)

    Zhang, Xinming; Li, Zhaoyang; Yuan, Xubo; Cui, Zhenduo; Yang, Xianjin

    2013-01-01

    The bare inert surface of titanium (Ti) alloy typically causes early failures in implants. Layer-by-layer self-assembly is one of the simple methods for fabricating bioactive multilayer coatings on titanium implants. In this study, a dopamine-modified hyaluronic acid/chitosan (DHA/CHI) bioactive multilayer was built on the surface of Ti–24Nb–2Zr (TNZ) alloy. Zeta potential oscillated between −2 and 17 mV for DHA- and CHI-ending layers during the assembly process, respectively. The DHA/CHI multilayer considerably decreased the contact angle and dramatically improved the wettability of TNZ alloy. Atomic force microscopy results revealed a rough surface on the original TNZ alloy, while the surface became smoother and more homogeneous after the deposition of approximately 5 bilayers (TNZ/(DHA/CHI) 5 ). X-ray photoelectron spectroscopy analysis indicated that the TNZ/(DHA/CHI) 5 sample was completely covered by polyelectrolytes. Pre-osteoblast MC3T3-E1 cells were cultured on the original TNZ alloy and TNZ/(DHA/CHI) 5 to evaluate the effects of DHA/CHI multilayer on osteoblast proliferation in vitro. The proliferation of osteoblasts on TNZ/(DHA/CHI) 5 was significantly higher than that on the original TNZ alloy. The results of this study indicate that the proposed technique improves the biocompatibility of TNZ alloy and can serve as a potential modification method in orthopedic applications.

  15. Compositional effects on the formation of a calcium phosphate layer and the response of osteoblast-like cells on polymer-bioactive glass composites.

    Science.gov (United States)

    Lu, Helen H; Tang, Amy; Oh, Seong Cheol; Spalazzi, Jeffrey P; Dionisio, Kathie

    2005-11-01

    Biodegradable polymer-ceramic composites are attractive systems for bone tissue engineering applications. These composites have the combined advantages of the component phases, as well as the inherent ease in optimization where desired material properties can be tailored in a well-controlled manner. This study focuses on the optimization of a polylactide-co-glycolide (PLAGA) and 45S5 bioactive glass (BG) composite for bone tissue engineering. The first objective is to examine the effects of composition or overall BG content on the formation of a Ca-P layer on the PLAGA-BG composite. It is expected that with increasing BG content (0%, 10%, 25%, 50% by weight), the required incubation time in a simulated body fluid (SBF) for the composite to form a detectable surface Ca-P layer will decrease. Both the kinetics and the chemistry will be determined using SEM+EDAX, FTIR, and mu-CT methods. Solution phosphorous and calcium concentrations will also be measured. The second objective of the study is to determine the effects of BG content on the maturation of osteoblast-like cells on the PLAGA-BG composite. It is hypothesized that mineralization will increase with increasing BG content, and the composite will support the proliferation and differentiation of osteoblasts. Specifically, cell proliferation, alkaline phosphatase activity and mineralization will be monitored as a function of BG content (0%, 10%, 50% by weight) and culturing time. It was found that the kinetics of Ca-P layer formation and the resulting Ca-P chemistry were dependent on BG content. The response of human osteoblast-like cells to the PLAGA-BG composite was also a function of BG content. The 10% and 25% BG composite supported greater osteoblast growth and differentiation compared to the 50% BG group. The results of this study suggest that there is a threshold BG content which is optimal for osteoblast growth, and the interactions between PLAGA and BG may modulate the kinetics of Ca-P formation and the

  16. Primary microglia isolation from mixed glial cell cultures of neonatal rat brain tissue.

    Science.gov (United States)

    Tamashiro, Tami T; Dalgard, Clifton Lee; Byrnes, Kimberly R

    2012-08-15

    Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted. Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study. The principle and protocol of this methodology have been described in the literature. Additionally, alternate methodologies to isolate primary microglia are well described. Homogenized brain tissue may be separated

  17. Pyrethroid insecticide accumulation in primary cultures of cortical neurons in vitro

    Science.gov (United States)

    Primary cultures of neurons have been widely utilized to study the actions of pyrethroids and other neurotoxicants, with the presumption that the media concentration accurately reflects the dose received by the cells. However, recent studies have demonstrated that lipophilic comp...

  18. Optimal Zn-Modified Ca–Si-Based Ceramic Nanocoating with Zn Ion Release for Osteoblast Promotion and Osteoclast Inhibition in Bone Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Jiangming Yu

    2017-01-01

    Full Text Available We investigated the slow release of Zn ion (Zn2+ from nanocoatings and compared the in vitro response of osteoblasts (MC3T3-E1 and proosteoclasts (RAW 264.7 cultured on Ca2ZnSi2O7 nanocoated with different Zn/Ca molar ratios on a Ti-6Al-4V (i.e., Ti substrate to optimize cell behaviors and molecule levels. Significant morphology differences were observed among samples. By comparing with pure Ti and CaSiO3 nanocoating, the morphology of Ca2ZnSi2O7 ceramic nanocoatings was rough and contained small nanoparticles or aggregations. Slow Zn2+ release from nanocoatings was observed and Zn2+ concentration was regulated by varying the Zn/Ca ratios. The cell-response results showed Ca2ZnSi2O7 nanocoating at different Zn/Ca molar ratios for osteoblasts and osteoclasts. Compared to other nanocoatings and Ti, sample Zn/Ca (0.3 showed the highest cell viability and upregulated expression of the osteogenic differentiation genes ALP, COL-1, and OCN. Additionally, sample Zn/Ca (0.3 showed the greatest inhibition of RAW 264.7 cell growth and decreased the mRNA levels of osteoclast-related genes OAR, TRAP, and HYA1. Therefore, the optimal Zn-Ca ratio of 0.3 in Ca2ZnSi2O7 ceramic nanocoating on Ti had a dual osteoblast-promoting and osteoclast-inhibiting effect to dynamically balance osteoblasts/osteoclasts. These optimal Zn-Ca ratios are valuable for Ca2ZnSi2O7 ceramic nanocoating on Ti-coated implants for potential applications in bone tissue regeneration.

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

    Science.gov (United States)

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

    2017-01-01

    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. PMID:28990069

  20. Microcracks induce osteoblast alignment and maturation on hydroxyapatite scaffolds

    Science.gov (United States)

    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.

  1. Cytokine expression in human osteoblasts after antiseptic treatment: a comparative study between polyhexanide and chlorhexidine.

    Science.gov (United States)

    Röhner, Eric; Hoff, Paula; Gaber, Timo; Lang, Annemarie; Vörös, Pauline; Buttgereit, Frank; Perka, Carsten; Windisch, Christoph; Matziolis, Georg

    2015-02-01

    Chlorhexidine and polyhexanide are frequently used antiseptics in clinical practice and have a broad antimicrobial range. Both antiseptics are helpful medical agents for septic wound treatment with a high potential for defeating joint infections. Their effect on human osteoblasts has, so far, not been sufficiently evaluated. The aim of this study was to investigate the activating potential of polyhexanide and chlorhexidine on inflammatory cytokines/chemokines in human osteoblasts in vitro. Human osteoblasts were isolated and cultivated in vitro and then treated separately with 0.1% and 2% chlorhexidine and 0.04% polyhexanide as commonly applied concentrations in clinical practice. Detection of cell structure and cell morphology was performed by light microscopic inspection. Cytokine and chemokine secretion was determined by using a multiplex suspension array. Cell shrinking, defective cell membrane, and the loss of cell adhesion indicated cell damage of human osteoblasts after treatment with both antiseptics was evaluated by using light microscopy. Polyhexanide, but not chlorhexidine, caused human osteoblasts to secrete various interleukins (1β, 6, and 7), interferon γ, tumor necrosis factor α, vascular endothelial growth factor, eotaxin, fibroblast growth factor basic, and granulocyte macrophage colony-stimulating factor as quantified by multiplex suspension array. Both antiseptics induced morphological cell damage at an optimum exposure between 1 and 10 min. But only polyhexanide mediated a pronounced secretion of inflammatory cytokines and chemokines in human osteoblasts. Therefore, we recommend a preferred usage of chlorhexidine in septic surgery to avoid the induction of an inflammatory reaction.

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

    Science.gov (United States)

    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.

  3. Measurement of oxygen consumption rate of osteoblasts from ...

    African Journals Online (AJOL)

    Jane

    2011-05-10

    May 10, 2011 ... E-mail: kedongsong@dlut.edu.cn. Tel: +86 411 ... the experiments, including inverted phase contrast microscope. (IX70-Olympus ... The pictures showed that the osteoblasts still had very high cellular viability. consumption of ...

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

    DEFF Research Database (Denmark)

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

    2003-01-01

    The propagation of mechanically induced intercellular calcium waves (ICW) among osteoblastic cells occurs both by activation of P2Y (purinergic) receptors by extracellular nucleotides, resulting in "fast" ICW, and by gap junctional communication in cells that express connexin43 (Cx43), resulting...... in "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 Cx...

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

    Science.gov (United States)

    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.

  6. Fabrication of poly(ethylene glycol) hydrogel micropatterns with osteoinductive growth factors and evaluation of the effects on osteoblast activity and function

    Energy Technology Data Exchange (ETDEWEB)

    Subramani, K [Institute for Nanoscale Science and Technology (INSAT), University of Newcastle upon Tyne, Newcastle upon Tyne, NE1 7AR (United Kingdom); Birch, M A [Institute for Nanoscale Science and Technology (INSAT), University of Newcastle upon Tyne, Newcastle upon Tyne, NE1 7AR (United Kingdom)

    2006-09-15

    The aims of this study were to fabricate poly(ethylene glycol) (PEG) hydrogel micropatterns on a biomaterial surface to guide osteoblast behaviour and to study how incorporating vascular endothelial growth factor (VEGF) within the adhered hydrogel influenced cell morphology. Standard photolithographic procedures or photopolymerization through a poly(dimethyl siloxane) (PDMS) mould were used to fabricate patterned PEG hydrogels on the surface of silanized silicon wafers. Hydrogel patterns were evaluated by light microscopy and surface profilometry. Rat osteoblasts were cultured on these surfaces and cell morphology investigated by fluorescence microscopy, scanning electron microscopy (SEM) and atomic force microscopy (AFM). Release of protein trapped in the polymerized PEG was evaluated and VEGF-PEG surfaces were characterized for their ability to support cell growth. These studies show that photopolymerized PEG can be used to create anti-adhesive structures on the surface of silicon that completely control where cell interaction with the substrate takes place. Using conventional lithography, structures down to 50 {mu}m were routinely fabricated with the boundaries exhibiting sloping sides. Using the PDMS mould approach, structures were fabricated as small as 10 {mu}m and boundaries were very sharp and vertical. Osteoblasts exhibiting typical morphology only grew on the silicon wafer surface that was not coated with PEG. Adding BSA to the monomer solution showed that protein could be released from the hydrogel for up to 7 days in vitro. Incorporating VEGF in the hydrogel produced micropatterns that dramatically altered osteoblast behaviour. At boundaries with the VEGF-PEG hydrogel, there was striking formation of cellular processes and membrane ruffling indicative of a change in cell morphology. This study has explored the morphogenetic properties of VEGF and the applications of nano/microfabrication techniques for guided tissue (bone) regeneration in dental and

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-04-28

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

  8. Comparison of gene expression profile in embryonic mesencephalon and neuronal primary cultures.

    Directory of Open Access Journals (Sweden)

    Dario Greco

    Full Text Available In the mammalian central nervous system (CNS an important contingent of dopaminergic neurons are localized in the substantia nigra and in the ventral tegmental area of the ventral midbrain. They constitute an anatomically and functionally heterogeneous group of cells involved in a variety of regulatory mechanisms, from locomotion to emotional/motivational behavior. Midbrain dopaminergic neuron (mDA primary cultures represent a useful tool to study molecular mechanisms involved in their development and maintenance. Considerable information has been gathered on the mDA neurons development and maturation in vivo, as well as on the molecular features of mDA primary cultures. Here we investigated in detail the gene expression differences between the tissue of origin and ventral midbrain primary cultures enriched in mDA neurons, using microarray technique. We integrated the results based on different re-annotations of the microarray probes. By using knowledge-based gene network techniques and promoter sequence analysis, we also uncovered mechanisms that might regulate the expression of CNS genes involved in the definition of the identity of specific cell types in the ventral midbrain. We integrate bioinformatics and functional genomics, together with developmental neurobiology. Moreover, we propose guidelines for the computational analysis of microarray gene expression data. Our findings help to clarify some molecular aspects of the development and differentiation of DA neurons within the midbrain.

  9. Intracortical osteoblastic osteosarcoma with oncogenic rickets

    Energy Technology Data Exchange (ETDEWEB)

    Hasegawa, T.; Hirohashi, Setsuo [Pathology Division, National Cancer Center Research Institute, Tokyo (Japan); Shimoda, Tadakazu [Clinical Laboratory Division, National Cancer Center Hospital, Tokyo (Japan); Yokoyama, Ryohei; Beppu, Yasuo [Orthopedic Division, National Cancer Center Hospital, Tokyo (Japan); Maeda, Shotaro [Department of Pathology, Nippon Medical School Hospital, Tokyo (Japan)

    1999-01-01

    Intracortical osteosarcoma is the rarest variant of osteosarcoma, occurring within, and usually confined to, the cortical bone. Oncogenic osteomalacia, or rickets, is an unusual clinicopathologic entity in which vitamin D-resistant osteomalacia, or rickets, occurs in association with some tumors of soft tissue or bone. We present a case of oncogenic rickets associated with intracortical osteosarcoma of the tibia in a 9-year-old boy, whose roentgenographic abnormalities of rickets disappeared and pertinent laboratory data except for serum alkaline phosphatase became normal after surgical resection of the tumor. Histologically, the tumor was an osteosarcoma with a prominent osteoblastic pattern. An unusual microscopic feature was the presence of matrix mineralization showing rounded calcified structures (calcified spherules). Benign osteoblastic tumors, such as osteoid osteoma and osteoblastoma, must be considered in the differential diagnosis because of the relatively low cellular atypia and mitotic activity of this tumor. The infiltrating pattern with destruction or engulfment of normal bone is a major clue to the correct diagnosis of intracortical osteosarcoma. The co-existing radiographic changes of rickets were due to the intracortical osteosarcoma. (orig.) With 8 figs., 25 refs.

  10. Bmp2 in osteoblasts of periosteum and trabecular bone links bone formation to vascularization and mesenchymal stem cells

    Science.gov (United States)

    Yang, Wuchen; Guo, Dayong; Harris, Marie A.; Cui, Yong; Gluhak-Heinrich, Jelica; Wu, Junjie; Chen, Xiao-Dong; Skinner, Charles; Nyman, Jeffry S.; Edwards, James R.; Mundy, Gregory R.; Lichtler, Alex; Kream, Barbara E.; Rowe, David W.; Kalajzic, Ivo; David, Val; Quarles, Darryl L.; Villareal, Demetri; Scott, Greg; Ray, Manas; Liu, S.; Martin, James F.; Mishina, Yuji; Harris, Stephen E.

    2013-01-01

    Summary We generated a new Bmp2 conditional-knockout allele without a neo cassette that removes the Bmp2 gene from osteoblasts (Bmp2-cKOob) using the 3.6Col1a1-Cre transgenic model. Bones of Bmp2-cKOob mice are thinner, with increased brittleness. Osteoblast activity is reduced as reflected in a reduced bone formation rate and failure to differentiate to a mature mineralizing stage. Bmp2 in osteoblasts also indirectly controls angiogenesis in the periosteum and bone marrow. VegfA production is reduced in Bmp2-cKOob osteoblasts. Deletion of Bmp2 in osteoblasts also leads to defective mesenchymal stem cells (MSCs), which correlates with the reduced microvascular bed in the periosteum and trabecular bones. Expression of several MSC marker genes (α-SMA, CD146 and Angiopoietin-1) in vivo, in vitro CFU assays and deletion of Bmp2 in vitro in α-SMA+ MSCs support our conclusions. Critical roles of Bmp2 in osteoblasts and MSCs are a vital link between bone formation, vascularization and mesenchymal stem cells. PMID:23843612

  11. Transfection of brain capillary endothelial cells in primary culture with defined blood-brain barrier properties.

    Science.gov (United States)

    Burkhart, Annette; Thomsen, Louiza Bohn; Thomsen, Maj Schneider; Lichota, Jacek; Fazakas, Csilla; Krizbai, István; Moos, Torben

    2015-08-07

    Primary brain capillary endothelial cells (BCECs) are a promising tool to study the blood-brain barrier (BBB) in vitro, as they maintain many important characteristics of the BBB in vivo, especially when co-cultured with pericytes and/or astrocytes. A novel strategy for drug delivery to the brain is to transform BCECs into protein factories by genetic modifications leading to secretion of otherwise BBB impermeable proteins into the central nervous system. However, a huge challenge underlying this strategy is to enable transfection of non-mitotic BCECs, taking a non-viral approach. We therefore aimed to study transfection in primary, non-mitotic BCECs cultured with defined BBB properties without disrupting the cells' integrity. Primary cultures of BCECs, pericytes and astrocytes were generated from rat brains and used in three different in vitro BBB experimental arrangements, which were characterised based on a their expression of tight junction proteins and other BBB specific proteins, high trans-endothelial electrical resistance (TEER), and low passive permeability to radiolabeled mannitol. Recombinant gene expression and protein synthesis were examined in primary BCECs. The BCECs were transfected using a commercially available transfection agent Turbofect™ to express the red fluorescent protein HcRed1-C1. The BCECs were transfected at different time points to monitor transfection in relation to mitotic or non-mitotic cells, as indicated by fluorescence-activated cell sorting analysis after 5-and 6-carboxylfluorescein diacetate succinidyl ester incorporation. The cell cultures exhibited important BBB characteristics judged from their expression of BBB specific proteins, high TEER values, and low passive permeability. Among the three in vitro BBB models, co-culturing with BCECs and astrocytes was well suited for the transfection studies. Transfection was independent of cell division and with equal efficacy between the mitotic and non-mitotic BCECs. Importantly

  12. The influence of surface chemistry and topography on the contact guidance of MG63 osteoblast cells.

    Science.gov (United States)

    Ismail, F S Magdon; Rohanizadeh, R; Atwa, S; Mason, R S; Ruys, A J; Martin, P J; Bendavid, A

    2007-05-01

    The purpose of the present study was to determine in vitro the effects of different surface topographies and chemistries of commercially pure titanium (cpTi) and diamond-like carbon (DLC) surfaces on osteoblast growth and attachment. Microgrooves (widths of 2, 4, 8 and 10 microm and a depth of 1.5-2 microm) were patterned onto silicon (Si) substrates using microlithography and reactive ion etching. The Si substrates were subsequently vapor coated with either cpTi or DLC coatings. All surfaces were characterized using atomic force microscopy (AFM), scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and contact angle measurements. Using the MG63 Osteoblast-Like cell line, we determined cell viability, adhesion, and morphology on different substrates over a 3 day culture period. The results showed cpTi surfaces to be significantly more hydrophilic than DLC for groove sizes larger than 2 microm. Cell contact guidance was observed for all grooved samples in comparison to the unpatterned controls. The cell viability tests indicated a significantly greater cell number for 8 and 10 microm grooves on cpTi surfaces compared to other groove sizes. The cell adhesion study showed that the smaller groove sizes, as well as the unpatterned control groups, displayed better cell adhesion to the substrate.

  13. Cultural significance of primary teeth for caregivers in Northeast Brazil.

    Science.gov (United States)

    Nations, Marilyn K; Calvasina, Paola Gondim; Martin, Michele N; Dias, Hilma Fontenele

    2008-04-01

    This anthropological study critically evaluates Brazilian caregivers' symbolic production and significance of their malnourished offspring's primary teeth, as well as their own, and describes popular dental practices. From January to June 2004, ethnographic interviews of 27 poor, low-literacy mothers were conducted at a public Malnutrition Treatment Center in Fortaleza, Ceará State. Participant observation of clinical pathways and home environments supplemented the data. Content analysis was performed. Results confirm that primary teeth are imbued with cultural significance in Northeast Brazil. Mothers examine mouths, perceive signs of decay, associate primary with permanent tooth healthiness, identify ethnodental illnesses, seek assistance, and perform rituals with exfoliated teeth. The mother's motivation to care for primary teeth is sparked by her memories of past toothache and attempts to avoid stigma and discrimination. Social determinants, not mothers' beliefs or behaviors, are the most critical obstacles to effective dental care. Legitimizing lay knowledge and empowering caregivers and children can improve oral health in Northeast Brazil.

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

    Science.gov (United States)

    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.

  15. Addition of Wollastonite Fibers to Calcium Phosphate Cement Increases Cell Viability and Stimulates Differentiation of Osteoblast-Like Cells

    Directory of Open Access Journals (Sweden)

    Juliana Almeida Domingues

    2017-01-01

    Full Text Available Calcium phosphate cement (CPC that is based on α-tricalcium phosphate (α-TCP is considered desirable for bone tissue engineering because of its relatively rapid degradation properties. However, such cement is relatively weak, restricting its use to areas of low mechanical stress. Wollastonite fibers (WF have been used to improve the mechanical strength of biomaterials. However, the biological properties of WF remain poorly understood. Here, we tested the response of osteoblast-like cells to being cultured on CPC reinforced with 5% of WF (CPC-WF. We found that both types of cement studied achieved an ion balance for calcium and phosphate after 3 days of immersion in culture medium and this allowed subsequent long-term cell culture. CPC-WF increased cell viability and stimulated cell differentiation, compared to nonreinforced CPC. We hypothesize that late silicon release by CPC-WF induces increased cell proliferation and differentiation. Based on our findings, we propose that CPC-WF is a promising material for bone tissue engineering applications.

  16. Protective Effect of Edaravone against Carbon Monoxide Induced Apoptosis in Rat Primary Cultured Astrocytes

    Directory of Open Access Journals (Sweden)

    Xiaodan Xu

    2017-01-01

    Full Text Available Objective. To observe the protective effect of edaravone (Eda on astrocytes after prolonged exposure to carbon monoxide (CO and further to investigate the potential mechanisms of Eda against CO-induced apoptosis. Methods. The rat primary cultured astrocytes were cultured in vitro and exposed to 1% CO for 24 h after being cultured with different concentrations of Eda. MTT assay was used to detect the cytotoxicity of CO. Flow cytometry was used to detect the apoptosis rate, membrane potential of mitochondria, and ROS level. The mRNA and protein expressions of Bcl-2, Bax, and caspase-3 were assessed by real-time PCR and Western blotting analysis, respectively. Results. Eda can significantly suppress cytotoxicity of CO, and it can significantly increase membrane potential of mitochondria and Bcl-2 expressions and significantly suppress the apoptosis rate, ROS level, Bax, and caspase-3 expressions. Conclusion. Eda protects against CO-induced apoptosis in rat primary cultured astrocytes through decreasing ROS production and subsequently inhibiting mitochondrial apoptosis pathway.

  17. Changes of muscarinic cholinergic receptors during aging process of primary cultured neutrons

    International Nuclear Information System (INIS)

    Fan Guohuang; Yi Ningyu; Xia Zongqin

    1996-01-01

    The dynamic changes of muscarinic receptor density and its reactivity during aging process in primary cultured neutrons were studied. Muscarinic receptor density was measured by 3 H-QNB binding assay, and muscarinic receptor reactivity was assessed by carbachol stimulation of cGMP formation, the latter was measured by RIA. After 2 weeks' incubation of neonatal rat brain cells, the nutrients began to rupture and the cell bodies shrank markedly showing senescent feature. The muscarinic receptor density reached peak at the 12th day in vitro (12 DIV), but the muscarinic receptor reactivity reached peak at 9 DIV and declined significantly at 12 DIV. The results demonstrated that during aging process of primary cultured neutrons, the decline of muscarinic receptor reactivity is likely prior to the decrease of receptor density

  18. Static and dynamic hypergravity responses of osteoblasts and osteoclasts in medaka scales

    OpenAIRE

    Yano, Sachiko; Kitamura, Kei-ichiro; Satoh, Yusuke; Nakano, Masaki; Hattori, Atsuhiko; Sekiguchi, Toshio; Ikegame, Mika; Nakashima, Hiroshi; Omori, Katsunori; Hayakawa, Kazuichi; Chiba, Atsuhiko; Sasayama, Yuichi; Ejiri, Sadakazu; Mikuni-Takagaki, Yuko; Mishima, Hiroyuki

    2013-01-01

    Fish scales are a form of calcified tissue similar to that found in human bone. In medaka scales, we detected both osteoblasts and osteoclasts and subsequently developed a new scale assay system. Using this system, we analyzed the osteoblastic and osteoclastic responses under 2-, 3-, and 4-gravity (G) loading by both centrifugation and vibration. After loading for 10 min, the scales from centrifugal and vibration loading were incubated for 6 and 24 hrs, respectively, after which the osteoblas...

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

    Science.gov (United States)

    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.

  20. Organizational culture in the primary healthcare setting of Cyprus.

    Science.gov (United States)

    Zachariadou, Theodora; Zannetos, Savvas; Pavlakis, Andreas

    2013-03-24

    The concept of organizational culture is important in understanding the behaviour of individuals in organizations as they manage external demands and internal social changes. Cyprus healthcare system is under restructuring and soon a new healthcare scheme will be implemented starting at the Primary Healthcare (PHC) level. The aim of the study was to investigate the underlying culture encountered in the PHC setting of Cyprus and to identify possible differences in desired and prevailing cultures among healthcare professionals. The population of the study included all general practitioners (GPs) and nursing staff working at the 42 PHC centres throughout the island. The shortened version of the Organizational Culture Profile questionnaire comprising 28 statements on organizational values was used in the study. The instrument was already translated and validated in Greek and cross-cultural adaptation was performed. Participants were required to indicate the organization's characteristic cultural values orientation along a five-point Likert scale ranging from "Very Much = 1" to "Not at all= 5". Statistical analysis was performed using SPSS 16.0. Student t-test was used to compare means between two groups of variables whereas for more than two groups analysis of variance (ANOVA) was applied. From the total of 306 healthcare professionals, 223 participated in the study (72.9%). The majority of participants were women (75.3%) and mean age was 42.6 ± 10.7 years. Culture dimension "performance orientation" was the desired culture among healthcare professionals (mean: 1.39 ± 0.45). "Supportiveness" and "social responsibility" were the main cultures encountered in PHC (means: 2.37 ± 0.80, 2.38 ± 0.83). Statistical significant differences were identified between desired and prevailing cultures for all culture dimensions (p= 0.000). This was the first study performed in Cyprus assessing organizational culture in the PHC setting. In the forthcoming health system reform