Dissertations / Theses on the topic 'Osteoblast'
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McManus, Lindsay L. "A study of human mesenchymal stem cells, human primary osteoblasts and osteoblast-like cells using Raman spectroscopy." Thesis, University of Ulster, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.551188.
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Raman spectroscopy is a vibrational spectroscopy technique that provides a global biochemical signature and has been shown to have utility in the analysis of biological cells for bone tissue engineering applications. Traditionally, sample analysis in this field employs destructive biological methods that require the use of biomarkers, however, Raman has since become an essential tool in various areas of bio-industry and by incorporating the technique into biological laboratories these perturbing methodologies are no longer the only means of analysis. Therefore the focus of this study was to investigate the capability of Raman spectroscopy as a tool for the in vitro characterisation of the sub-cellular composition and osteogenic potential of human mesenchymal stem cells. As with most biological samples a high degree of heterogeneity is often found, therefore in order to extract the desired information from the biological studies multivariate analysis tools were utilised. The reliability and consistency of the vibrational analysis was confirmed by means of comparison with current gold-standard techniques such as, alizarin red staining, real-time polymerase chain reaction and immunocytochemistry. A further novelty was introduced with the use of Raman spectroscopy to determine the suitability of the U20S osteoblast-like cell line for use as a model for human primary osteoblasts with emphasis on the ability of these cell types to replicate their tissue of origin. Investigation of the U20S osteoblast-like cell line provided evidence of dense multilayered mineralised regions that corresponded more closely to native bone, which has not been previously reported on. This finding contradicts previous reports on U20S osteoblast-like cells which have consistently been described as non-osteoinductive. When Raman spectroscopy is coupled with biological and multivariate analyses techniques, it shows further novelty when employed to monitor mineralisation of human mesenchymal stem cells, human primary osteoblasts and osteoblast-like cells. This body of work culminates the success of a truly multidisciplinary approach and provides the potential for further work on bone cell analysis and the applications of spectroscopy for biological studies and bone tissue engineering applications.
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Hempel, Ute, Carolin Preissler, Sarah Vogel, Stephanie Möller, Vera Hintze, Jana Becher, Matthias Schnabelrauch, Martina Rauner, Lorenz C. Hofbauer, and Peter Dieter. "Artificial Extracellular Matrices with Oversulfated Glycosaminoglycan Derivatives Promote the Differentiation of Osteoblast-Precursor Cells and Premature Osteoblasts." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-165309.
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Sulfated glycosaminoglycans (GAG) are components of the bone marrow stem cell niche and to a minor extent of mature bone tissue with important functions in regulating stem cell lineage commitment and differentiation. We anticipated that artificial extracellular matrices (aECM) composed of collagen I and synthetically oversulfated GAG derivatives affect preferentially the differentiation of osteoblast-precursor cells and early osteoblasts. A set of gradually sulfated chondroitin sulfate and hyaluronan derivatives was used for the preparation of aECM. All these matrices were analysed with human bone marrow stromal cells to identify the most potent aECM and to determine the influence of the degree and position of sulfate groups and the kind of disaccharide units on the osteogenic differentiation. Oversulfated GAG derivatives with a sulfate group at the C-6 position of the N-acetylglycosamine revealed the most pronounced proosteogenic effect as determined by tissue nonspecific alkaline phosphatase activity and calcium deposition. A subset of the aECM was further analysed with different primary osteoblasts and cell lines reflecting different maturation stages to test whether the effect of sulfated GAG derivatives depends on the maturation status of the cells. It was shown that the proosteogenic effect of aECMwasmost prominent in early osteoblasts. [ABSTRACT FROM AUTHOR]
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Hempel, Ute, Carolin Preissler, Sarah Vogel, Stephanie Möller, Vera Hintze, Jana Becher, Matthias Schnabelrauch, Martina Rauner, Lorenz C. Hofbauer, and Peter Dieter. "Artificial Extracellular Matrices with Oversulfated Glycosaminoglycan Derivatives Promote the Differentiation of Osteoblast-Precursor Cells and Premature Osteoblasts." Hindawi, 2014. https://tud.qucosa.de/id/qucosa%3A28669.
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Sulfated glycosaminoglycans (GAG) are components of the bone marrow stem cell niche and to a minor extent of mature bone tissue with important functions in regulating stem cell lineage commitment and differentiation. We anticipated that artificial extracellular matrices (aECM) composed of collagen I and synthetically oversulfated GAG derivatives affect preferentially the differentiation of osteoblast-precursor cells and early osteoblasts. A set of gradually sulfated chondroitin sulfate and hyaluronan derivatives was used for the preparation of aECM. All these matrices were analysed with human bone marrow stromal cells to identify the most potent aECM and to determine the influence of the degree and position of sulfate groups and the kind of disaccharide units on the osteogenic differentiation. Oversulfated GAG derivatives with a sulfate group at the C-6 position of the N-acetylglycosamine revealed the most pronounced proosteogenic effect as determined by tissue nonspecific alkaline phosphatase activity and calcium deposition. A subset of the aECM was further analysed with different primary osteoblasts and cell lines reflecting different maturation stages to test whether the effect of sulfated GAG derivatives depends on the maturation status of the cells. It was shown that the proosteogenic effect of aECMwasmost prominent in early osteoblasts. [ABSTRACT FROM AUTHOR]
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Duque, Gustavo. "Molecular changes in the aging osteoblast." Thesis, McGill University, 2003. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=19466.
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Aging is the consequence ofan array of phenotypic variations that appear to involve intrinsic or constitutional properties in all cells and systems, including qualitative and quantitative alterations in development, maturational structure and function. The aging process in bone involves a set ofchanges in bone cells differentiation, interaction and premature death. Osteoblasts are the cells most affected during the aging process in bone due to their complex mechanisms ofdifferentiation, their interaction with honnones and growth factors and their progression to apoptosis.
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Townsend, Paul Andrew. "The molecular basis of osteoblast adhesion." Thesis, University College London (University of London), 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.263651.
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Boonphayak, Piyanan. "Substituted hydroxyapatite analysis of osteoblast response." Thesis, University of Manchester, 2017. https://www.research.manchester.ac.uk/portal/en/theses/substituted-hydroxyapatite-analysis-of-osteoblast-response(71ddb64d-03b9-4825-875e-61d68bd2d3e6).html.
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Ceramics used for medical purposes are known as bioceramics, such as hydroxyaptite (HA), which is one of the most well studied bioceramics because of its similar composition to human bone and also good biocompatibility, is bioactive and has excellent osteoconductivity. In addition many properties of HA can be improved by the addition of specific elements into its structure. The research in this thesis investigates the substitution of some selected elements into the structure of HA and subsequent characterisation in terms of physical, mechanical and biological responses. Si/S-HA and Sr/B/S-HA was obtained from Lucideon and Ho/HA was synthesised in house. Initially a cell response to a variety of element oxides was performed to identify elements to avoid or potentially use for substitution. Dysprosium (Dy), erbium (Er), europium (Eu), gadolinium (Gd), holmium (Ho) and praseodymium (Pr) at 100 ppm showed better results for alkaline phosphatase activity than 25 ppm in culture medium.2.5 and 5 mol.% of Ho was substituted into HA structure using a wet chemical method of synthesis. The samples were sintered at 1200°C for 10 hours. There was high crystallinity when 2.5 mol.% of Ho was added into the HA structure. Substitution of Ho in HA structure had the effect of shortening in a axis and elongation in c axis along with the higher concentration of Ho ion.2 mol.% of Si and S was also substituted into HA structure, where both elements Si and S replace PO4 site in HA structure. Si substituted in SiO4 form and S substituted in SO4 form for PO4 as confirmed by FT-IR results. Osteoblast-like cells cultured on Si/S-HA showed an increase in alkaline phosphatase, collagen type I and ostecalcin on samples sintered at high temperature. Sr/B/S-HA was also made where 2 mol.% of each element was substituted into the HA structure. The best condition of sintering temperature for Sr/B/S-HA was 1100°C for 10 hrs due to the resulting small grain size, improved cell adhesion and more collagen and osteocalcin production. These results suggest that the substitution of elements into the HA structure can provide novel bioceramics for control of physical mechanical and cell responses properties.
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Li, Bo. "The role of BK channel in cellular proliferation and differentiation in human osteoblast and osteoblast-like cells." Thesis, Cardiff University, 2012. http://orca.cf.ac.uk/35876/.
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Both excitable and non-excitable cells possess plasma membrane ion channels and evidence has accumulated over the last 30 or so years that these channels perhaps play key roles in the cell life and death. This Thesis investigated the characteristics and putative functions of one class of potassium channel, the BK channel in osteoblast-like cells and primary osteoblasts from human, rat and mouse. The properties and functions were defined in vitro using a combination of patch-clamp, reverse transcription-polymerase chain reaction (RT-PCR) and functional assays for cell growth and mineralisation. RT-PCR showed the presence of KCNMA1, KCNMB1, KCNMB2, KCNMB3 and KCNMB4, the gene for BK channel α, β1, β2, β3 and β4 subunits respectively. The channel was voltage-dependent with a mean unitary conductance of 315 pS in cell-attached patches, a conductance of 124 pS in excised outside-out and 151 pS in inside-out patches. The channel was blocked by TEA (0.3 mM), TBuA (1 mM), TPeA (1-10 μM), THeA (1-3 μM), tetrandrine (5-30 μM) and paxilline (10 μM) and was activated by isopimaric acid (20 μM). Notably iberiotoxin (IbTX) (90 nM) only blocked a proportion of the channels tested (2/5). Osteoblast-like MG63 cell number changed in response to BK channel modulators. It increased significantly with TEA and tetrandrine at low concentrations (1 mM, 3 μM respectively), and reduced at high concentrations (>10 mM, >10 μM respectively). It was not affected by IbTX (20-300 nM) or slotoxin (300 nM). The increase in cell number by TEA was blocked by isopimaric acid. In addition, TPeA and THeA caused a decrease of osteoblast-like SaOS2 cell mineralisation at the concentrations (3 and 0.3 μM, respectively) increased MG63 cell numbers. The BK channel has a distinctive pharmacology and represents a new target for therapeutic strategies in modulating osteoblast proliferation.
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Park, Jung Hwa. "The role of surface chemistry and wettability of microtextured titanium surfaces in osteoblast differentiation." Diss., Georgia Institute of Technology, 2012. http://hdl.handle.net/1853/44732.
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Biomaterial surface energy, chemical composition, charge, wettability and roughness all play an important role in determining the degree of the direct bone-to-implant interface, termed osseointegration. Surface chemistry, which is influenced by surface energy, wettability, and composition, is another factor that determines osteoblast phenotype and regulates osteoblast maturation. Increased surface energy is desirable for bone implants due to enhanced interaction between the implant surface and the biological environment. The extent of bone formation in vivo is also increased with increasing water wettability of implants.
The physiological role of implant surface chemistry is important in determining the success of implant osseointegration because of molecular rearrangements, surface reactions, contamination, and release of toxic or biologically active ions that are determined by the starting chemistry. However, the role of surface chemistry on osteoblast response is not fully studied.
Therefore, the overall goal of this dissertation is to understand how the surface chemistry, including wettability, chemical composition, and charge density, of titanium biomaterials impacts osteoblast maturation (in vitro). This study focuses on the general hypothesis that modifications of surface chemistry of titanium surfaces with sterilization or polyelectrolyte coating on titanium surfaces regulate osteoblast response.
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Huesa, Carmen. "Mechanotransduction in cells of the osteoblast lineage." Thesis, Available from the University of Aberdeen Library and Historic Collections Digital Resources, 2008. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?application=DIGITOOL-3&owner=resourcediscovery&custom_att_2=simple_viewer&pid=25468.
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Allen, Matthew Robert. "Mechanisms of impaired osteoblast function during disuse." Diss., Texas A&M University, 2003. http://hdl.handle.net/1969.1/1056.
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Prolonged periods of non-weightbearing activity result in a significant loss of bone mass which increases the risk of fracture with the initiation of mechanical loading. The loss of bone mass is partially driven by declines in bone formation yet the mechanisms responsible for this decline are unclear. To investigate the limitations of osteoblasts during disuse, marrow ablation was superimposed on hindlimb unloaded mice. Marrow ablation is a useful model to study osteoblast functionality as new cancellous bone is rapidly formed throughout the marrow of a long bone while hindlimb unloading is the most common method used to produce skeletal unloading. The specific hypotheses of this study were aimed at determining if changes in osteoblast functionality, differentiation, and/or proliferation were compromised in non-weightbearing bone in response to a bone formation stimulus. Additionally, the influence of having compromised osteoblast functionality at the time of stimulation was assessed in non-weightbearing bones. Key outcome measures used to address these hypotheses included static and dynamic cancellous bone histomorphometry, bone densitometry, and real-time polymerase chain reaction (PCR) analyses of gene expression. The results document similar ablation-induced increases of cancellous bone in both weightbearing and unloaded animals. Similarly, there was no influence of load on ablation-induced increases in cancellous bone forming surface or mineral apposition rate. Unloading did significantly attenuate the ablation-induced increase in bone formation rate, due to reduced levels of total surface mineralization. When osteoblast functionality was compromised prior to marrow ablation, bone formation rate increases were also attenuated in ablated animals due to reduced mineralization. Additionally, increases in forming surface were attenuated as compared to unloaded animals having normal osteoblast function at the time of ablation. Collectively, these data identify mineralization as the limiting step in new bone formation during periods of disuse. The caveat, however, is that when bone formation is stimulated after a period of unloading sufficient to compromise osteoblast functionality, increases in osteoblast recruitment to the bone surface are compromised.
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Somayajula, Dilip Ayyala. "Biocompatibility of osteoblast cells on titanium implants." Cleveland, Ohio : Cleveland State University, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=csu1207322725.
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Thesis (M.S.)--Cleveland State University, 2008.Abstract. Title from PDF t.p. (viewed on May 8, 2008). Includes bibliographical references (p. 72-76). Available online via the OhioLINK ETD Center. Also available in print.
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Rickard, David J. "Modulation of human osteoblast function by oestradiol." Thesis, University of Bath, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.293240.
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Ball, Michael David. "The optimisation of hydroxyapatite for osteoblast growth." Thesis, University of Nottingham, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.312229.
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Berger, Christine Elizabeth Marie. "Superoxide anion in osteoclast and osteoblast function." Thesis, University of Newcastle Upon Tyne, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.265210.
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Yellowley, Clare Elizabeth. "Electrophysiological characterisation of human osteoblast-like cells." Thesis, University of Bristol, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.240717.
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Ayyala, Somayajula Dilip. "Biocompatibility of osteoblast cells on titanium implants." Cleveland State University / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=csu1207322725.
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Osman, M. "Investigation of molecular regulators in osteoblast differentiation." Thesis, University of Liverpool, 2017. http://livrepository.liverpool.ac.uk/3009617/.
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Bones provide mechanical support for movement and normal daily functions, which can only be provided when healthy. Bone diseases such as osteoporosis and cancer are a worldwide problem affecting millions of people and causing significant financial impact. This study should indicate possible solutions that might contribute to combatting bone diseases by understanding the expression of bone markers that are known to be expressed differentially at different stages of osteoblastic differentiation. Thus a reliable, easily available and easy to maintain bone cell model was selected using osteosarcoma cell lines (MG-63, TE-85 and SaOS-2). The effect of ATP and PTH treatments on the expression of these markers was also tested to determine differences with primary osteoblasts (HOBs). We also determined the differential expression of miRNAs between the cell lines and HOBs and identified miRNAs involved in regulating expression of sclerostin, examining also their effect on the properties of trabecular bone in a mouse model. We hypothesized that these models were appropriate for studying the biology of osteoblastic cells and that miRNAs could control bone cell phenotype. In the first objective of this study, the expression of seven bone markers actively involved in bone development (OPG, ALP, COL1A2, COL6A3, SOST, OSX and RANKL) were investigated. The expression of OPG, COL1A2 and COL6A3 were significantly higher in the least differentiated cell line, MG-63, while expression of ALP, SOST, OSX and RANKL were highest in SaOS-2, the most differentiated. ATP and PTH exerted the expected inhibitory or inductive effects on the cells at the osteoblastic maturation stage confirming that the cell lines were a suitable model for the study of osteoblasts. MicroRNA profiling of cell lines and HOBs revealed hundreds of differentially expressed miRNAs. Six that were predicted to target the regulation of SOST were chosen for further validation. Mimics and antagomirs of these were transfected into TE-85 and SaOS-2 cells to confirm their involvement in SOST expression regulation. SOST gene expression and protein production were quantified by qPCR and ELISA, revealing that miR-1231, miR-1254 and miR-1914 decreased SOST protein expression in TE-85 and SaOS-2 cells and miR-378a-3p in just TE-85 cells, but these effects were not reversed by the corresponding antagomir. Adult and elderly mice were treated with miR-378a-3p mimic and antagomir by injection into the tail vein. Few differences could be observed in trabecular bone three weeks following treatment, except for significantly increased trabecular pattern factor and decreased trabecular separation in elderly mice treated with miR-378a-3p mimic. In conclusion, this thesis demonstrates that ATP and PTH regulate osteosarcoma cell lines similarly to HOBs and that miRNAs can be used to control SOST protein expression. Potential future studies should investigate miRNAs as novel therapies for bone diseases.
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Bhangu, P. S. "Vesicular 'pre-synaptic' glutamatergic signalling mechanisms in bone." Thesis, University of York, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.288814.
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Clare, Matthew David. "An investigation into novel mechanisms of osteoblast differentiation." Thesis, University of Bristol, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.422569.
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McHenry, S. M. "Synergistic interactions between osteoblast cells and endothelial cells." Thesis, Queen's University Belfast, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.403243.
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Liu, Fina. "Molecular and cellular analysis of the osteoblast lineage." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0008/NQ28291.pdf.
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Addison, William. "Molecular Determinants of mineralization in osteoblast cell cultures." Thesis, McGill University, 2010. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=92158.
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Mineralization of the extracellular matrix of bone is a cell-mediated process, which is tightly regulated by a delicate balance between stimulatory and inhibitory molecules. A disruption in the metabolism or levels of these mediating factors results in pathologically hypomineralized or hypermineralized bone. The experimental results presented in this thesis describe the effects of key proteins, peptides and small-molecule ions on an osteoblast cell-culture model of bone mineralization.This study presents evidence that pyrophosphate inhibits mineralization by at least three different mechanisms that include direct binding to hydroxyapatite crystals, induction of osteopontin expression, and inhibition of alkaline phosphatase activity. The data presented also demonstrate that inhibition of mineralization by mineral-binding osteopontin and MEPE ASARM peptides is phosphorylation-dependent and that this inhibition can be rescued by enzymatic degradation of the peptides by PHEX. This result identifies a novel mechanism by which loss of PHEX activity can lead to extracellular matrix accumulation of ASARM resulting in the osteomalacia of X-linked hypophosphatasia. Finally, the thesis describes the novel effects of a potentially physiologic modulator of mineralization, inositol hexakisphosphate, on osteoblast activity namely, that inositol hexakisphosphate inhibits osteoblast culture mineralization, adsorbs to mineral and induces expression of osteopontin.
In summary, the findings herein suggest a model whereby regulation of crystal propagation/growth within the extracellular matrix is maintained by the enzymatic removal (e.g. by alkaline phosphatase and PHEX) of mineralization inhibitors (e.g. pyrophosphate and ASARM peptides) and that these inibitors regulate mineralization by acting on both the mineral phase and on the cellular expression of mineral-regulating genes.
Minéralisation de la matrice extracellulaire de l'os est un processus de médiation cellulaire, qui est rigoureusement réglementé par un délicat équilibre entre les molécules stimulatoires et inhibitrices. Un dysfonctionnement dans le métabolisme de ces facteurs donnera soit une déficience ou un excès des minéraux dans les os. Les résultats expérimentaux présentés dans cette thèse, décrivent les effets des protéines et des ions principaux sur un modèle de cellule-culture d'osteoblaste de minéralisation d'os.
Cette étude présente les éléments de preuve que le pyrophosphate inhibe la minéralisation par au moins trois différents mécanismes qui incluent la liaison directe aux cristaux d'hydroxyapatite, à l'induction de expression d'osteopontin et à l'inhibition de l'activité de la phosphatase alcaline. Les données présentées démontrent également que l'inhibition de la minéralisation par la liaison de minéraux osteopontin/MEPE ASARM peptides est phosphorylation dépendante et que cette inhibition peut être sauvée par dégradation enzymatique avec PHEX. Ce résultat identifie un nouveau mécanisme par lequelle une perte d'activité de PHEX peut conduire à l'accumulation d'ASARM dans la matrice ce qui se traduit par la ostéomalacie de X-linked hypophosphatasia.
Enfin, la thèse décrit les effets d'un nouveau modulateur de minéralisation, l'inositol hexakisphosphate, de l'activité d'ostéoblaste. Nous avons trouver que l'inositol hexakisphosphate inhibe la minéralisation de culture d'ostéoblastes, s'attache aux minéraux et augmente l'expression d'osteopontin.
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Bensaud, Nabila. "ATP-mediated mineralization of MC3T3-E1 osteoblast cultureS." Thesis, McGill University, 2013. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=117162.
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ABSTRACTExtracellular matrix mineralization is a physiological process where hydroxyapatite is deposited onto collagenous matrix. Mineralization is initiated by an increase in phosphate concentration in the matrix and one major regulator of this process is alkaline phosphatase (TNAP). Absence of TNAP in hypophosphatasia patients and TNAP knockout mouse models significantly decreases matrix mineralization; however some mineral is deposited indicating that additional mechanisms exist to increase phosphate levels. In this study, we have used MC3T3-E1 osteoblast cultures to investigate ATP (adenosinetriphosphate) as a source of phosphate in osteoblast matrix mineralization. ATP hydrolysis by an ATPase enzyme can provide three phosphate ions. Osteoblasts also have receptors for ATP, ADP and AMP - latter two resulting from partial hydrolysis of ATP. To investigate ATP-mediated mineralization in osteoblasts, we used MC3T3-E1 preosteoblast cell line. Cells were cultured for 9 or 12 days and treated with ascorbic acid to induce collagen matrix formation and phosphate sources; β-glycerophosphate (βGP)(common source of phosphate in vitro), sodium monophosphate (SMP) and ATP. ATP added at level of 4mM resulted in mineral deposition, increased calcium and phosphate levels in the matrix and collagen deposition similar to that following conventional treatment with SMP and βGP. Non-hydrolyzable ATP analogue did not allow mineral deposition, demonstrating that mineralization directly coming from phosphate-ions of ATP. Other nucleotides, UTP and GTP also allowed mineral deposition, but were not as potent as ATP. When asking what enzyme could be responsible for ATP hydrolysis, it was noted that ATP treatment significantly downregulated TNAP. Downregulation was reversed by suramin, a P2 purinergic receptor (ATP and ADP receptor) antagonist, but was not affected by ATP receptor agonist indicating that the effect might arise from ADP receptor engagement. Furthermore, ATP-mediated mineralization was completely resistant to TNAP inhibition (by levamisole) indicating that ATP-mediated mineralization is mediated by another phosphate releasing enzyme(s). Interestingly, ATP-treatment increased general ATPase activity and the presence of a 64 kDa transglutaminase 2 (TG2) fragment in the culture medium. We have previously shown that a TG2 fragment in the extracellular space has increased ATPase activity. Treating the cultures with an extracellular ATPase inhibitor completely abolished not only ATP-, but also βGP- and SMP-induced mineralization, without affecting collagen deposition or cell density. This indicates that ATPase activity could contribute to general mineralization mechanisms, perhaps to initiation of the process.ABRÉGÉLa minéralisation de la matrice extracellulaire est un processus physiologique qui consiste dans le dépôt des cristaux d'hydroxyapatite sur les fibres de collagène de la matrice extracellulaire. Le processus de minéralisation est initié par l'augmentation de la concentration des ions phosphate dans la matrice. Un facteur majeur de la régularisation des ions phosphates est la phosphatase alkaline (TNAP). L'absence du TNAP dans de patients atteints de l'hypophosphatasie se concrétise dans la diminution significative de la minéralisation de la matrice et ce a aussi été démontré par de modèles de souris knockout. Une certaine quantité de minéral en ayant été toutefois aussi déposée, indiquent que d'autre facteurs doivent exister en lien avec l'augmentation du niveau de phosphate. Dans cette étude nous avons utilisé des cultures de cellules ostéoblastes MC3T3-E1 pour investiguer l'adénosine-triphosphate (ATP) comme source de phosphate dans la minéralisation de la matrice des ostéoblastes. L'hydrolyse de l'ATP par une enzyme ATPase peut générer trois groupes phosphate. Les ostéoblastes possèdent aussi de récepteurs pour ATP, ADP et AMP - les deux derniers résultant par l'hydrolyse partielle de l'ATP. La croissance des ostéoblastes MC3T3-E1 a été faite pour 9-12 jours et les cellules ont été traités avec acide ascorbique (pour accélérer la formation de la matrice), β-glycerol phosphate (βGP) (source commune de phosphate dans les expérimentations in vitro), monophosphate de sodium (SMP) et l'ATP. L'adition d'ATP à une concentration de 4 mM induit le dépôt du minéral, l'augmentation du niveau de calcium et phosphate dans la matrice ainsi que le dépôt du collagène dans une manière similaire à celui observé pendant le traitement conventionnel avec βGP. En utilisant un agent analogue à l'ATP non-hydrolysé où le largage des ions phosphate est bloqué, le dépôt du minéral n'est plus observé, ce qui démontre que la minéralisation est directement induite par les ions phosphate d'ATP. D'autres nucléotides comme UTP et GTP aussi permettent le dépôt du minéral, mais à un niveau qui n'est pas aussi élevé que celui observé dans le cas de l'ATP. En se questionnant sur quelle enzyme pourrait être responsable pour l'hydrolyse de l'ATP, on note que le traitement avec ATP diminue significativement le TNAP. Au contraire, le TNAP augmente par le traitement avec suramin, un récepteur (d'ATP et d'ADP) purinérgique antagoniste P2, mais n'est pas affecté par le récepteur ATP agoniste, indiquant que l'effet pourrait être du au recepteur ADP. De plus, la minéralisation régulée par l'ATP s'est avérée résistante à l'inhibiteur TNAP levamisole, indiquant que la minéralisation en présence d'ATP doit être régulée par une autre enzyme libératrice de phosphate. A remarquer, le traitement ATP augmente l'activité générale d'ATPase en présence d'un fragment de 64 kDa de la transglutaminase 2 (TG2) dans le millieu de culture. Nous avons déjà montré qu'un fragment TG2 dans l'espace extracellulaire augment l'activité ATPase. Le traitement des cellules avec un inhibiteur ATPase extracellulaire bloque non seulement la minéralisation induit par l'ATP, mais aussi la minéralisation régulée par βGP et SMP sans toutefois altérer le dépôt du collagène ou la densité cellulaire. Ce résultat indique que l'activité ATPase pourrait contribuer aux mécanismes de minéralisation en général, au moins en jouant un rôle important à l'initiation du processus.
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Stewart, E. A. "Tyrosine phosphorylation during osteoblast adhesion to biomaterial surfaces." Thesis, University of Nottingham, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.493347.
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The number of hip replacement operations performed is currently increasing due to the ageing population and the need for revision surgery after around 15 years. Improving the materials used for hip replacements would decrease the need for revision surgery and have an important economic effect as well as increasing quality of life for patients. To ensure the success and longevity of a bone implant, osteoblast cells must adhere and differentiate on the implant material to form a tight junction with the surface.
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Kelly, Jonathan M. "Osteoblast response to oxygen functionalised plasma polymer surfaces." Thesis, University of Sheffield, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.246918.
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Das-Gupta, Victoria. "Investigating the control of osteoblast nitric oxide production." Thesis, Royal Veterinary College (University of London), 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.412918.
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Faibish, Dan. "The actions of resolvin E1 on osteoblast function." Thesis, Boston University, 2012. https://hdl.handle.net/2144/12368.
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Thesis (Ph.D.)--Boston UniversityResolvins are endogenous anti-inflammatory I pro-resolving lipid mediators derived from omega-3 fatty acids. Resolvin E1 (RvE1) reverses periodontitis and promotes regeneration of alveolar bone in vivo. The goal of this project was to determine the mechanism of RvE1 impact on bone formation. RvE1 significantly enhanced bone formation relative to a vehicle control in a mouse craniotomy model of bone healing. Since RvE1 is reported to act through receptors expressed by cells of the innate immune system, the initial hypothesis tested was that RvE1 actions are mediated through bone macrophages. The hypothesis was rejected, as no impact of RvE1 on macrophage mediated bone formation was demonstrable. The alternative hypothesis was that RvE1 acts directly on osteoblasts. Using mouse neonatal osteoblasts, calcification of osteoblast cultures was demonstrated. Osteoblasts express the RvE1 receptor, ChemR23, at the mRNA and protein level. Examination of intracellular signaling by RvE1 demonstrated increased phosphorylation of rpS6 through the AKT-mTOR pathway. The specificity of RvE1 signaling through ChemR23 was demonstrated with ChemR23 specific blocking antibody that abrogated the phosphorylation of rpS6. Rapamycin, an inhibitor of mTOR, also blocked rpS6 phosphorylation. To examine the mechanism of RvE1 treated osteoblast enhanced bone formation, secretion of bone specific proteins by osteoblasts after pro-inflammatory stimulation (IL-6) was examined with a focus on the osteoprotegerin (OPG) and receptor activator of NF-κB ligand (RANKL) axis, which regulates osteoclast differentiation. Secretion of RANKL and OPG by mouse neonatal osteoblasts stimulated with IL-6 and treated with RvE1 was measured by ELISA. IL-6 stimulation did not impact RANKL levels but decreased OPG production, thereby changing the RANKL/OPG to favor osteoclast activation and bone resorption. RvE1 blocked OPG changes, however, maintaining a RANKL/OPG more favorable to bone formation. In conclusion, RvE1 has anabolic actions in a mouse model of bone healing mediated through RANKL/OPG. RvE1 signals the receptor ChemR23 on the osteoblast surface through the mTOR pathway and phosphorylation of rpS6. Functionally, RvE1 shifts the balance between OPG and RANKL to favor bone formation. Mediators of innate immunity thus also directly regulate bone cells.
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Vörös, Pauline [Verfasser]. "Human osteoblast damage after antiseptic treatment / Pauline Vörös." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2015. http://d-nb.info/1067442197/34.
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Al-Ajmi, Nada Muhammad Zayd. "The effect of clinostat rotation on osteoblast behaviour." Thesis, University of Manchester, 1997. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.668169.
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Hoflack, Bernard, Pierre Jurdic, Thilo Riedl, Anne Gallois, and Maria Arantzazu Sanchez-Fernandez. "Osteoclasts control osteoblast chemotaxis via PDGF-BB/PDGF receptor beta signaling." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-184120.
Full textAbstract:
BACKGROUND:
Bone remodeling relies on the tightly regulated interplay between bone forming osteoblasts and bone digesting osteoclasts. Several studies have now described the molecular mechanisms by which osteoblasts control osteoclastogenesis and bone degradation. It is currently unclear whether osteoclasts can influence bone rebuilding.
METHODOLOGY/PRINCIPAL FINDINGS:
Using in vitro cell systems, we show here that mature osteoclasts, but not their precursors, secrete chemotactic factors recognized by both mature osteoblasts and their precursors. Several growth factors whose expression is upregulated during osteoclastogenesis were identified by DNA microarrays as candidates mediating osteoblast chemotaxis. Our subsequent functional analyses demonstrate that mature osteoclasts, whose platelet-derived growth factor bb (PDGF-bb) expression is reduced by siRNAs, exhibit a reduced capability of attracting osteoblasts. Conversely, osteoblasts whose platelet-derived growth factor receptor beta (PDGFR-beta) expression is reduced by siRNAs exhibit a lower capability of responding to chemotactic factors secreted by osteoclasts.
CONCLUSIONS/SIGNIFICANCE:
We conclude that, in vitro mature osteoclasts control osteoblast chemotaxis via PDGF-bb/PDGFR-beta signaling. This may provide one key mechanism by which osteoclasts control bone formation in vivo.
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Lefebvre, Céline. "Transglutaminase expression and activity in MC3T3-E1 osteoblast cultures." Thesis, McGill University, 2004. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=81353.
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Transglutaminases are enzymes that stabilize extracellular matrices by catalyzing the formation of protease-resistant isopeptide crosslinks between and within their substrate proteins. Two transglutaminase isoforms, tissue transglutaminase (tTG) and factor XIIIa (FXIIIa), are expressed in skeletal tissues. Tissue transglutaminase has been previously localized in bone to osteoblasts, the osteoid layer and the pericellular matrix of osteocytes. Factor XIIIa has been localized to mineralizing chondrocytes, however, its expression in osteoblasts has not been reported yet. In order to understand the role of transglutaminases in hard tissue formation, we have investigated tTG and FXIIIa expression and activity during osteoblast differentiation and mineralization using the well-characterized MC3T3-E1 preosteoblast cell line. We show that tTG and FXIIIa are expressed at all time points examined in the cell cultures, and that their expression and activity does not appear to be significantly changed by the differentiation or mineralization state of the cells. We show that tTG localizes homogeneously throughout the cells and the extracellular matrix, whereas FXIIIa distribution is polarized to one side of the cells at earlier time points, but becomes incorporated into the matrix produced by differentiating cells in mature cultures. These expression profiles suggest a dual role, initially in cell adhesion and attachment and, later, in protein assembly, where the transglutaminases likely stabilize the matrix and assist in the formation of an extracellular matrix competent for subsequent mineralization. The identification and characterization of two transglutaminase isoforms in osteoblasts suggest a potentially overlapping roles for these two enzymes in the MC3T3-E1 cell culture system.
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Jones, Gemma. "Optimisation and characterisation of osteoblast : osteoclast growth in biomaterials." Thesis, Keele University, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.505664.
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This investigation aims to utilise the cell-cell communications between osteoblasts and osteoclasts to create a functional tissue engineered construct that is closer to physiological remodelling than current single cell tissue engineered constructs. A ratio of osteoblasts:osteoclasts was optimised as well as a culture medium that supports both cell types. Four different materials, each with excellent properties for tissue engineering including biocompatible and biodegradable, were compared for their ability to support co-cultures. These materials are; silk fibroin, from Bombyx mori (water vapour and methanol stabilised), chitosan and Poly (1-lactin acid) (PLLA). Silk fibroin and chitosan were shown to support the growth and differentiation of both osteoblasts and osteoclasts in both mono and co-cultures, PLLA did not support osteoclast growth as determined by Tartrate resistant acid phosphatase stain and DNA concentration. The 2D films showed signs of degradation after 10 days culture according to differential scanning calorimetry, fourier transform infra red and gel permeation chromatography. Silk fibroin 3D sponges were manufactured to determine if the co-cultures adhere to, proliferate and differentiate in a 3D environment. Static and dynamic (rotary bioreactor) conditions were compared to determine if the bioreactor conditions enhanced the co-culture of osteoblasts and osteoclasts. The cells were shown to adhere to and proliferate on the sponges by scanning electron microscopy and DNA analysis. The sponges also showed early signs of degradation. The use of silk fibroin and a co-culture system appear to provide excellent potential for bone tissue engineering.
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Kirmizidis, George. "Osteoblast responsiveness to VEGF : potential applications in tissue engineering." Thesis, University of Newcastle Upon Tyne, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.499329.
Full textAbstract:
Vascular endothelial growth factor (VEGF) has a multifaceted role in bone remodelling influencing early osteoblastogenesis, promoting angiogenesis to aid bone repair and chemo-attraction of several cell types needed for bone formation. Consequently, VEGF is a good candidate for therapeutic strategies in bone healing and sue engineering. The aim of this study was to characterise osteoblast responsiveness to VEGT during osteogenesis.
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Chen, M. "Electrical signalling controls the mobility behaviour of osteoblast cells." Thesis, University of Aberdeen, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.590956.
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Direct current (DC) electric fields (EF) as a guidance cue to influence the migration behaviours of osteoblasts were studied in detail for the first time. Direct current electric fields induced human primary osteoblast cells to migrate towards the anode and the osteoblast -like cell line TE85 migrated towards the cathode. In an EF, TE85 cells re-orientated and the long axis of cells were perpendicular to the vector of the electric field. This reorienting response showed voltage-dependence and could be blocked by the MAPK inhibitor U0126. Both growth factor bFGF and VEGF played an important role in the observed EF-induced migration. bFGF increased the migration speed and the directedness of migration in a physiological EF at a certain concentration of bFGF (l0ng/ml). VEGF increased the migration speed but reduced cathodal directedness. Immunofluorescence staining and quantitative confocal microscopy analysis showed that directed migration and perpendicular orientation of the TE85 cells were associated with the re-arrangement of F-actin filaments. U0126 blocked cell orientation in an EF with the disassembly of F-actin filaments. Electric fields failed to redistribute FGF receptors (FGFR) and bFGF in cells. MAPK signal transduction pathways were involved in the EF-induced cell migrations and Western blot revealed that EFs can increase the expression of active ERK1/2 but not of total ERKl/2. Further experiments focused on the effects of an applied EF on the murine preosteoblast cell line MC3T3. MC3T3 cells migrated towards cathode. In addition the applied EF increased the expression of the early osteogenic marker collagen I and reduced the expression of alkaline phosphatase and osteocalcin, with no change of osteopontin and runx-2. These novel findings on osteoblast cell responses to a physiological EF could lead to new insights into the concept of bone development and new clinical treatment for bone repair.
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Fan, Ngo-yin, and 樊傲賢. "The role of protein kinase D in osteoblast differentiation." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2008. http://hub.hku.hk/bib/B41508488.
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Humphrey, Emma Louise. "The control of osteoprotegerin production in osteoblast-like cells." Thesis, University of Liverpool, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.422110.
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Ning, Jian. "The cytoxity of chromium VI in osteoblast derived cells." Thesis, University of Strathclyde, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.366889.
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Jenkins, Alison L. "The thrombin receptor in neutrophils and osteoblast-like cells." Thesis, University of Cambridge, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.338075.
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Nedelcescu, Mihai. "Effects of DP and CRTH2 activation on osteoblast function." Mémoire, Université de Sherbrooke, 2011. http://savoirs.usherbrooke.ca/handle/11143/4067.
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Modulation of PGs by inhibition or stimulation is a promising approach for the management of pain and inflammation in patients with rheumatic disease. Based on recent results from our laboratories as well as on the literature, we hypothesise that Prostaglandin D[subscript 2] (PGD[subscript 2]) is an important anabolic agent for osteoblasts. Our results show that the PGD[subscript 2] decreases the osteoblasts proliferation acting probably through the CRTH2 receptor. Surprisingly, when DK-PGD[subscript 2] was used alone or with Naproxen, although the proliferation decreased with the dose, it seemed to be restored to the control level at higher concentrations of DK-PGD[subscript 2] . Thus, we hypothesise the existence of other compensatory mechanisms. The PGD[subscript 2] had no relevant effect alone or when used with Naproxen, but seemed to decrease the osteoblast differentiation when used with Diclofenac at a higher concentration only. When vitamin D was added to all conditions, PGD[subscript 2] had an inhibitory effect on the differentiation (dose-response), but this could not be replicated when Naproxen was used. In a test with Diclofenac, we can assume a decreasing trend-line for differentiation when augmenting the PGD[subscript 2] dose, but the effect is not statistically relevant. In a competition test with PGD[subscript 2] and DP/CRTH2 antagonists, blocking DP receptor yielded no effect on differentiation, and blocking the CRTH2 receptor showed a relevant decrease at high concentration of PGD[subscript 2]. The effect was similar in a test with PGD[subscript 2] and PPAR[gamma] antagonist suggesting that it might have a compensatory, positive effect that reversed DP activation. The PGD[subscript 2] has a slight positive effect on the osteoblast matrix mineralisation (with Naproxen), but not through its receptors since use of DP/CRTH2 antagonists did not abrogate this. In a competition test with PGD[subscript 2] and DP/CRTH2 antagonists we had no response. When we used the PGD[subscript 2] in the presence of PPAR[gamma] antagonist, the calcification decreased significantly, indicating that the positive effect of PGD[subscript 2] on calcification works rather through this receptor.
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Fan, Ngo-yin. "The role of protein kinase D in osteoblast differentiation." Click to view the E-thesis via HKUTO, 2008. http://sunzi.lib.hku.hk/hkuto/record/B41508488.
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Weivoda, Megan Moore. "The isoprenoid biosynthesis pathway and regulation of osteoblast differentiation." Diss., University of Iowa, 2011. https://ir.uiowa.edu/etd/1106.
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Statins, drugs commonly used to lower serum cholesterol, have been shown to stimulate osteoblast differentiation and bone formation. By inhibiting HMG-CoA reductase (HMGCR) statins deplete the cellular isoprenoid biosynthetic pathway products farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). Current thought in the field is that statins stimulate bone formation through the depletion of GGPP, since exogenous GGPP prevents the effects of statins on osteoblasts in vitro.
We hypothesized that direct inhibition of GGPP synthase (GGPPS) would similarly stimulate osteoblast differentiation. Digeranyl bisphosphonate (DGBP), a specific inhibitor of GGPPS, decreased GGPP levels in MC3T3-E1 pre-osteoblasts and calvarial osteoblasts leading to impaired protein geranylgeranylation. In contrast to our hypothesis, DGBP inhibited the matrix mineralization of MC3T3-E1 cells and the expression of osteoblast differentiation markers in calvarial osteoblasts. The effect on mineralization was not prevented by exogenous GGPP. By inhibiting GGPPS, DGBP led to an accumulation of the GGPPS substrate FPP. We show that FPP and GGPP levels decreased during MC3T3-E1 and calvarial osteoblast differentiation, which correlated with decreased expression of HMGCR and FPP synthase. The decrease in FPP during differentiation was prevented by DGBP treatment. The accumulation of FPP following 24 h DGBP treatment correlated with activation of the glucocorticoid receptor, suggesting a potential mechanism by which DGBP-induced FPP accumulation may inhibit osteoblast differentiation.
To further investigate whether FPP inhibits osteoblast differentiation, we utilized the squalene synthase (SQS) inhibitor zaragozic acid (ZGA), which causes a greater accumulation of FPP than can be achieved with GGPPS inhibition. ZGA treatment decreased osteoblast proliferation, gene expression, alkaline phosphatase (ALP) activity, and matrix mineralization of calvarial osteoblasts. Prevention of ZGA-induced FPP accumulation with HMGCR inhibition prevented the effects of ZGA on osteoblast differentiation. Treatment of osteoblasts with exogenous FPP similarly inhibited matrix mineralization. These results suggest that the accumulation of FPP negatively regulates osteoblast differentiation.
While we did not find that specific depletion of GGPP stimulates osteoblast differentiation, we obtained evidence that GGPP does negatively regulate the differentiation of these cells. Exogenous GGPP treatment inhibited primary calvarial osteoblast gene expression and matrix mineralization. Interestingly, GGPP pre-treatment increased markers of insulin signaling, despite reduced phosphorylation of the insulin receptor (InsR). Inhibition of osteoblast differentiation by GGPP led to the induction of PPARã and enhanced adipogenesis in osteoblastic cultures, suggesting that GGPP may play a role in the osteoblast versus adipocyte fate decision. Adipogenic differentiation of primary bone marrow stromal cell (BMSC) cultures was prevented by DGBP treatment.
Altogether these data present novel roles for the isoprenoids FPP and GGPP in the regulation of osteoblast differentiation and have intriguing implications for the isoprenoid biosynthetic pathway in the regulation of skeletal homeostasis.
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Hoflack, Bernard, Pierre Jurdic, Thilo Riedl, Anne Gallois, and Maria Arantzazu Sanchez-Fernandez. "Osteoclasts control osteoblast chemotaxis via PDGF-BB/PDGF receptor beta signaling." PLOS one, 2008. https://tud.qucosa.de/id/qucosa%3A28994.
Full textAbstract:
BACKGROUND:
Bone remodeling relies on the tightly regulated interplay between bone forming osteoblasts and bone digesting osteoclasts. Several studies have now described the molecular mechanisms by which osteoblasts control osteoclastogenesis and bone degradation. It is currently unclear whether osteoclasts can influence bone rebuilding.
METHODOLOGY/PRINCIPAL FINDINGS:
Using in vitro cell systems, we show here that mature osteoclasts, but not their precursors, secrete chemotactic factors recognized by both mature osteoblasts and their precursors. Several growth factors whose expression is upregulated during osteoclastogenesis were identified by DNA microarrays as candidates mediating osteoblast chemotaxis. Our subsequent functional analyses demonstrate that mature osteoclasts, whose platelet-derived growth factor bb (PDGF-bb) expression is reduced by siRNAs, exhibit a reduced capability of attracting osteoblasts. Conversely, osteoblasts whose platelet-derived growth factor receptor beta (PDGFR-beta) expression is reduced by siRNAs exhibit a lower capability of responding to chemotactic factors secreted by osteoclasts.
CONCLUSIONS/SIGNIFICANCE:
We conclude that, in vitro mature osteoclasts control osteoblast chemotaxis via PDGF-bb/PDGFR-beta signaling. This may provide one key mechanism by which osteoclasts control bone formation in vivo.
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Bell, Bryan Frederick. "Mechanisms regulating osteoblast response to surface microtopography and vitamin D." Diss., Atlanta, Ga. : Georgia Institute of Technology, 2009. http://hdl.handle.net/1853/31711.
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Thesis (Ph.D)--Materials Science and Engineering, Georgia Institute of Technology, 2010.Committee Chair: Barbara Boyan; Committee Member: Andres Garcia; Committee Member: Anthony Norman; Committee Member: Nael McCarty; Committee Member: Zvi Schwartz. Part of the SMARTech Electronic Thesis and Dissertation Collection.
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Wilson, Cameron. "Mediation of Osteoblast Responses to Titanium Roughness by Adsorbed Proteins." Queensland University of Technology, 2005. http://eprints.qut.edu.au/16096/.
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Stable fixation of implants such as artificial teeth depends on the direct apposition of bone to the implanted material. While endosseous implants were traditionally allowed to "osseointegrate" over several months without carrying load, clinical and experimental data show that prostheses with roughened surfaces allow successful integration when subject to earlier loading and more challenging implant sites. However, to design implant surfaces for an optimal biological response requires an understanding of the mechanism by which roughened surfaces promote osseointegration. Research into this mechanism has, to date, focussed primarily on the response of osteoblastic cells to surface topography in vitro. While these have demonstrated some consistent trends in cell behaviour, the fundamental means by which cells sense and respond to roughness remain unclear. It has been suggested that cell responses to changes in topography may relate to differences in the proteins adsorbed from serum (in vitro). While experimental evidence indirectly suggests that physical features can affect protein adsorption, few studies have examined this with respect to surface roughness, particularly as a mediator of cell responses. To address this issue, cell culture and protein adsorption experiments were conducted on a limited range of surface textures. Titanium samples were ground to produce morphologically similar surfaces with three grades of roughness. A duplicate set of specimens were heated at 600°C for one hour, with the aim of masking potential variations in physicochemical properties with differing degrees of grinding. Osteoblast attachment and proliferation studies were conducted over a short time-frame of 48 hours or less, to highlight the effects of proteins adsorbed from serum rather than secreted by adherent cells. Gel electrophoresis provided a profile of the proteins adsorbed to each surface after 15 minutes, corresponding to the time by which the cells had settled onto the surface. Finally, confocal microscopy was used to examine cell morphology on each surface, and to visualize specific interactions between cellular structures and adsorbed adhesion-mediating proteins. Although the effects were inconsistent, attachment assays showed some indications that fewer cells attached in the first 90 minutes as roughness increased. This inverse cell number-roughness trend was significant at 48 hours; however, the variability in attachment assays prevented reliable separation of attachment and proliferation rate effects. While the reduction in cell number with increasing roughness is consistent with previous reports, it is typically observed at later time points, and thus may be increasingly confounded by contact inhibition and differentiation. Thermal oxidation of the titanium did not impact on osteoblast responses to roughness, although it significantly slowed cell proliferation. The latter result was unexpected on the basis of previous reports. One-dimensional gel electrophoresis revealed no significant differences in the composition of adsorbed layers with variations in roughness. However, as expected on account of wettability changes, the heat-treatment did correspond to significant changes in the adsorption profile. While this was not a highly sensitive analysis, it suggests that the cell responses to roughness changes were not governed by broadscale differences in the proteins initially available to adhering cells. In addition to the composition of the adsorbed layer, the distribution of proteins may also vary with topography. The immunofluorescence methods were not sufficiently sensitive to reveal the distribution of adsorbed adhesion proteins (vitronectin and fibronectin). However, the lack of clear labelling does suggest an absence of large accumulations due to specific topographic features. Further work is required to address this issue conclusively. Observations of cell morphology were consistent with widely-reported contact guidance phenomena on grooved surfaces, with elongation and alignment (with topography) increasing with groove depth. Cell elongation was also enhanced on the more hydrophilic, heat-treated titanium, but this effect diminished over time. Although increased elongation at 90 minutes corresponded to lower cell numbers at 48 hours, no causal relationship has yet been established.
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Al-Jallad, Hadil. "Role of transglutaminase enzymes in osteoblast differentiation and matrix deposition." Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=106326.
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Bone formation is an osteoblast-mediated process that is controlled by systemic factors such as hormones, growth factors and local cues that arise from the extracellular matrix (ECM). Bone ECM is elaborated by osteoblasts and therefore they can control their own activity. The ultimate goal of bone matrix formation is to elaborate an extracellular network, consisting mainly of fibronectin and collagen type I, that is capable of mineralizing and forming a strong tissue with appropriate tensile and elastic properties. This thesis describes studies that link transglutaminases (TGs), the protein cross-linking enzymes to type I collagen matrix deposition, osteoblast differentiation and bone formation. Findings here show that MC3T3-E1 osteoblasts require TG-activity for differentiation and proper production of collagenous matrices. We also show that osteoblasts produce two transglutaminase enzymes, transglutaminase 2 (TG2) and Factor XIIIA (FXIIIA), which are both expressed during osteoblast differentiation. The work further defines the roles of the two TGs in osteoblasts and shows that FXIIIA is the main TG-enzyme with transamidating activity in osteoblasts' ECM. Production of FXIIIA is induced during osteoblast differentiation and is externalized to the cell surface, then secreted to the ECM. TG2 was mainly found on the cell surface of osteoblasts with no transamidating activity; however, it is co-localized with FXIIIA on the cell surface. Studies conducted with chemical inhibitors, TG-substrates and activity probes suggest that TG-activity is required for osteoblast differentiation at three different levels. First, by positively affecting microtubule dynamics, delivery and fusion of secretory vesicles carrying cellular collagen type I to the plasma membrane. Second, by promoting fibronectin matrix deposition and collagen type I secretion. And third, by stabilizing the interaction between fibronectin and collagen type I in the ECM. Furthermore, we demonstrated that tubulin and fibronectin are candidate substrates for FXIIIA in osteoblasts. In summary, our studies are the first to describe FXIIIA transglutaminase expression in osteoblasts in vitro and in vivo, and first to link it to collagen secretion and osteoblast differentiation. Furthermore, these studies were the first to suggest a role for cellular FXIIIA in microtubule dynamics. We conclude that transglutaminase activity arising from FXIIIA can regulate osteoblast differentiation affecting extracellular matrix deposition.La formation et le développement de l'os est un processus complexe dirigé par les ostéoblastes. Contrôlés par des hormones systémiques, des cytokines et d'autres facteurs locaux, les ostéoblastes sécrètent et assemblent la matrice extracellulaire (MEC) des tissus osseux. L'aboutissement de ce processus sera la génération d'un réseau extracellulaire constitué notamment de la fibronectine et du collagene de type I qui va se minéraliser en formant le tissu dur de l'os avec d'excellent propriétés mécaniques. Cette thèse présente des études liées aux transglutaminases (TGs) – une classe des enzymes responsables de la polymérisation (cross-linking) des protéines et d'autres composée biomacromoléculaires - en relation avec le collagène de type I secrété pendant l'élaboration de la MEC, la différentiation des ostéoblastes et l'élaboration du tissu osseux. Les principaux résultats de ces études portent sur l'observation que l'activité polymérisante de la TG est un facteur crucial pour la différentiation des cellules ostéoblastiques MC3T3-E1 et pour la production normale de la matrice collagénique. Un résultat essentiel de la présente recherche porte sur la découverte que les ostéoblastes synthétisent deux types d'enzymes TG, i.e. la transglutaminase 2 (TG2) et le facteur XIIIA (FXIIIA), qui sont tous les deux secrétés pendant la différentiation des ostéoblastes. Les résultats suivants éclaircirent les rôles des deux enzymes TG (TG2 et FXIIIA) dans l'activité des ostéoblastes en montrant que c'est le FXIIIA qui est l'enzyme TG dominante avec une activité de transamidation importante dans la MEC des ostéoblastes. FXIIIA est produit pendant la différentiation des ostéoblastes en s'externalisant vers la surface des cellules pour être par la suite sécrété dans la MEC. L'enzyme TG2 a été localisé seulement à la surface des cellules osteoblastiques. Même si le TG2 a été trouvé colocalisé avec le FXIIIA à la surface des cellules, aucune activité de transamidation n'est identifiée pour le TG2. Des études comportant des inhibiteurs chimiques, de substrats TG et de sondes d'activité TG suggèrent que l'activité TG est nécessaire pour la différentiation des ostéoblastes sur trois plans distincts, à savoir : (i) par une action bénéfique sur la dynamique des microtubules, l'acheminement et la fusion des vésicules sécrétoires qui transportent le collagène I cellulaire vers la membrane plasmatique; (ii) par l'accélération du dépôt de la matrice de fibronectine et la sécrétion du collagène de type I; (iii) par la stabilisation de l'interaction de la fibronectine avec le collagène I dans la MEC. De plus, nous avons démontré que la tubuline and la fibronectine ce sont de candidats substrat pour le facteur FXIIIA dans les ostéoblastes. En résumé, notre recherche décrit pour la première fois l'expression de l'enzyme transglutaminase FXIIIA dans les ostéoblastes, tant in vitro qu'in vivo, en corrélant l'expression du FXIIIA à la sécrétion et la différentiation des ostéoblastes. De plus, notre étude est la première en attribuant un rôle au facteur FXIIIA relative à la dynamique des microtubules. On conclut de notre étude que l'activité transglutaminase du facteur FXIIIA exerce une influence décisive dans les processus de différentiation des ostéoblastes avec un effet régulateur crucial à la sécrétion et au dépôt de la matrice extracellulaire.
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Horgan, Fergal G. "Osteoblast response to sputter deposited calcium phosphate thin film coatings." Thesis, University of Ulster, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.400963.
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Langeveldt, Carmen Ronel. "Alternative insulin mitogenic signaling pathways in immature osteoblast cell lines." Thesis, Stellenbosch : Stellenbosch University, 2002. http://hdl.handle.net/10019.1/52646.
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Thesis (MSc)--University of Stellenbosch, 2002.ENGLISH ABSTRACT: Insulin is a mitogen for many cells and commonly signals through the classical, mitogenic Raf- MEK-ERK or metabolic PB-kinase pathways. Insulin deficiency or type I diabetes causes severe osteopenia. Obese patients with type II diabetes or insulin resistance, a disease associated with defective insulin signaling pathways and high levels of circulating insulin, have increased or normal bone mineral density. The question of whether hyperinsul inemia preserves bone mass is frequently raised. However, there is still a lot of controversy on the role of insulin as an osteoanabolic agent and this question still remains unanswered. A critical role for insulin signaling in bone building osteoblasts has recently been demonstrated with IRS-l knock-out mice. These mice developed low-turnover osteopenia due to impaired proliferation and differentiation, stressing the importance of osteoblastic IRS-l for maintaining normal bone formation. In the present study it was found that insulin does function in vitro as an osteoblast mitogen. This was illustrated in three relatively immature osteoblast (MBA-15.4, -15.6 mouse and MG- 63 human) cell lines, which responded to insulin with significant increases in proliferation. In the MBA -15.4 preosteoblasts insulin stimulation of proliferation was comparable to the welldescribed mitogen, TPA. The UMR-I06 cell line expresses markers of differentiated osteoblasts, and was much less responsive to insulin treatment. The difference in proliferative potential may be due to differences between spontaneously transformed cell lines, or the stage of cell differentiation. UOI26, a MEKI/2 inhibitor and wortmannin, a PB-kinase inhibitor, were used to investigate the pathway used by insulin to signal and activate ERK and osteoblast proliferation. In MBA-15.4 mouse preosteoblasts, GF-containing FCS was completely dependent on MEK for DNA synthesis. In contrast, in both MBA-15.4 and more mature MBA-15.6 osteoblasts, insulininduced proliferation was resistant to the inhibitors alone or in combination. Higher MEKinhibitor concentrations had no effect, and proliferation was also increased by the inhibitors in several experiments. This indicated that the classical, insulin mitogenic pathway was not involved in MBA-15.4 proliferation. Wortmannin had no effect on either insulin- or 20% FCSstimulated proliferation, but inhibited activation of Akt/PKB, the metabolic downstream target of PI3-kinase. Insul in signal ing to ERK was both MEK-and PI3-kinase- dependent, but this had no effect on proliferation. In contrast, FCS-stimulated ERK activation and proliferation was almost completely dependent on MEK-ERK activation. Proliferative signaling in the MG-63 human osteoblastic cell line in response to insulin was partially dependent on MEK and partially dependent on PB-kinase. In contrast, signaling in response to the phorbol ester, TPA, was partially dependent on PI3K but totally dependent on MEK-ERK. This indicates that the signal converges on ERK, suggesting the involvement of a PB-kinase upstream of a dominant MEK-ERK pathway. The differences found here between mouse and human insulin mitogenic signaling pathways indicate that there may be species differences between osteoblast signaling pathways, with mouse cells being independent and human cells being dependent on MEK for DNA synthesis in response to insulin. The effects of glucocorticoids on insulin mitogenic signaling in osteoblasts were also investigated, because chronic long-term steroid use results in excessive bone loss. The PTP inhibitor, sodium orthovanadate, reversed GC-impaired TPA- and FCS- induced proliferation in MBA-1SA and MG-63 preosteoblasts. PTPs, such as SHP-l and PTP-IB, dephosphorylate and inactivate phosphorylated kinases. Both SHP-l and PTPlB associated with kinases in the mitogenic signaling cascade of MBA-lS.4 preosteoblasts growing rapidly in 10% FCS. Further, SHP-I co-irnmunoprecipitated with active, tyrosine phosphorylated ERK, which may indicate that it can dephosphorylate and inactivate ERK. However, since the MEK-ERK or PB-kinase pathways are not important in insulin-induced proliferation in mouse osteoblasts, the PTPs are unlikely to be role players in the negative regulation of this signaling pathway. This was confirmed by the finding that vanadate was unable to reverse GC-induced decreases in insulinstimulated DNA synthesis. This suggests that vanadate-sensitive PTPs may not be important in the negative regulation of insulin-induced mouse osteoblast proliferation, and provides further evidence of a novel insulin mitogenic pathway in the MBA-lSA but not MG-63 osteoblastic cell line.
AFRIKAANSE OPSOMMING: Insulien is 'n mitogeen vir baie selle en gelei na binding aan die insulien reseptor, intrasellulêre seine via die klassieke, mitogeniese Raf-MEK-ERK of die metaboliese PB-kinase seintransduksie pad. 'n Insulien gebrek of tipe I diabetes veroorsaak osteopenie. Vetsugtige pasiënte met insulien weestandigheid of tipe II diabetes, 'n siekte wat geassosieer word met foutiewe insulien seintransduksie en hoë vlakke van sirkuierende insulien, het verhoogde of normale been mineraal digtheid (BMD). Die vraag of hiper insulin ernie 'n verlies aan beenmassa teëwerk word dikwels gevra. Teenstrydigheid oor die rol van insulien as 'n osteo-anaboliese stof bestaan egter steeds en hierdie vraag bly dus onbeantwoord. Dat insulien seintransduksie wel 'n kritiese rol speel in beenvormende osteoblaste is onlangs bevestig in studies met muise waarvan die geen vir IRS-l uitgeslaan is. Hierdie muise ontwikkel 'n lae omset osteopenie weens verswakte proliferasie en differensiasie. fn hierdie studie is gevind dat insulien wel in vitro as 'n osteoblast mitogeen kan funksioneer. Dit is in drie relatief onvolwasse (MBA-15.4, -15.6 muis en MG-63 mens) sellyne geillistreer, deur betekenisvolle verhogings in insulien-geaktiveerde proliferasie. In MBA-15.4 preosteoblaste is die persentasie verhoging in insulien-gestimuleerde proliferasie vergelykbaar met dié van die bekende mitogeniese forbolester, TPA. Die UMR-I06 sellyn het kenmerke van gedifferensieerde osteoblaste, en was baie minder responsief op insulien behandeling. Die verskil in die proliferasie vermoë van die verskillende sellyne kan die gevolg wees van verskille wat bestaan tussen spontaan getransformeerde sellyne of die stadium van sel differensiasie. 'n MEK 1/2 inhibitor, UO126 en 'n PB-kinase inhibitor, wortmannin, is gebruik om die insulien seintransduksie pad noodsaaklik vir die aktivering van ERK en osteoblast proliferasie te bepaal. In MBA-1S.4 muis pre-osteoblaste, was fetale kalf SenlTI1(FKS)-geinduseerde DNA sintese totaal afhanklik van MEK. Beide die MBA-15.4 en die meer volwasse MBA-15.6 muis osteoblaste was weerstandig teen die inhibitors op hulle eie, of in kombinasie. Verhoogde MEK-inhibitor konsentrasies het geen verdere effek gehad nie en in verskeie eksperimente is 'n verhoging in preliferasie selfs waargeneem met MEK-inhibisie. Hierdie resultate dui aan dat die klassieke insulien mitogeniese pad nie betrokke is in MBA-I5.4 gestimuleerde selproliferasie nie. Wortmannin het geen effek gehad op insulien- of20% FKS-gestimuleerde DNA sintese nie, maar het wel die aktivering van PB-kinase se metaboliese teiken, AktJPKB geinhibeer. Insulien seintransduksie aktiveer dus ERK deur beide MEK en PB-kinase, maar het geen effek op proliferasie gehad nie. FKS-gestimuleerde ERK aktivering en proliferasie was totaal afhanlik van MEK-ERK aktivering. Insulien-geaktiveerde DNA sintese in die mens MG-63 osteoblaste was gedeeltelik afhanklik van beide MEK en PB-kinase. Alhoewel IPA ook PB-kinase kon aktiveer, was dit totaal afhanklik van MEK vir DNA sintese. Dit dui aan dat daar 'n PB-kinase stroom-op van 'n dominante MEK-ERK seintransduksie pad voorkom. Die verskille wat ons dus waargeneem het in insulien mitogeniese seintransduksie tussen muis en mens, kan aandui dat insuliengestimuleerde seintranduksie paaie kan verskil van spesie tot spesie. Dit is bevestig met die muisselle wat onafhanklik is en mens selle wat afhanklik is van MEK aktivering vir insuliengeaktiveerde DNA sintese. Kroniese, langtermyn steroied behandeling kan beenverlies veroorsaak en die effek van glukokortikoide (GK) op die insulien mitogeniese pad in osteoblaste is dus ook ondersoek. Natrium-ortovanadaat, 'n proteien tirosien fosfatase (PIP) inhibitor het GK-verlaagde proliferasie in repons tot beide IPA- en FKS behandeling herstel in MBA-lSA en MG-63 preosteoblaste. PIPs soos SHP-l en PIP-l B funksioneer deur gefosforileerde kinases te defosforileer en dus te inaktiveer. Beide SHP-l and PIP-lB kon assosieer met kinases in die mitogeniese insulien seintransduksie pad van vinnig groeiende MBA-IS A preosteoblaste in 10% FKS. Verder het SHP-I ook geko-immunopresipiteer met aktiewe, tirosien-gefosforileerde ERK, wat aandui dat SHP-I met ERK assosieer om dit te defosforileer en inaktiveer. Die MEKERK of PB-kinase paaie is nie belangrik vir insulien-geaktiveerde seintransduksie in muis osteoblaste nie. Dit is dus onwaarskynlik dat die PIPs 'n rol sal speel in die negatiewe regulering van hierdie seintransduksie paaie. Die ontdekking dat vanadaat nie glukokortikoiedverlaagde insulien-geaktiveerde DNA sintese kan herstel nie, toon dat vanadaat-sensitiewe PIPs nie 'n rol speel in insulien-geaktiveerde proliferasie in muisselle nie. Hierdie bevinding het verder bevestig dat 'n nuwe insulien mitogeniese pad in die MBA-ISA, maar nie die MG-63 selle moontlik bestaan.
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Whited, Bryce Matthew. "Osteoblast Response to Zirconia-Hybridized Pyrophosphate Stabilized Amorphous Calcium Phosphate." Thesis, Virginia Tech, 2005. http://hdl.handle.net/10919/32699.
Full textAbstract:
Biodegradable polyesters, such as poly(DL-lactic-co-glycolic acid) (PLGA), have been used to fabricate porous bone scaffolds to support bone tissue development. These scaffolds allow for cell seeding, attachment, growth and extracellular matrix production in vitro and are replaced by new bone tissue when implanted into bone sites in vivo. Hydroxyapatite (HAP) and Æ Ã -tricalcium phosphate (Æ Ã -TCP) ceramics have been incorporated into PLGA bone scaffolds and have been shown to increase their osteoconductivity (support cell attachment). Although HAP, Æ Ã -TCP, and biodegradable polyesters are osteoconductive, there is no evidence that these scaffold materials are osteoinductive (support cell differentiation). Calcium and phosphate ions, in contrast, have been postulated to be osteogenic factors that enhance osteoblast differentiation and mineralization. Recently, a zirconia-hybridized pyrophosphate stabilized amorphous calcium phosphate (Zr-ACP) has been synthesized which permits controlled release of calcium and phosphate ions and thus is hypothesized to be osteoinductive. Incorporation of Zr-ACP into a highly porous poly(DL lactic-co-glycolic acid) (PLGA) scaffold could potentially increase the osteoinductivity of the scaffold and therefore promote osteogenesis when implanted in vivo.
To determine the osteoinductivity of Zr-ACP, a MC3T3-E1 mouse calvarial-derived osteoprogenitor cell line was used to measure cell response to Zr-ACP. To accomplish this objective, Zr-ACP was added to cell culture at different stages in cell maturation (days 0, 4 and 11). DNA synthesis, alkaline phosphatase (ALP) activity, osteopontin synthesis and collagen synthesis were determined. Results indicate that culture in the presence of Zr-ACP significantly increased cell proliferation, ALP activity and osteopontin synthesis but not collagen synthesis. To determine the feasibility of incorporating Zr-ACP into a PLGA scaffold, PLGA/Zr-ACP composite foams (5% or 10% (w/v) polymer:solvent with 25 wt% or 50 wt% Zr-ACP) were fabricated using a thermal phase inversion technique. Scanning electron microscopy revealed a highly porous structure with pores ranging in size from a few microns to about 100 Æ Ã m. The amorphous structure of the Zr-ACP was maintained during composite fabrication as confirmed by X-ray diffraction measurements. Composite scaffolds also showed significantly greater compressive yield strengths and moduli as compared to pure polymer scaffolds.
The results of this study indicate that Zr-ACP enhances the osteoblastic phenotype of MC3T3-E1 cells in vitro and can be incorporated into a porous PLGA scaffold. Porous PLGA/Zr-ACP composites are promising for use as bone scaffolds to heal bone defects.Master of Science
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Brister, Aaron B. "OASIS AND XBP-1 ACTIVITY IN OSTEOBLAST DIFFERENTIATION AND OSTEOSARCOMA." Case Western Reserve University School of Graduate Studies / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=case1196287582.
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Wilson, Cameron John. "Mediation of Osteoblast Responses to Titanium Roughness by Adsorbed Proteins." Thesis, Queensland University of Technology, 2005. https://eprints.qut.edu.au/16096/1/Cameron_Wilson_Thesis.pdf.
Full textAbstract:
Stable fixation of implants such as artificial teeth depends on the direct apposition of bone to the implanted material. While endosseous implants were traditionally allowed to "osseointegrate" over several months without carrying load, clinical and experimental data show that prostheses with roughened surfaces allow successful integration when subject to earlier loading and more challenging implant sites. However, to design implant surfaces for an optimal biological response requires an understanding of the mechanism by which roughened surfaces promote osseointegration. Research into this mechanism has, to date, focussed primarily on the response of osteoblastic cells to surface topography in vitro. While these have demonstrated some consistent trends in cell behaviour, the fundamental means by which cells sense and respond to roughness remain unclear. It has been suggested that cell responses to changes in topography may relate to differences in the proteins adsorbed from serum (in vitro). While experimental evidence indirectly suggests that physical features can affect protein adsorption, few studies have examined this with respect to surface roughness, particularly as a mediator of cell responses. To address this issue, cell culture and protein adsorption experiments were
conducted on a limited range of surface textures. Titanium samples were ground to
produce morphologically similar surfaces with three grades of roughness. A duplicate set of specimens were heated at 600°C for one hour, with the aim of masking potential variations in physicochemical properties with differing degrees of grinding. Osteoblast attachment and proliferation studies were conducted over a short time-frame of 48 hours or less, to highlight the effects of proteins adsorbed from serum rather than secreted by adherent cells. Gel electrophoresis provided a profile of the proteins adsorbed to each surface after 15 minutes, corresponding to the time by which the cells had settled onto the surface. Finally, confocal microscopy was used to examine cell morphology on each surface, and to visualize specific interactions between cellular structures and adsorbed adhesion-mediating proteins. Although the effects were inconsistent, attachment assays showed some indications that fewer cells attached in the first 90 minutes as roughness increased. This inverse cell number-roughness trend was significant at 48 hours; however, the variability in attachment assays prevented reliable separation of attachment and proliferation rate effects. While the reduction in cell number with increasing roughness is consistent with previous reports, it is typically observed at later time points, and thus may be increasingly confounded by contact inhibition and differentiation. Thermal oxidation of the titanium did not impact on osteoblast responses to roughness, although it significantly slowed cell proliferation. The latter result was unexpected on the basis of previous reports. One-dimensional gel electrophoresis revealed no significant differences in the composition of adsorbed layers with variations in roughness. However, as expected on account of wettability changes, the heat-treatment did correspond to significant changes in the adsorption profile. While this was not a highly sensitive analysis, it suggests that the cell responses to roughness changes were not governed by broadscale differences in the proteins initially available to adhering cells. In addition to the composition of the adsorbed layer, the distribution of proteins may also vary with topography. The immunofluorescence methods were not sufficiently sensitive to reveal the distribution of adsorbed adhesion proteins (vitronectin and fibronectin). However, the lack of clear labelling does suggest an absence of large accumulations due to specific topographic features. Further work is required to address this issue conclusively. Observations of cell morphology were consistent with widely-reported contact guidance phenomena on grooved surfaces, with elongation and alignment (with topography) increasing with groove depth. Cell elongation was also enhanced on the more hydrophilic, heat-treated titanium, but this effect diminished over time. Although increased elongation at 90 minutes corresponded to lower cell numbers at 48 hours, no causal relationship has yet been established.