7 results on '"Cool SM"'
Search Results
2. Temporal and functional changes in glycosaminoglycan expression during osteogenesis.
- Author
-
Nurcombe V, Goh FJ, Haupt LM, Murali S, and Cool SM
- Subjects
- 3T3 Cells, Animals, Calcification, Physiologic genetics, Calcification, Physiologic physiology, Cell Differentiation drug effects, Cell Differentiation genetics, Cell Differentiation physiology, Cell Proliferation drug effects, Extracellular Matrix drug effects, Extracellular Matrix metabolism, Fluorescent Antibody Technique, Glucuronidase genetics, Glucuronidase metabolism, Heparan Sulfate Proteoglycans genetics, Heparan Sulfate Proteoglycans metabolism, Heparitin Sulfate isolation & purification, Heparitin Sulfate metabolism, Heparitin Sulfate pharmacology, Immunoblotting, Mice, Osteogenesis genetics, Phenotype, Reverse Transcriptase Polymerase Chain Reaction, Sulfotransferases genetics, Glycosaminoglycans metabolism, Osteogenesis physiology, Sulfotransferases metabolism
- Abstract
Heparan sulfate proteoglycans (HSPGs) are complex and labile macromolecular moieties on the surfaces of cells that control the activities of a range of extracellular proteins, particularly those driving growth and regeneration. Here, we examine the biosynthesis of heparan sulfate (HS) sugars produced by cultured MC3T3-E1 mouse calvarial pre-osteoblast cells in order to explore the idea that changes in HS activity in turn drive phenotypic development during osteogenesis. Cells grown for 5 days under proliferating conditions were compared to cells grown for 20 days under mineralizing conditions with respect to their phenotype, the forms of HS core protein produced, and their HS sulfotransferase biosynthetic enzyme levels. RQ-PCR data was supported by the results from the purification of day 5 and day 20 HS forms by anionic exchange chromatography. The data show that cells in active growth phases produce more complex forms of sugar than cells that have become relatively quiescent during active mineralization, and that these in turn can differentially influence rates of cell growth when added exogenously back to preosteoblasts.
- Published
- 2007
- Full Text
- View/download PDF
3. Isolation of a native osteoblast matrix with a specific affinity for BMP2.
- Author
-
Grünert M, Dombrowski C, Sadasivam M, Manton K, Cool SM, and Nurcombe V
- Subjects
- 3T3 Cells, Analysis of Variance, Animals, Binding Sites genetics, Bone Morphogenetic Protein 2, Bone Morphogenetic Proteins genetics, Cell Line, Cells, Cultured, Chondroitinases and Chondroitin Lyases metabolism, Chromatography, Ion Exchange, Extracellular Matrix ultrastructure, Glycosaminoglycans metabolism, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, Heparin Lyase metabolism, Humans, Mesenchymal Stem Cells cytology, Mesenchymal Stem Cells metabolism, Mice, Microscopy, Electron, Scanning, Microscopy, Fluorescence, Osteoblasts cytology, Protein Binding, Proteoglycans metabolism, Reverse Transcriptase Polymerase Chain Reaction, Transforming Growth Factor beta genetics, Bone Morphogenetic Proteins metabolism, Extracellular Matrix metabolism, Osteoblasts metabolism, Transforming Growth Factor beta metabolism
- Abstract
During their commitment and differentiation toward the osteoblast lineage, mesenchymal stem cells secrete a unique extracellular matrix (ECM) that contains large quantities of glycosaminoglycans (GAGs). Proteoglycans (PGs) are major structural and functional components of the ECM and are composed of a core protein to which one or more glycosaminoglycan sugar chains (GAGs) attach. The association of BMP2, a member of the TGF-beta super-family of growth factors, and a known heparin-binding protein, with GAGs has been implicated as playing a significant role in modulating the growth factor's in vitro bioactivity. Here we have characterised an osteoblast-derived matrix (MX) obtained from decellularised MC3T3-E1 cell monolayers for its structural attributes, using SEM and histology, and for its functional ability to maintain cell growth and viability. Using a combination of histology and anion exchange chromatography, we first confirmed the retention of GAGs within MX following the decellularisation process. Then the binding specificity of the retained GAG species within the MX for BMP2 was examined using a BMP2-HBP/EGFP (BMP2 Heparin-Binding Peptide/Enhanced Green Fluorescent Protein) fusion protein. The results of this study provide further evidence for a central role of the ECM in the regulation of BMP2 bioactivity, hence on mesenchymal stem cell commitment to the osteoblast lineage.
- Published
- 2007
- Full Text
- View/download PDF
4. Sustained release and osteogenic potential of heparan sulfate-doped fibrin glue scaffolds within a rat cranial model.
- Author
-
Woodruff MA, Rath SN, Susanto E, Haupt LM, Hutmacher DW, Nurcombe V, and Cool SM
- Subjects
- Alkaline Phosphatase genetics, Alkaline Phosphatase metabolism, Animals, Bone Regeneration physiology, Core Binding Factor Alpha 1 Subunit genetics, Core Binding Factor Alpha 1 Subunit metabolism, Female, Glycosaminoglycans metabolism, Heparitin Sulfate chemistry, Immunohistochemistry, Microscopy, Confocal, Parietal Bone metabolism, Parietal Bone surgery, Rats, Rats, Wistar, Reverse Transcriptase Polymerase Chain Reaction, Tomography, X-Ray Computed, Wound Healing, Fibrin Tissue Adhesive metabolism, Heparitin Sulfate metabolism, Osteogenesis physiology, Parietal Bone physiopathology
- Abstract
This paper explores the potential therapeutic role of the naturally occurring sugar heparan sulfate (HS) for the augmentation of bone repair. Scaffolds comprising fibrin glue loaded with 5 microg of embryonically derived HS were assessed, firstly as a release-reservoir, and secondly as a scaffold to stimulate bone regeneration in a critical size rat cranial defect. We show HS-loaded scaffolds have a uniform distribution of HS, which was readily released with a typical burst phase, quickly followed by a prolonged delivery lasting several days. Importantly, the released HS contributed to improved wound healing over a 3-month period as determined by microcomputed tomography (microCT) scanning, histology, histomorphometry, and PCR for osteogenic markers. In all cases, only minimal healing was observed after 1 and 3 months in the absence of HS. In contrast, marked healing was observed by 3 months following HS treatment, with nearly full closure of the defect site. PCR analysis showed significant increases in the gene expression of the osteogenic markers Runx2, alkaline phosphatase, and osteopontin in the heparin sulfate group compared with controls. These results further emphasize the important role HS plays in augmenting wound healing, and its successful delivery in a hydrogel provides a novel alternative to autologous bone graft and growth factor-based therapies.
- Published
- 2007
- Full Text
- View/download PDF
5. Selection using the alpha-1 integrin (CD49a) enhances the multipotentiality of the mesenchymal stem cell population from heterogeneous bone marrow stromal cells.
- Author
-
Rider DA, Nalathamby T, Nurcombe V, and Cool SM
- Subjects
- Adipose Tissue cytology, Adult, Antigens, CD genetics, Antigens, CD metabolism, Bone Marrow Cells cytology, Bone and Bones cytology, Cartilage cytology, Cell Differentiation genetics, Cell Differentiation physiology, Cell Separation, Cells, Cultured, Endoglin, Female, Flow Cytometry, Gene Expression, Humans, Integrin alpha1 genetics, Mesenchymal Stem Cells cytology, Multipotent Stem Cells cytology, Multipotent Stem Cells metabolism, Polymerase Chain Reaction, Receptors, Cell Surface genetics, Receptors, Cell Surface metabolism, Stromal Cells cytology, Thy-1 Antigens genetics, Thy-1 Antigens metabolism, Bone Marrow Cells metabolism, Integrin alpha1 metabolism, Mesenchymal Stem Cells metabolism, Stromal Cells metabolism
- Abstract
Bone marrow-derived mesenchymal stem cells consist of a developmentally heterogeneous population of cells obtained from colony forming progenitors. As these colonies express the alpha-1 integrin (CD49a), here we single-cell FACS sorted CD49a+ cells from bone marrow in order to create clones and then compared their colony forming efficiency and multilineage differentiation capacity to the unsorted cells. Following selection, 40% of the sorted CD49a+ cells formed colonies, whereas parental cells failed to form colonies following limited dilution plating at 1 cell/well. Following ex vivo expansion, clones shared a similar morphology to the parental cell line, and also demonstrated enhanced proliferation. Further analysis by flow cytometry using a panel of multilineage markers demonstrated that the CD49a+ clones had enhanced expression of CD90 and CD105 compared to unsorted cells. Culturing cells in adipogenic, osteogenic or chondrogenic medium for 7, 10 and 15 days respectively and then analysing them by quantitative PCR demonstrated that CD49a+ clones readily underwent multlineage differentiation into fat, bone and cartilage compared to unsorted cells. These results thus support the use of CD49a selection for the enrichment of mesenchymal stem cells, and describes a strategy for selecting the most multipotential cells from a heterogeneous pool of bone marrow mononuclear stem cells.
- Published
- 2007
- Full Text
- View/download PDF
6. Glycosaminoglycan and growth factor mediated murine calvarial cell proliferation.
- Author
-
Manton KJ, Haupt LM, Vengadasalam K, Nurcombe V, and Cool SM
- Subjects
- Animals, Bone Morphogenetic Protein 2, Bone Morphogenetic Proteins pharmacology, Cell Differentiation drug effects, Cells, Cultured, Dose-Response Relationship, Drug, Fibroblast Growth Factor 2 pharmacology, Humans, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, Organ Culture Techniques, Simvastatin pharmacology, Skull cytology, Transforming Growth Factor beta pharmacology, Transforming Growth Factor beta1 pharmacology, Cell Proliferation drug effects, Glycosaminoglycans pharmacology, Intercellular Signaling Peptides and Proteins pharmacology
- Abstract
Understanding the complex mechanisms underlying bone remodeling is crucial to the development of novel therapeutics. Glycosaminoglycans (GAGs) localised to the extracellular matrix (ECM) of bone are thought to play a key role in mediating aspects of bone development. The influence of isolated GAGs was studied by utilising in vitro murine calvarial monolayer and organ culture model systems. Addition of GAG preparations extracted from the cell surface of human osteoblasts at high concentrations (5 microg/ml) resulted in decreased proliferation of cells and decreased suture width and number of bone lining cells in calvarial sections. When we investigated potential interactions between the growth factors fibroblast growth factor-2 (FGF2), bone morphogenic protein-2 (BMP2) and transforming growth factor-beta1 (TGFbeta1) and the isolated cell surface GAGs, differences between the two model systems emerged. The cell culture system demonstrated a potentiating role for the isolated GAGs in the inhibition of FGF2 and TGFbeta1 actions. In contrast, the organ culture system demonstrated an enhanced stimulation of TFGbeta1 effects. These results emphasise the role of the ECM in mediating the interactions between GAGs and growth factors during bone development and suggest the GAG preparations contain potent inhibitory or stimulatory components able to mediate growth factor activity.
- Published
- 2007
- Full Text
- View/download PDF
7. A novel use of TAT-EGFP to validate techniques to alter osteosarcoma cell surface glycosaminoglycan expression.
- Author
-
Kumarasuriyar A, Dombrowski C, Rider DA, Nurcombe V, and Cool SM
- Subjects
- Biological Transport drug effects, Cell Line, Tumor, Chondroitin Lyases pharmacology, Chondroitin Sulfates pharmacology, Flow Cytometry, Glycosides pharmacology, Green Fluorescent Proteins genetics, Heparin pharmacology, Heparin Lyase pharmacology, Humans, Microscopy, Confocal, Osteosarcoma genetics, Osteosarcoma metabolism, Osteosarcoma pathology, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, tat Gene Products, Human Immunodeficiency Virus genetics, Cell Membrane metabolism, Glycosaminoglycans metabolism, Green Fluorescent Proteins metabolism, tat Gene Products, Human Immunodeficiency Virus metabolism
- Abstract
Several methods to alter cell surface glycosaminoglycan (GAG) expression have previously been described, including treatments with chlorate to reduce the addition of charged sulfate groups, xyloside compounds to displace GAGs from their core proteins, and GAG lyases, such as heparinase and chondroitinase, to release GAG fragments from the cell layer. While these methods are useful in identifying cellular mechanisms which are dependent on GAGs, they must be stringently validated to assess results in the appropriate context. To determine the most useful technique for the evaluation of GAG function in osteogenesis, MG-63 osteosarcoma cells were systematically treated with these agents and evaluated for changes in cell surface GAGs using a TAT-EGFP fusion protein. TAT, a protein transduction domain from the HIV-1 virus, requires cell surface GAGs to traverse cell membranes. The EGFP component provides a method to assess protein entry into cells in both qualitative and quantitative tests. Here, TAT-EGFP transduction analysis confirmed radiochemical and physiological data that chlorate effectively disrupts GAG expression. TAT-EGFP entry into cells was also inhibited by the exogenous application of commercial heparin and GAGs extracted from MG-63 cells as well as by the pre-treatment of cells with chondroitinase ABC. However, neither heparinase III treatment nor the addition of exogenous chondroitin-6-sulfate affected TAT-EGFP entry into cells. In addition, xyloside-beta-D-naphthol and xyloside-beta-D-cis/trans-decahydro-2-naphthol treatment could not induce significant phenotypic change in these cells, and the unaffected TAT-EGFP transduction confirmed that this was due to an inability to efficiently prime GAG synthesis. The use of TAT-EGFP is thus a useful technique to specifically evaluate cell surface GAG expression in a simple, quantifiable manner, and avoids the complications involved with conventional radiochemical assays or analytical chromatography.
- Published
- 2007
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.