The addition of silicon ions (10-500 μM) to the culture medium of MG63 osteoblast-like cells showed no changes in cell viability, metabolic activity, proliferation or morphology. Silicon ions resulted in a concentration-dependent increase in the expression of vascular endothelial growth factor (VEGF) by the MG63 cells. Addition of magnesium ions (1-50 mM) to the culture medium of MG63 cells caused a dose-dependent decrease in cell viability, metabolic activity and proliferation at each time point. In general, silicon and magnesium ions had no effect on the viability of a human endothelial cell line (HUVEC). A slight decreasing trend to the metabolic activity of the HUVECs was observed with increasing concentrations of silicon ions at all time points, but this decreasing trend was more pronounced with the addition of magnesium ions. The highest magnesium ion concentration studied (50 mM) caused a change in HUVEC morphology from a typical cobblestone appearance to a fibroblast-like shape. Lastly, the effect of silicon ions on angiogenesis <i>in vitro</i> was studied using two different <i>in vitro</i> assays. The first, using Matrigel as an extracellular matrix coating for the guidance of endothelial cells to form tube-like structures (an indicator of angiogenesis), proved unreliable for studying the promotion of angiogenesis. Additionally, tube-like structures were also observed with osteoblasts cells, raising questions about the efficiency of this assay. The second assay, AngioKit, was a suitable model for studying stimulation and inhibition of tube-like formation. Results obtained using this assay showed that silicon ions alone (500 μM) did not stimulate tube-like formation, but a significant increase in tube-like formation was observed with MG63 cell-conditioned media, with (500 μM) and without silicon ions, when compared to the control medium.

Microarray analysis of gene expression in osteoblasts from patients with osteoporosis (OP) and osteoarthritis (OA) showed that 115 genes were robustly differentially expressed (P<0.05). Functional annotation clustering revealed cell adhesion to be the gene ontology classification most likely to be associated with this gene list. In addition scrutiny of the list revealed several genes with strong biological support for the involvement in bone homeostasis (FOSL1, BMPR2 and TGFBR1). Real -time PCR validated the trends seen in the microarray analysis, but failed to reach statistical significance for any of the genes examined. This analysis supports the value and potential of larger scale comparison of gene expression in OA and OP osteoblasts as a method for identifying novel factors involved in bone homeostasis. The cannabinoid system has recently been identified as involved in the regulation of bone homeostasis. In vitro investigation revealed that although cannnabinoid receptor agonists N-arachidonoylethanolamine (AEA), 2-arachidonoylglycerol (2-AG) and JWH015 had no effect on metabolic activity, cell number, or alkaline phosphatase activity of calvarial mouse osteoblasts there were changes in gene expression. RankL expression was reduced relative to Opg expression by both JWH015 and AEA. Preliminary results indicate that JWH015 was also capable of increasing PPARγ expression which could alter the balance of osteoblastic and adipocytic differentiation of mesenchymal stem cells (MSC). This could have implications for use of these drugs in vivo. Using the 3T3-F442A cell line to develop a model of MSC differentiation highlighted difficulties associated with using cell models. Necessary additional factors required to induce differentiation of a cell line compared to a primary cell make interpretation of results more complicated. This model also highlighted that alkaline phosphatase and osteocalcin (markers usually used to identify osteogenic differentiation) were expressed during adipocytic differentiation. Future use of such markers in MSC models should be closely scrutinized.

Bisphosphonates are the most widely prescribed anti-resorptive agents and work by preventing the post-translational modification (prenylation) of small GTPases in osteoclasts, subsequently leading to cell death by apoptosis.  Phosphonocarboxylate analogues of bisphosphonates also have anti-resorptive activity and work by inhibiting the enzyme Rab GGTase, thereby preventing the prenylation of Rab GTPases specifically.  Rab GTPases comprise a large family of related proteins that coordinate vesicular trafficking, which involves the processing, transportation and delivery of cellular cargo in a strict temporal and spatial manner.  In osteoclasts, vesicular trafficking is vital for the formation of the ruffled border (the resorptive organelle of the cell), the delivery of lytic enzymes and acid into the resorption space, and the uptake and disposal of bone degradation products.  However, the role that specific Rabs play in this functionally unique cell type remains poorly defined, and the Rab expression profile in osteoclasts is incomplete.  The work presented here aimed to increase our understanding of the role that Rabs play in osteoclasts.  Results indicate that the 70% reduction of Rab GGTase activity observed in <i>gunmetal </i>mice is detrimental to the activity of osteoclasts and osteoblasts <i>in vitro</i>, therefore highlighting the importance of Rabs for bone resorption and deposition.  Furthermore, this study is the first to determine the Rab expression profile of human osteoclasts, following a proteomic approach, and describes the transfection methods devised to characterise these candidate Rabs in osteoclasts.  Finally, this study details the characterisation of Rab18 in human osteoclasts, following its discovery during proteomic analysis.

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No description available.

The purpose of the study was to examine the relationship and comparisons of athletic amenorrhea and bone mineral density in adolescent, cross-country runners. Subjects: Twenty-eight female adolescent cross-country runners (Mean Age + SD = 15.0 + 1.3 years); consisting of seventeen eumenorrheics & eleven amenorrheics. Design: The design consisted of a sixmonth longitudinal design in which the subjects were measured before and after cross-country season for height, weight, and lean tissue (LT), body fat (BF), bone mineral content (BMC), and bone mineral density (BMD) using whole-body scan densitiometry with a Lunar Dual-energy X-ray Absorptiometer (DXA). Run performance, weekly training volumes, menstrual dysfunction, menarchal age, nutritional information, and stress fractures were reported by the subjects. Statistical analyses consisted of Pearson product-moment and partial correlations to examine the associations of the variables, paired t-tests to measure seasonal body composition changes, multivariate analysis (MANOVA & MANCOVA) to investigate the subgroup differences of variables, and simple linear regression to determine the best body composition predictor variable for BMD. Results: The eumenorrheic subgroup's BMD was significantly greater than the amenorrheic subgroup's BMD (F(1, 54) = 16.22, p<.05, partial h² =.231). The eumenorrheic subgroup's bodyweight (F(1, 54) = 7.65, p<.05, partial h² =.124), BF (F(1, 54) = 8.56, p<.05, partial h² =.137), and BMC (F(1, 54) = 8.52, p<.05, partial h² =.136) were significantly greater than the amenorrheic subgroup. There was also a significant seasonal increase in BMD (t(27) = -4.01, p <.05) for the overall group. Bodyweight was the body composition component that best predicted BMD (F(1, 26) = 46.434, p<.05, R² =.641). There were no significant subgroup differences with respect to run performance, stress fractures, and nutritional supplementation. Conclusions: Athletic amenorrhea was highly associated with lower levels of BMD in adolescent, cross-country runners. Athletic amenorrhea was also highly associated with lower levels of bodyweight, BF, and BMC in adolescent cross-country runners. Finally, cross-country running was highly associated with increased BMD in adolescent athletes. Implications: The long-term implication of the study is that subjects with lower levels of BMD may be at a greater risk of osteoporosis. Recommendations: Educate and instruct runners to utilize proper training methods so the healthful benefits of crosscountry running, as well as improved performance, are obtained.

The development of bone regeneration materials to support new bone formation is an active field of research. This report describes the development and characterization of a novel composite scaffold made of a chitosan-alginate co-polymer hydrogel matrix and eggshell (ES) particles. Scaffolds with ES particles or with nanotextured ES (NTES) particles following treatment with phosphoric acid were compared to scaffolds without particles. The scaffolds with particles exhibited a higher porosity and a larger median pore size. Their mechanical strength remained low, but both scaffold types were more resistant to deformation following compression than the scaffolds without particles. The osteogenic potential of the scaffolds was then evaluated with human bone-marrow derived mesenchymal stem cells (MSCs) from four different donors. Results showed that the inclusion of ES or NTES particles significantly increased MSC adherence and viability, as well as alkaline phosphatase activity in the scaffolds. A change of cell morphology and a small, although not statistically significant, increase of osteogenic protein expression (RUNX2 and osteopontin) were also observed at later time points (days 14 and 21). Overall, this research highlights the potential of ES for bone regeneration applications, opening the door for a high-value repurposing of a current industrial waste product.

Bone is a highly dynamic organ system comprised of various cell types that are constantly working to maintain the health and stability of bone. The main cells involved are the osteoblasts that form bone, the osteoclasts that degrade bone, and the osteocytes that act as sensors of the microenvironment and coordinate a response. An imbalance of the interactions between the cell types can potentially result in pathological states in bone at the microscopic level that can then affect the entire skeleton. Moreover, a number of genetic mutations can also lead to pathogenic changes in bone. An example of such is the development of sclerotic bone lesions in patients with the disease tuberous sclerosis complex. Tuberous sclerosis complex, or TSC, is an autosomal dominant disorder affecting approximately 1.5 million people worldwide. It is caused by a mutation in one of the genes encoding either member of the TSC1-TSC2 complex. Molecularly, TSC1-TSC2 negatively regulate the mechanistic target of rapamycin (mTOR) kinase in the mutli-protein complex mTORC1. Activation of mTORC1 leads to an upregulation of protein synthesis and cell growth. Tuberous sclerosis patients are heterozygous for TSC1 or TSC2, and post-natal loss of the second allele results in the development of multiple, benign, tumor-like hamartomas in various organ systems, most notably affecting the brain, kidneys, lungs, skin, and heart. Additionally, CT scans of patients reveal multiple loci of dense, compact bone termed sclerotic bone lesions. The bone lesions were most commonly seen in the posterior elements of the vertebrae and while they are asymptomatic, a remarkably high frequency of patients express them. To further investigate and better understand the mechanisms of tuberous sclerosis complex in bone, we analyzed a mouse model with heterozygous deletion in Tsc2. Initial examination showed the Tsc2+/- mice recapitulated tumors in various organ systems, most notably the kidney, and presented bone lesions in the pelvis and elements of the vertebrae. To further investigate the mechanism driving the disease state, we used a Cre driver thought to be specific for osteoclast (Cathepsin K-Cre, or Ctsk-Cre) to selectively delete Tsc2. Cathepsin K-Cre; Tsc2fl/fl mice exhibit a remarkably high bone mass. This study examined three specific aspects of this high bone mass phenotype. First, we sought to verify that the increased bone mass caused by Ctsk-Cre driven Tsc2 deletion was dependent on mTORC1 upregulation. This was done by generating Ctsk-Cre;Tsc2fl/fl mice lacking Raptor, a mTORC1 component essential for function. Next, we investigated the cell of origin driving the increase bone density by utilizing additional Cre drivers specific for osteoclasts and osteocytes. Additionally, we used radiation chimeras to assess if donated wild type cells could rescue the observed phenotype. We lastly explored the role of a secreted signaling molecule, CTHRC1, that has been proposed as a candidate to mediate osteoclast-osteoblast interaction, in the high bone mass phenotype of Ctsk-Cre;Tsc2fl/fl mice. Selective deletion of Tsc2 in bone cells provides an excellent model to investigate pathways regulating bone mass and strength and may provide new candidate targets for treating diseases of low bone mass, such as osteoporosis.

Faculty of Health Sciences, Degree of Master of Science in Medicine. 9208366a / The initiation of new bone formation within the porous spaces of hydroxyapatite (HA) implants involves the expression of osteogenic markers belonging to the TGF-β superfamily. To study the genetic expression of these osteogenic markers in relation to the type of HA implant used and implantation period, five different types of porous HA biomaterials were implanted in the rectus abdominis muscles of adult baboons Papio ursinus, and were harvested at two, three and 12 months. The total RNA of all harvested samples was extracted and analysed using the Northern blot technique. The results showed that Collagen type IV, GDF-10 and BMP-7 were expressed at the early time points at relatively high levels, and their expression levels were significantly reduced at 12 months. The expression of these makers was not affected by the type of porous HA implant used. The histological sections of these specimens at two and three months showed vascularised connective tissue within the porous spaces of the implants with no bone formation. However, at 12 months there were substantial amounts of bone formed in all the studied implants. The down-regulation of the expressed osteogenic markers at 12 months correlates to the amount of bone formed, suggesting some negative feedback mechanism which may be acting via inhibitory Smads proteins in relation to the amount of bone formed. Neither TGF-β1 nor BMP-3 messages were detected in any of the studied samples, It is possible that these bone markers are not expressed locally within the vicinity of the porous HA implants but are adsorbed to the HA implants from the circulatory system.

Thesis. 1977. M.S.--Massachusetts Institute of Technology. Dept. of Physics. / Microfiche copy available in Archives and Science. / Includes bibliographical references. / by Patricia Canty. / M.S.