Thesis (Ph.D.)--Aberdeen University, 2008. / Title from web page (viewed on June 3, 2009). Includes bibliographical references.

The aim of this thesis was to study mechanotransduction in bone, focusing first on the mechanical stimulus <i>per sé</i> and then looking at the regulation of response to mechanical stimuli in osteoblastic cells, specifically looking at the regulation of nitric oxide. A new system was designed to produce fluid flow and eliminate, or at least reduce, strain and pressure. A system to induce only hydrostatic pressure was also designed. Using these systems, cells were tested for response to pressure and fluid flow. The responses measured were the early expression of the transcription factor c-Fos, and the translocation of β-catenin to the nucleus. Although both types of mechanical stimuli induced the translocation of β-catenin, pressure did not produce an increases in expression of <i>c-fos</i>. This showed the differences in response as a result of different types of stimuli, and is likely caused by the initiation of different signalling pathways by each stimulus. A comprehensive study of the distribution and quantity of caveolae in cells of the osteoblastic lineage was conducted, and this revealed that osteocytes have significantly more caveolae than osteoblasts. The connection between eNOS and mechanotransduction was studied with eNOS null mice, in whole bones and in cultured bone cells. The mechanical and material properties of eNOS null bones were compared to wild type bones. Differences between these were found but were few, indicating the role of eNOS in bone might be minimal and suggesting that the enzyme is only involved in osteoblasts differentiation during development. Nitric oxide production was monitored in bone cells from eNOS null mice. Surprisingly they produced an increased in nitric oxide in response to stimulation by fluid flow. This nitric oxide was inhibited with a specific inducible nitric oxide synthase (iNOS) inhibitor, pointing to iNOS as the enzyme responsible for nitric oxide synthesis.

Thesis (M.S.)--California Polytechnic State University, 2009. / Title from PDF title page; viewed on January 6, 2010. Major professor: Scott Hazelwood, Ph.D. "Presented to the faculty of California Polytechnic State University, San Luis Obispo." "In partial fulfillment of the requirements for the degree [of] Master of Science in Engineering With a Specialization in Biomedical Engineering." "December 2009." Includes bibliographical references.

No description available.

Thesis (M.S.)--California Polytechnic State University, 2010. / Title from PDF title page; viewed on May 15, 2010. Major professor: Scott Hazelwood, Ph.D. "Presented to the faculty of California Polytechnic State University, San Luis Obispo." "In partial fulfillment of the requirements for the degree [of] Master of Science in Engineering, with a Specialization in Biomedical Engineering." "April 2010." Includes bibliographical references (p. 73-78).

Bisphosphonates (BPs) are stable analogues of pyrophosphate widely used for the treatment of bone diseases characterised by increased bone resorption. Studies over the years have shown that the pharmacological potencies of BPs are dependent both on their binding affinities for bone mineral and on their inhibitory actions on osteoclasts. In addition, potential effects on other cell types present locally in the environment of skeletal tissues have been reported. The present study systematically evaluated the relative mineral-binding affinities of individual BPs of clinically relevance in mixtures of these compounds and the changes with elution pH by using column chromatography with ceramic hydroxyapatite and fluoroapatite combined with mass spectrometric identification and quantitation of the individual BPs. The results indicate that pH has a profound effect on the ionisation of the phosphonate and R2 functional groups, with BPs having greater affinities at lower pH as shown by increased retention times. Moreover, two other approaches, namely using Langmuir adsorption isotherms and competition assays based on fluorescent BP, have been developed to assess the mineral-binding capacities and dissociation constants of BPs. These results suggest that there are substantial differences among BPs in their binding to hydroxyapatite. From the cellular aspect of my study, I present evidence for the anti-apoptotic effects of BPs in osteocytes and osteoblasts. However, the study of prosurvival signalling pathways involved in these cells needs to be optimised. The work described in this thesis provides novel insights into the physiological and biological mechanisms of BP action. My project has provided a better knowledge of the physicochemical properties of BPs, which are highly relevant to their differential distributions within bone, their biological potencies, and their durations of action. Additionally, the cell culture studies may provide new information on the cellular effects of BPs on osteocytes and osteoblasts.

Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2007. / Vita. Includes bibliographical references.

Osteoporosis and low bone mass are devastating and costly diseases affecting over half the US population over 50-years old. Peak bone mass is reached around age 30 and begins to decline in the following years, leading to a multi-decade disease. While there are several treatment options, including both ant-resorptive agents -- preventing bone loss -- and anabolic agents -- promoting bone formation -- they are inadequate due to either the limited scope of approval or decreased patient compliance from the perceived high risk of complications. Fracture risk increases significantly in osteopenic individuals with vertebral, hip, wrist, and pelvic fractures being among the most common. While there is a significant annual cost of osteoporotic fractures – on the order of $25 billion annually – the increased rate of mortality after osteoporotic fracture is halting. Risk of mortality one year after hip fracture ranges from 20% to 40%. Therefore, there is a clinical and economic need to develop the next-generation of bone saving therapeutics. Bone has the innate ability to respond to mechanical loading -- forming new bone to accommodate increased mechanical loads, and resorbing bone when there is a period of low mechanical loading or disuse. If we can determine how mechanical loads are detected on the cellular level, we can open up a new avenue for drug development. The osteocyte, a terminally differentiated cell embedded in the bone mineral matrix, is accepted as a key bone mechanotransducer -- detecting mechanical loading and translating it into biochemical signals promoting bone formation. There are several hypothesized models of how the osteocyte detects mechanically loading – fluid flow through the canalicular environment stimulates the osteocyte's dendritic processes; fluid flow results in deformation of the primary cilium within the lacunar cavity; or matrix deformation is directly transduced through integrin attachments at the lacunar wall. The primary cilium is a solitary antenna-like organelle that forms a distinct signaling domain with a unique protein pool, which makes it an attractive therapeutic target. In this thesis, we seek to unravel the role of the primary cilium in bone mechanotransduction in order to open new avenues for drug development. We examine if the osteocyte primary cilium contributes to load-induced bone formation, determine if adenylyl cyclase 3 (AC3) -- a cAMP catalyzing enzyme that localizes to the primary cilium -- contributes to osteocyte mechanotransduction, and investigate if the primary cilium coordinates actin adaptation in response to mechanical loading. Using a Cre-lox system to knockout Ift88, a gene encoding a critical protein for cilia formation, we find that load-induced bone formation is dependent on whether one or two alleles of Ift88 are present globally, but not if they are only deleted in the osteocyte cell population. We also find that knocking down AC3 mRNA expression leads to an increased response to mechanical stimulation and altered primary cilia length, likely through decreased cAMP production. Finally, we determine that inhibiting primary cilia formation dysregulates actin adaptation to mechanical loading and prevents the actin-dependent mechanoresponse of Taz, a transcriptional co-regulator. This action is likely through alterations in the expression of actomyosin components and in the activation of focal adhesion kinases. Together, this work demonstrates that the primary cilium plays a role in load-induced bone formation, but this effect is not localized to the osteocyte cell population. We also show that adenylyl cyclases play a role in osteocyte mechanobiology, and that whole cell mechanosensitivity may be determined through the primary cilium in its function regulating the actin cytoskeleton.

BACKGROUND: Human mesenchymal progenitor cells are multipotent cells that can be harvested from various adult and fetal tissues. They exhibit the potential to differentiate into several cell lineages, most notably osteogenic, chondrogenic, and adipogenic lineages. Conditions such as osteoporosis, metabolic disease, and arthritis are examples of dysfunction of tissues formed by the mesenchyme. The inability of these conditions to be healed by the body’s own mechanisms has raised considerable interest in the potential of using mesenchymal progenitor cells as a therapeutic intervention. This concept opens the possibility of harvesting mesenchymal progenitor cells from an individual, growing them into the desired tissue, and implanting them back into the individual. Treatment of this nature is much less invasive than current methods, overcomes rejection by the immune system, and could potentially demonstrate better outcomes in individuals suffering from degenerative disease of the mesenchyme. AIMS/OBJECTIVES: The aims of this study were to determine and to characterize the differentiation of human adipose-derived mesenchymal progenitor cells into osteocytes, chondrocytes, and adipocytes. The differentiation capacity of the mesenchymal progenitor cells was evaluated through cell staining, immunofluorescence, and RNA sequencing. METHODS: Subcutaneous adipose tissue was collected from patients undergoing elective panniculectomies. The abdominal panniculus was liposuctioned, and small explants of fat were embedded in Matrigel. Mesenchymal progenitor cells were extracted from the explants and plated for differentiation into osteogenic, chondrogenic, and adipogenic lineages. Control cells were grown in parallel in basal media to confirm differentiation. Dye staining for differentiation was performed with Alizarin Red S, Alcian Blue, and Oil Red O, and immunofluorescence staining was performed to indicate lineage-specific markers for differentiation. RNA sequencing was also completed on the different cell lineages. RESULTS: Human adipose-derived mesenchymal progenitor cells displayed the capacity to differentiate into osteogenic, chondrogenic, and adipogenic lineages as evidenced by dye staining. Osteogenic differentiation was confirmed with Alizarin Red S staining of calcium deposits in the differentiated cells, whereas staining in the control resulted in no calcium deposits. Alcian Blue staining confirmed chondrogenic differentiation as glycoproteins secreted by the differentiated cells were evident and different in morphology compared with the control cells. Oil Red O staining indicated adipogenic differentiation by showing lipid droplets in the differentiated cells and no lipid droplets in the control. RNA sequencing provided support that lineage differentiation was successful. Immunofluorescence staining further proved that differentiated cells expressed lineage-specific proteins and demonstrated morphological differences. CONCLUSIONS: This study demonstrates that mesenchymal progenitor cells harvested from human adipose tissue have the potential to differentiate into adipogenic, chondrogenic, and osteogenic cell lineages when induced with differentiation media. The differentiation of these cells can be assessed with dye staining, RNA sequencing, and immunofluorescence staining methods. Further studies should be done to investigate the potential of mesenchymal progenitor cells for therapeutic interventions in the treatment of various illnesses related to the mesenchyme.

Osteocytes are the most abundant bone cells, however, they are also the least understood. They sense mechanical stress within the bone matrix to control remodelling, but there is debate about the way that this occurs. The bone matrix experiences changes in electrical charge through stress generated potentials in the canaliculi, and piezoelectricity of the collagen-hydroxyapatite junctions. External electrical stimulation (ES) has been shown to increase bone formation, indicating that the cells involved in remodelling are electrically sensitive. However, the effects of ES on osteocytes specifically are under-researched. Before applying ES in vitro the electrical characteristics of the culture media need to be understood to see if it will negatively impact cells in culture. ES in culture media causes pH changes and gas formation as well as precipitate formation directly on the electrode surface. The resistance of the media increases rapidly upon application of the electrical stimulus and plateaus after 100 - 200 minutes. The pH gradient disperses around the same time frame, with most stimulating currents causing no permanent change to the media pH. Stimulation parameters that cause minimal side effects will be better for the health of cells in culture. This should also make it more clear which outcomes are a result of the electrical stimulation and which come from the electrochemical reactions that are present in the media due to the ES. / Thesis / Master of Applied Science (MASc) / Osteocytes are the least understood bone cells and the way that they communicate with the bone matrix and other cells is widely debated. It is assumed that osteocytes sense mechanical stresses within the bone matrix through electrical charges that develop in areas of increased strain. External electrical stimuli have been shown to increase bone formation indicating that the cells are electrically sensitive. The electrical sensitivity of the osteocytes specifically is under-researched causing a gap in knowledge of the behaviour of the cell in the remodelling process. To study the effects of electrical stimulation on osteocytes, an experimental apparatus must be designed to deliver stimulation to the cells in vitro and maintain a stable environment. A culture medium is needed to keep cells alive in vitro To do this, the electrical characteristics of the cell culture medium must be understood in attempts to maintain homeostatic conditions for the cells.