Thesis (doctoral)--Lund University, 1992. / Added t.p. with thesis statement inserted. Booklet of related papers inserted.

Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted.

Thesis (M.A.)--University of Missouri-Columbia, 2006. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on September 14, 207) Includes bibliographical references.

4 p. / Originally published: 2000. / Osteoporosis means “porous bones.” It is a condition where the skeleton becomes fragile and results in broken bones under normal use. Osteoporosis is a “silent” condition that happens slowly over years. The rate of bone loss (“resorption”) exceeds the rate of new bone formation (“acretion”). Many times neither a person nor a doctor is aware of weakened bones until one breaks unexpectedly. Originally published: 2000

Investigations into silicate-substituted hydroxyapatite (Si-HA) were performed. The aqueous precipitation method produced phase pure Si-HA with modi cations to the method causing impurities in the material. A novel study using Raman spectroscopy followed the behaviour of the silicate ions and provided a new interpretation regarding silicate substitution. The silicate ions created interactions with hydroxyl ions, initially, which reduced upon sintering of the material. As the silicate ions do not behave inde- pendently in the HA structure initially, suggests that these interactions may contribute to the bioactivity of Si-HA. Also industrial aspects of Si-HA were investigated regarding the silicate reagent (TEOS). A small di erence of 1% in the percentage concentration of TEOS was not negligible and caused a decrease in the amount of silicate substituted into HA. Di erent brands and grades of TEOS did produce Si-HA with similar structural properties. Therefore, a variety of brands and grades of TEOS can be used and thus the most cost e ective choice can be made. The rst analytical investigations into the molecular arrangement of fully mineralised osteoarthritic (OA) and osteoporotic (OP) bone were performed through small angle neutron scattering (SANS) studies. This study provided a description for the molecular arrangement of collagen molecules, along the lateral plane, regarding the molecular di- ameter and the packing of these molecules into the bril by the development of a model based on SANS theory. The collagen molecules behave like a 2-dimensional liquid-like system. Through the development of the model, the rst written solution for the struc- ture factor for a system of hard-disks was stated. This study provided an understanding into how collagen molecules are arranged in OA and OP bone. Also, compositional studies iterated possible di erences between the organic content of OA and OP bone. Thus the organic content of bone may play a role in the bone disorders.

This thesis describes a toolbox of complementary techniques that together measure and mechanical properties of bone. Three-point bending is used to measure the mechanical properties of bone; micro computed tomography provides cortical geometry and parameters describing trabecular bone.  The material properties, elastic modulus and density, are measured directly using ultrasound and Archimedes’ principle, while composition and bone chemistry are investigated by ashing and Raman microscopy.  These methods are used to characterise bone from the naturally occurring Gunmetal mouse and the engineered neuronal nitric oxide synthase (nNOS) knockout mouse. Comparison was also made between femora and tibiae and cortical and trabecular bone from wild type mice. Gunmetal mice had inferior mechanical properties, but unaffected material and chemical properties.  Cortical area but not second moment of area was also reduced.  nNOS knockouts had superior bone mechanically, due to increased mineralisation and geometrical parameters.  Femora and tibiae had different mechanical and material properties that were not linked to the size or shape of the bones.  Cortical bone  had characteristics of older bone compared to trabecular material, possibly due to the lower turnover rate. These results show the necessity for measuring material properties directly, rather than inferring them from mechanical and geometrical properties.  The differences in femora and tibiae suggest testing only femur or tibia may result in the risk of missing important results.  Application of this toolbox of methods provides a comprehensive description of bone’s overall fitness for purpose and an understanding of the origin of any defect or enhancement in its properties.

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.

The effects of endocannabinoids on human, mouse and rabbit bone cells were investigated.  At high concentrations anandamide and 2-arachidonyl glycerol (2-AG) inhibited human osteoclast formation with no effects at lower concentrations. The inhibition was not attenuated by antagonists for the CB₁, CB₂ or TRPV1 receptors, indicating a non-receptor mediated effect.  Conversely, anandamide and 2-AG increased mouse osteoclast formation.  The effect of anandamide was enhanced in cells from fatty acid amide hydrolase (FAAH)-null mice and abolished in cells from CB_1/2 knockout mice.  The effect of 2-AG was not eliminated in CB_1/2 knockout cells, indicating a non-CB₁/CB₂ action.  The CB₁ antagonist, AM251, and the CB₂ antagonist, AM630, both inhibited mouse osteoclast formation.  These effects were not rescued in the CB_1/2-knockout mouse cells.  Both anandamide and 2-AG stimulated actin ring formation and osteoclast activity in human and rabbit osteoclast.  This was prevented in the presence of AM630 but not AM251, indicating a CB₂-mediated response.  The endocannabinoids and the cannabinoid receptor antagonists do not have a regulatory action on osteoblast activity. The effects of the novel cyclooxygenase-2 (COX-2) metabolite of 2-AG, prostaglandin E₂-glycerol ester (PGE₂-G), on human osteoclasts were examined.  Treatment with PGE₂-G inhibited formation and ERK phosphorylation of human osteoclasts.  These effects were attenuated by a selective EP₄ antagonist and mimicked by PGE₂ alone, indicating that PGF₂-G is rapidly metabolised into PGE₂ in human osteoclast cultures.  However, PGE₂-G treatment elevated intracellular calcium levels in human osteoclasts, through a phospholipase C (PLC)- and IP₃- dependent mechanism, indicative of a G-protein coupled receptor effect.  This was not mimicked by PGE₂, or prevented by the EP₄ antagonist, but blocked by a putative PGE₂-G receptor antagonist, PDA-94 indicating that PDA-94 may be a PGE₂-G receptor antagonist.

LD2668 .T4 1950 B83 / Master of Science