No description available.

No description available.

The microstructural impact of diagenetic or post mortem alteration has been assessed in predominately human skeletal tissues. The method of assessment selected was microscopical analysis, mainly using backscattered electron imaging in a scanning electron microscope and, to a lesser extent, confocal reflection microscopy. The microstructural morphologies of post mortem alteration were investigated in archaeological material, both normal and pathological, from terrestrial and marine contexts. Further studies were undertaken on a case-by-case basis on skeletal material which offered some unique pathology, environmental context, spatial relationship, time variable, or mortuary practice. Additionally, the effect of diagenetic change on mitochondrial DNA (mtDNA) recovery and the potential location of DNA within the skeletal tissues were investigated. Two quantitative studies were undertaken to validate and measure the observed mineral density changes. The investigations showed that post mortem alteration or diagenetic change to skeletal material can be extensive, and can occur shortly after death. Diagenesis did not represent a post burial phenomenon as the term diagenesis suggests, but was found to have begun above ground in a range of exposural contexts. The implication of gut bacteria in the promotion of early bacterially-related microstructural change was strong, and it is proposed that body status at the point of, or soon after, death is important. Post mortem alteration to skeletal microstucture can provide environmental information, since terrestrial and marine contexts exhibited distinct morphologies. It may also provide localized environmental information within a stratigraphic matrix. Characterizing the post mortem microstructural and density changes to bone has helped to elucidate the preservational status of mtDNA in terms of its relative retrieval in archaeological specimens, and the potential location of mtDNA in bone. It is proposed that the shift in mineral density that was found in bacterially-remodelled specimens from terrestrial contexts, relative to the excellent preservation of marine specimens, may help to explain why marine vertebrates far outnumber terrestrial ones in the fossil record, since bacterially driven microstructural change is here considered to be a destructive form of fossilisation.

No description available.

No description available.

Despite advances in investigating functional aspects of osteoblast (OB) differentiation, especially studies on how bone proteins are deposited and mineralized, there has been little research on the intracellular trafficking of bone proteins during OB differentiation. Collagen synthesis and secretion is the major function of OBs and is markedly upregulated upon ascorbic acid (AA) stimulation, significantly more so than in fibroblast cells. Understanding the mechanism by which collagen is mobilized in specialized OB cells is important for both basic cell biology and bone disease studies. Cellular organelles and vesicles in the exocytic and endocytic pathways have a distinctive spatial distribution and their trafficking is aided by many molecules, Rab GTPases being a master regulator. In this work, I identified the Rab GTPases that are upregulated during OB differentiation using microarray analysis, namely Rab1, Rab3d, and Rab27b, and investigated their role in regulating the trafficking of collagen from the site of synthesis in the ER to the Golgi and ultimately to the plasma membrane (PM) utilizing their dominant negative (DN) expression. The experimental halting of biosynthetic trafficking by these mutant Rabs initiated proteasome-mediated degradation of procollagen and ceased global protein translation. Acute expression of Rab1 and Rab3d DN constructs resulted in impaired ER to Golgi trafficking of procollagen. Similar expression of Rab27b DN constructs resulted in dispersed collagen vesicles which may represent failed secretory vesicles sequestered in the cytosol. A significant and strong reduction in extracellular collagen levels also was observed showing roles of Rab1, Rab3d and Rab27b in the specific function of these major collagen producing cells in the body. I further observed that a fraction of procollagen colocalized with lysosomes which was markedly increased when procollagen was experimentally misfolded. Lysosomes, essential organelles for intracellular degradation, are generally sequestered near the cell centre to receive vesicles with contents targeted for destruction. During AA-induced differentiation of OB cells, I saw a marked increase in total degradative lysosome organelles in addition to an enhanced endocytic rate. Interestingly, lysosomes were dispersed toward the cell periphery in differentiating OBs without being secreted. This required intact microtubules for long range transport and was kinesin motor-dependent but did not involve cytosolic acidification. Moreover, impairment of lysosome dispersion markedly reduced AA-induced OB differentiation. Taken together, this study provides an important general mechanism for cell secretion phenomena that may ultimately lead to clinical targets for treatments of diseases driven by aberrant collagen processing and secretion including Osteogenesis Imperfecta (OI).

Despite advances in investigating functional aspects of osteoblast (OB) differentiation, especially studies on how bone proteins are deposited and mineralized, there has been little research on the intracellular trafficking of bone proteins during OB differentiation. Collagen synthesis and secretion is the major function of OBs and is markedly upregulated upon ascorbic acid (AA) stimulation, significantly more so than in fibroblast cells. Understanding the mechanism by which collagen is mobilized in specialized OB cells is important for both basic cell biology and bone disease studies. Cellular organelles and vesicles in the exocytic and endocytic pathways have a distinctive spatial distribution and their trafficking is aided by many molecules, Rab GTPases being a master regulator. In this work, I identified the Rab GTPases that are upregulated during OB differentiation using microarray analysis, namely Rab1, Rab3d, and Rab27b, and investigated their role in regulating the trafficking of collagen from the site of synthesis in the ER to the Golgi and ultimately to the plasma membrane (PM) utilizing their dominant negative (DN) expression. The experimental halting of biosynthetic trafficking by these mutant Rabs initiated proteasome-mediated degradation of procollagen and ceased global protein translation. Acute expression of Rab1 and Rab3d DN constructs resulted in impaired ER to Golgi trafficking of procollagen. Similar expression of Rab27b DN constructs resulted in dispersed collagen vesicles which may represent failed secretory vesicles sequestered in the cytosol. A significant and strong reduction in extracellular collagen levels also was observed showing roles of Rab1, Rab3d and Rab27b in the specific function of these major collagen producing cells in the body. I further observed that a fraction of procollagen colocalized with lysosomes which was markedly increased when procollagen was experimentally misfolded. Lysosomes, essential organelles for intracellular degradation, are generally sequestered near the cell centre to receive vesicles with contents targeted for destruction. During AA-induced differentiation of OB cells, I saw a marked increase in total degradative lysosome organelles in addition to an enhanced endocytic rate. Interestingly, lysosomes were dispersed toward the cell periphery in differentiating OBs without being secreted. This required intact microtubules for long range transport and was kinesin motor-dependent but did not involve cytosolic acidification. Moreover, impairment of lysosome dispersion markedly reduced AA-induced OB differentiation. Taken together, this study provides an important general mechanism for cell secretion phenomena that may ultimately lead to clinical targets for treatments of diseases driven by aberrant collagen processing and secretion including Osteogenesis Imperfecta (OI).

With increasing demands in bone repair and replacement, this research investigates the microstructure, properties and performance of bovine bone, human bone, and synthetic materials. Doing so, experimental approaches were used to exam and compare bones, as well as mimicking nature by developing a synthetic material to repair bones. Experimentally, bovine bone, tumor-free human bone, and cancerous human bone were studied via the small scale mechanical loading test. Failure analysis was conducted via optical and electronic microscopic techniques. Characterization results were used to develop a synthetic material that possesses strength and strain needed as a bone material. Characterizing techniques include a small punch test, scanning electron microscope (SEM), optical microscope and x-ray diffraction (XRD) were used for experimental approach. The results showed that small punch tests in longitudinal and tangential directions showed different mechanical properties and failure mechanisms. Cancer cells in human bone caused the bone softening and lowered the density. Synthesized epoxy-silicone-geopolymer material had higher deformability than bone. Understanding obtained in this research helps us to develop better synthetic bone materials in future. This thesis is composed of six chapters. The first chapter covers as an introduction to understand the purpose and motivation of present studies, and this section followed by the details of the motivation and objectives of this research. The third chapter explains experimental approaches that were conducted to meet the objectives. The fourth chapter describes the results and the major discovery of the experiments, and the results will be discussed in the Chapter IV. Finally, the last chapter provides the conclusions and recommendations for future work.

Thesis (M.S.)--University of Hawaii at Manoa, 2007. / Includes bibliographical references (leaves 92-95). / ix, 95 leaves, bound ill. (some col.) 29 cm

Failure of a primary hip arthroplasty is often caused by osteolysis which compromises the patient’s periprosthetic bone stock. Impaction allografting involves the use of morselized allograft bone and cement to stabilize the implant and restore this periprosthetic bone stock. Although clinical results of impaction allografting are favourable, regions of necrotic bone graft have been shown to exist for many years post-operatively and may ultimately lead to implant failure. Previous laboratory research has identified a correlation between mechanical stimuli and bone growth; therefore, the purpose of this study was to develop an in vivo device that would enable the investigation of the effect of mechanical load on bone graft incorporation in impacted allograft hip prostheses. An actuator was developed with a finite volume to enable its subcutaneous implantation along the tibia (20mm x 10mm x 10mm) and spine (35mm x 25mm x 15mm) in a rat bone chamber model. The actuator was designed to deliver a dynamic, (1Hz), compressive, (-6N), load that was controlled telemetrically throughout a 6-week long in vivo study. Independent validations of the mechanical actuator and the electrical control system were performed prior to an electromechanical validation of the integrated system. The responsiveness, quantity and magnitude of the load were investigated. The mechanical actuator was motor-driven and the electrical control system was based on radio frequency signal transmission. The electromechanical actuator conformed to the volumetric restrictions of the rat bone chamber model (tibia: 13mm x 17mm x 10mm; spine: 35mm x 30mm x 11mm). A wide range of operating frequencies (0.5 to 3.0 ± 0.05Hz) was achieved and a telemetrically controlled load was produced for 20 seconds per day throughout a simulated 6 week in vivo study. Due to inefficiencies of the mechanical actuator and voltage limitations of the control system, the magnitude of the compressive load produced by the actuator (-1.67 ± 0.10N) was less than specified by the design criteria. Future work to optimize the actuator design and fabrication is warranted in order to increase the maximum load magnitude; however, the current design provides a novel means to begin the investigation of the role of mechanical load on bone graft incorporation in impaction allografting.