Osteocytes, the most abundant cell within bone, have been linked to the processes of mechanosensation and transduction. Based upon relatively limited empirical evidence, variations in their abundance and morphology have been linked to sex, age, biomechanics and disease. In order to better elucidate lacunar variation within a healthy cohort, samples from 30 women aged 20-86 and 36 men aged 18-92 were studied utilizing synchrotron radiation micro-CT. Initial studies of normal variation within the femoral proximal shaft cross-section found high variation in lacunar density (up to ~54%) and associated morphological differences linked to biomechanical regions. In women, a non-significant trend in lacunar density reduction was apparent with age; however, a significant reduction in lacunar volume with age (~30%) was observed. Also noted were differences in lacunar morphology, with the lacunae of younger women characterized as flatter and less equant than their older counterparts. The males, who demonstrated lacunar density decline with age and a tendency towards more equant and less elongate lacunae, did not share these characteristics. Intriguingly, the previously noted reductions in lacunar volume were not observed in males. The results of this research indicate that normal variation in osteocyte lacunar parameters is high. To our knowledge the observation that lacunar volume differs in women with age is novel, potentially resulting from preferential surface infilling within the extracellular space. The functional impact of this infilling is unclear but such a change in scale likely impacts the mechanosensing function of the osteocyte network. This hypothesis warrants further investigation as, if confirmed, it would represent a profound negative impact on the osteocyte network and may provide new insights into age-related bone loss.

Supporting videos attached / The nanoscale structure of fully dense human cortical bone is explored using advanced transmission electron microscopy (TEM) techniques. Studies of fully dense cortical bone are rare because of the sample preparation challenges. In this work, cryogenic ion milling is compared favourably with traditional ultramicrotoming methods because of the clearer imaging results and better preservation of biological structures in the ion milled samples. Ion milled samples were prepared parallel, perpendicular and at a 45 degree angle to the long axis of a human femur. The samples are cooled with liquid nitrogen while being milled to prevent heating damage to the bone structure. Bright-field and dark-field imaging show that mineral mainly occurs as 65 nm wide, 5 nm thick mineral structures, external to the collagen fibrils, and with the long axis of the mineral running parallel to the fibrils. In samples cut parallel to the long axis of the bone, the mineral structures have their c-axes aligned with the collagen fibril long axis. In these sections the mineral structures extend up to 200 nm and are grouped into stripe-like bunches, 53 nm apart. Samples cut perpendicular to the long axis of the bone show open areas roughly 45 nm in diameter. These open areas are assumed to be the location of collagen fibrils within the structure and are tangentially surrounded by 65 nm wide, 5 nm thick mineral structures. On average, there are 22 nm of mineral structures between adjoining collagen fibrils. Samples cut at 45 degrees to the long axis of the bone confirm that the open structure seen in the perpendicular section is not an artefact of sample preparation. By tilting the sample, the 45 degree sample shows the structure of both the parallel and perpendicular sections. The parallel structure strongly resembles images of embryonic bone and other mineralized tissues seen in the literature, so the perpendicular open structure is not caused by sample preparation. An examination of ultramicrotoming’s effect on mineral structure size compared with that of ion milling shows that the mineral structures in ion milled samples are twice as long as in ultramicrotomed samples, indicating that bone mineral may be damaged by the forces applied to the complex composite structure existing in fully dense cortical bone. Using energy dispersive X-ray spectroscopy (EDXS) results and a simplified model of the locations of mineral within the collagen/mineral framework, a calculation of the percentage of external mineral was performed. The result showed that 80+_ 6 % of the mineral in fully dense cortical bone must be external to the collagen fibrils to obtain the EDXS results. Finally, Z-contrast tomography, based on the use of high angle annular darkfield (HAADF) imaging, was used to prepared tomographic reconstructions of the external mineral in fully dense cortical bone. Unlike bright-field tomography, the Z-contrast technique allows examination of crystalline materials as the contrast in HAADF images is mass-thickness dependent instead of diffraction based. These reconstructions again showed the mineral tangentially surrounding 50 nm diameter cylindrical holes, assumed to be the location of collagen fibrils in all directions. This work shows the importance of mineral that is external to the collagen fibrils to the nanoscale structure of fully dense cortical bone. / Thesis / Doctor of Philosophy (PhD)

Il est estimé qu'une fracture se produit toutes les trois secondes autour du monde, accompagné par un risque élevé d'invalidité ou même de mortalité. La connaissance des mécanismes de fractures dans une configuration de chargement représentatif d'une chute semble être d'un intérêt majeur pour le développement de méthodes dédiées à la prédiction du risque de fracture. La ténacité est un paramètre approprié lorsqu'on s'intéresse à ces mécanismes de fracture, elle détermine l'énergie nécessaire pour propager une fissure à travers l'architecture du tissu. L'objectif de cette étude est d'évaluer la ténacité de l'os cortical humain, considérant à la fois des conditions chargement quasi-statique et représentatif d'une chute sur sites anatomiques appariés. L'acquisition d'images en micro-tomographie ainsi qu'une mesure des cross-links ont été réalisées afin d'évaluer leur influence sur les mécanismes fracture du tissu. Les résultats ont montré que dans des conditions quasi-statiques, les différents sites anatomiques présentent des propriétés mécaniques différentes : le radius résiste mieux à une propagation de fissure. Dans des conditions de chute, il n'y a plus de différences entre ces sites, mais la ténacité décroit de façon significative par rapport au chargement standard. L'os cortical résiste mieux à une propagation de fissure dans des conditions quasi-statiques. Les analyses structurales et biochimiques ont montré des différences entre les sites anatomiques qui expliquent les différences mécaniques. Les caractéristiques architecturales du tissu sont déterminantes vis-à-vis des mécanismes de fracture dans des conditions quasi-statiques. Mais leur rôle lors d'une chute est moins évident. Ces résultats impliquent que la microstructure de l'os cortical n'est pas un déterminant majeur vis-à-vis du risque de fracture. De futures études doivent être réalisées afin de déterminer les paramètres décisifs dans des conditions représentatives d'une chute / A fracture is estimated every three seconds in the world, leading to an increased risk of impairment or even mortality. The biomechanical knowledge of bone fracture mechanisms in a fall configuration of loading is of great interests for the development of clinical method for the prediction of the risk of fracture. Toughness seems to be a good candidate to investigate this fracture process as it corresponds to the energy needed to propagate a crack through cortical bone complex microstructure. The aim of this study was thus to evaluate human cortical bone toughness parameter under both quasi-static and fall-like loading conditions paired anatomical locations. Micro-computed tomography images using synchrotron radiation and collagen cross-links maturation measurements were performed to investigate the influence of the tissue architecture on crack propagation. Results found showed that under quasi-static condition, the different anatomical locations present different mechanical behavior. Radius significantly better resist crack propagation than the other studied location. Considering a fall-like loading condition, no more difference is observed between the locations but a significant decreased is measured compare to the first configuration. Human cortical bone has a better capacity to resist crack propagation under a standard quasi-static loading condition. By investigating the tissue morphometric and biochemical parameters, we observed different organization from a location to another that explains the mechanical differences. The architectural features appear to be determinant for crack propagation mechanisms under quasi-static condition, but they play a lesser role under fall-like condition. These results imply that the tissue microstructure is not a determinant when dealing with the prediction of the risk of fracture. Further work has to be done to reach out which parameters are more determinants under a specific fall-like loading condition