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)

Bone is a 3D dynamic and unique tissue that structurally adapts in response to mechanical stimuli. Comparative skeletal morphology is commonly utilized to infer ancient hominins' modes of locomotion; however, instances of remarkable gross similarity despite different modes of locomotion do occur. A common cited example is the similarity between the skeletal elements of bipedal human (Homo sapiens) hands/feet and quadrupedal black bear (Ursus americanus) front/hind paws. Through novel 3D Micro-CT and 2D histomorphology analysis, this thesis tests the hypothesis that a 3D microscopic analysis of biomechanically regulated cortical bone structures provides a more representative and accurate means to infer a species' mode of locomotion. Micro-CT data were collected at the mid-diaphysis of human (n=5) and bear (n=5) third metacarpal/metatarsal pairs and compared with independent and paired t-tests, Pearson correlation coefficients and Bland-Altman plots. Bone microarchitecture is quantifiable in 3D and accessible through non-destructive Micro-CT. Interspecies variation was present, however no significant cortical differences between elements of humans and bears was found. Histological inspection revealed further variation between and within species and element. A key limitation was sample size and further investigation of the relationship between mechanical loading and mode of locomotion is warranted.

Bone is a 3D dynamic and unique tissue that structurally adapts in response to mechanical stimuli. Comparative skeletal morphology is commonly utilized to infer ancient hominins' modes of locomotion; however, instances of remarkable gross similarity despite different modes of locomotion do occur. A common cited example is the similarity between the skeletal elements of bipedal human (Homo sapiens) hands/feet and quadrupedal black bear (Ursus americanus) front/hind paws. Through novel 3D Micro-CT and 2D histomorphology analysis, this thesis tests the hypothesis that a 3D microscopic analysis of biomechanically regulated cortical bone structures provides a more representative and accurate means to infer a species' mode of locomotion. Micro-CT data were collected at the mid-diaphysis of human (n=5) and bear (n=5) third metacarpal/metatarsal pairs and compared with independent and paired t-tests, Pearson correlation coefficients and Bland-Altman plots. Bone microarchitecture is quantifiable in 3D and accessible through non-destructive Micro-CT. Interspecies variation was present, however no significant cortical differences between elements of humans and bears was found. Histological inspection revealed further variation between and within species and element. A key limitation was sample size and further investigation of the relationship between mechanical loading and mode of locomotion is warranted.

Cortical thickness and entheseal robusticity were used to measure the effects of activity and age in a group of 77 adult males from the site of Lisieux-Michelet in northern France. There was no known age at death for this population; age was determined using a series of osteological age estimation methods. Based on the currently available dates for this sample, the skeletal remains were primarily from the Late Roman Period (3rd-7th century AD).The adults were divided into three age categories based on these estimation results. Trends in cortical and entheseal development were measured within and between age categories. Results showed that entheses increased with age while cortical thickness decreased. However, low correlation between these two factors suggests that while entheseal robusticity responds to age, it is highly influenced by physical activity. Activity levels also affect cortical thickness which causes variation within age groups. A comparison of the Lisieux-Michelet entheseal and cortical measurements to both modern and archaeological populations indicated that these males engaged in physically demanding occupations. The degree of activity experienced by these individuals decreased during the middle adult years likely due to a shift to less physically demanding occupations. However, cortical and entheseal data suggest that the old adults from Lisieux-Michelet were not particularly frail and continued to be active even after the decrease in activity during the middle years. / Thesis / Master of Arts (MA)

No description available.

Osteoporosis is an age-related skeletal disease characterised by an increased incidence of fragility fractures. In this thesis I develop a new technique capable of measuring the thickness of the previously unmeasured endocortical region, and providing an improved measure of the cortical bone mineral density (cBMD) from in-vivo clinical CT scans. These features are of interest as both have been linked to fracture risk. \\ The new technique is developed within the cortical bone mapping (CBM) framework so that it provides localised architectural measurements over a bone's surface. Its performance is assessed using simulated QCT data from three simulated phantoms with differing bone architecture, and two paired datasets of ex-vivo QCT and HR-pQCT scans across the proximal femur and the lumbar spine. The simulated data allows for inaccuracies in CBM measurements caused by beam hardening effects to be considered for the first time: I show that beam hardening leads to an underestimation in cortical thickness and an overestimation in trabecular BMD and that these inaccuracies can be reduced through adjustments to the CBM optimisation process. \\ A new technique of analysing HR-pQCT scans is also developed, for the validation of the new CBM method. It was used in place of other established HR-pQCT techniques for its ability to provide localised measurements of the endocortical region. A comparison with the well known full-width half-maximum (FWHM) method shows that it is less susceptible to errors caused by beam hardening. It also measures the mean cBMD, which has a greater clinical relevance than the peak cBMD measured by the FWHM method as it includes the impact of porosity. I demonstrate that the endocortical thickness can be measured to an accuracy of \(-0.15\pm0.71\thinspace\mathrm{mm}\), and that local cBMD measurements are possible down to \(300\thinspace\mathrm{mg/cm}^3\) from QCT scans over the proximal femur. I also validate CBM methods over the vertebrae for the first time and demonstrate that the cortical thickness and endocortical thickness can be measured with accuracies of \(0.10\pm0.30\thinspace\mathrm{mm}\) and \(-0.20\pm0.53\thinspace\mathrm{mm}\). \\ Two clinical trials involving Teriparatide are used to demonstrate that the new CBM method is able to detect significant regional changes in the dense cortical and endocortical bone over the proximal femur and lumbar spine, which can be attributed to changes in intracortical remodelling and endosteal apposition. The analysis of a cross-sectional fracture discrimination trial shows that fracture incidence is associated with significant decreases in endocortical thickness over specific regions.

No description available.

[EN] Cortical bone tissue is the responsible of giving support and structure to vertebrates. For that reason, understanding and analyzing its behavior is needed from each different hierarchical level that composes it. The lower the structural scale is, the greater the complexity and scarcity of studies in literature. These studies are relevant for understanding, preventing and solving important health problems that affect human beings. From a mechanical point of view is interesting to evaluate and apply engineering numerical tools to analyze complex materials as biological tissues, increasing the state of the art in different disciplines that can be applied in numerous fields as material science, biomechanics, numerical methods, medicine and more. In this Thesis the mechanical behavior of cortical bone at microstructural level is analyzed, with finite element models of its basic structure, the osteon. The microstructure of osteons, composed of mineralized collagen fibrils in layers with different orientations disposed concentrically around blood vessels is considered in the models for the calculation of elastic properties and failure criteria definition. For obtaining elastic properties, the use of micromechanical finite element models is considered, with heterogeneous composition for both mineralized collagen fibrils (at nanostructural level) and lamellar level (at sub-microstructural level). The failure analysis for realistic geometries is applied after comparing different models that involve, on one hand the growth of microcracks with contact conditions and on the other, degradation of elastic material properties by user subroutines of the finite element code, the latter being the one that brings better results from a computational cost viewpoint. Therefore an interesting alternative is here presented that can be used to evaluate the damage propagation at three-dimensional level, which with other methods as X-FEM can be computationally unaffordable. Composite materials failure criteria are applied to osteon analysis and the results are related with experimental tests from bibliography, showing the relevance of shear stresses between lamellae for failure initiation and propagation. In a two-dimensional study it is also shown the important role of osteocyte lacunae in the failure initiation, what is interesting from a cellular mechanotransduction approach. / [ES] El tejido óseo cortical es el encargado de dar soporte y estructura a los vertebrados. Existe por tanto una necesidad de conocer y analizar mecánicamente su comportamiento desde los distintos niveles jerárquicos que lo componen, siendo mayor la complejidad y más escasos los estudios disponibles en la literatura cuanto más pequeña es la escala estructural que se analiza. Estos estudios son relevantes para comprender, prevenir y solucionar problemas de salud importantes que afectan al ser humano. Desde el punto de vista mecánico es interesante evaluar y aplicar herramientas numéricas ingenieriles para el análisis de materiales más complejos como son los biológicos, incrementando el estado del arte en distintas disciplinas que pueden ser aplicadas en numerosos campos como la ciencia de los materiales, la biomecánica, los métodos numéricos o la medicina, entre otras. En esta Tesis se analiza el comportamiento mecánico del hueso cortical a nivel microestructural, donde se modela mediante el método de los elementos finitos su unidad básica, la osteona. Para la obtención de las propiedades elásticas se considera en los modelos la microestructura compuesta por capas de colágeno mineralizado con diferentes orientaciones, dispuestas de manera concéntrica alrededor de los canales vasculares. Se incluye además la utilización de modelos micromecánicos de elementos finitos que tienen en cuenta la composición heterogénea tanto para el nivel del fibrilo de colágeno mineralizado (nivel nanoestructural) como para el nivel de lamela (nivel sub microestructural). El análisis del fallo para geometrías realistas se aplica tras comparar varios modelos que involucran por un lado el crecimiento de grietas mediante condiciones de contacto y por otro, degradación de las propiedades elásticas del material mediante subrutinas de usuario del código de elementos finitos, siendo este último el que mejores resultados presenta desde el punto de vista del coste computacional. De esta manera se presenta una alternativa interesante que permite evaluar la propagación del daño a nivel tridimensional, lo que con otros métodos como el X-FEM puede ser computacionalmente inabordable. Se aplican criterios de fallo utilizados para materiales compuestos en ingeniería estructural a las osteonas y los resultados se relacionan con los de los ensayos experimentales disponibles en la bibliografía, mostrando la relevancia de las tensiones de cortadura entre lamelas para la iniciación y propagación del daño. En un estudio bidimensional, también se muestra la participación importante en la fase de inicio de daño de las lagunas de osteocitos lo que es interesante desde un enfoque de mecanotransducción celular. / [CA] El teixit ossi cortical és l'encarregat de donar suport i estructura als vertebrats. Existeix per tant una necessitat de conèixer i analitzar mecànicament el seu comportament des dels diferents nivells jeràrquics que ho componen, sent major la complexitat i més escassos els estudis disponibles en la literatura com més xicoteta és l'escala estructural que s'analitza. Aquests estudis són rellevants per a comprendre, prevenir i solucionar problemes de salut importants que afecten a l'ésser humà. Des del punt de vista mecànic és interessant avaluar i aplicar eines numèriques ingenieriles per a l'anàlisi de materials més complexos com són els biològics, incrementant l'estat de l'art en diferents disciplines que poden ser aplicades en nombrosos camps com la ciència dels materials, la biomecànica, els mètodes numèrics o la medicina, entre altres. En aquesta Tesi s'analitza el comportament mecànic de l'os cortical a nivell microestructural, on es modela mitjançant el mètode dels elements finits la seua unitat bàsica, la osteona. Per a l'obtenció de les propietats elàstiques es considera en els models la microestructura composta per capes de col·làgen mineralitzat amb diferents orientacions, disposades de manera concèntrica al voltant dels canals vasculars. S'inclou a més la utilització de models micromecànics d'elements finits que tenen en compte la composició heterogènia tant per al nivell del fibril de col·làgen mineralitzat (nivell nanoestructural) com per al nivell de lamela (nivell submicroestructural). L'anàlisi de la fallada per a geometries realistes s'aplica després de comparar diversos models que involucren d'una banda el creixement de clavills mitjançant condicions de contacte i per un altre, degradació de les propietats elàstiques del material mitjançant subrutines d'usuari del codi d'elements finits, sent aquest últim el que millors resultats presenta des del punt de vista del cost computacional. D'aquesta manera es presenta una alternativa interessant que permet avaluar la propagació del dany a nivell tridimensional, la qual cosa amb altres mètodes com el X-FEM pot ser computacionalment inabordable. S'apliquen criteris de fallada utilitzats per a materials compostos en enginyeria estructural a les osteones i els resultats es relacionen amb els de els assajos experimentals disponibles en la bibliografia, mostrant la rellevància de les tensions de cisallament entre lameles per a la iniciació i propagació del dany. En un estudi bidimensional, també es mostra la participació important en la fase d'inici de dany de les llacunes d'osteòcits el que és interessant des d'un enfocament de mecanotransducción cel·lular. / Arango Villegas, C. (2016). Study of the mechanical behavior of cortical bone microstructure by the finite element method [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/67570

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.

It is well established that bone is capable of adapting to changes in its environment; however, little is known regarding how environmental stimuli, specifically loading, are associated with the internal 3D microarchitecture of cortical bone. The aim of this thesis was to develop a small animal model that can be used to experimentally test hypotheses regarding bone adaptation. High resolution micro-CT was validated and employed as a novel method for the visualization and quantification of rat cortical bone microarchitecture in 3D. The use of this imaging method allowed for the measurement of primary vascular canal orientation in 3D, which had never been achieved before. Using this measure along with an immobilization model for unloading allowed me to test how loading is associated with the orientation of these vascular canals. Normally ambulating rat bones (from 10 female rats) had a canal structure that was 9.9° more longitudinal than their immobilized counterparts. This finding that loading has an effect on primary canal orientation brought to light the need to induce remodeling and therefore, secondary vascular canals, in the rat to increase its novelty as a model for looking at bone adaptation. Remodeling was induced by increasing the calcium demands of female rats, either through a calcium restricted diet (n=2) or pregnancy and lactation coupled with a calcium restricted diet (n=2). Mean cortical thickness for the calcium restricted rats and the pregnant and lactating rats that were on a calcium restricted diet were 622 µm and 419 µm, respectively. The mean BMU count for calcium restricted rats seemed to be higher than that of the pregnant and lactating rats; however, the calcium restricted rats seemed to have a lower BMU density. Once this full-scale study is executed the rat will provide a more representative model for studying human bone adaptation.