Fractal-like hierarchical organization of bone begins at the nanoscale

INTRODUCTION: The components of bone assemble hierarchically to provide stiffness and toughness. Deciphering the specific organization and relationship between bone’s principal components—mineral and collagen—requires answers to three main questions: whether the association of the mineral phase with collagen follows an intrafibrillar or extrafibrillar pattern, whether the morphology of the mineral building blocks is needle- or platelet-shaped, and how the mineral phase maintains continuity across an extensive network of cross-linked collagen fibrils. To address these questions, a nanoscale level of three-dimensional (3D) structural characterization is essential and has now been performed. RATIONALE: Because bone has multiple levels of 3D structural hierarchy, 2D imaging methods that do not detail the structural context of a sample are prone to interpretation bias. Site-specific focused ion beam preparation of lamellar bone with known orientation of the analyzed sample regions allowed us to obtain imaging data by 2D high-resolution transmission electron microscopy (HRTEM) and to identify individual crystal orientations. We studied higher-level bone mineral organization within the extracellular matrix by means of scanning TEM (STEM) tomography imaging and 3D reconstruction, as well as electron diffraction to determine crystal morphology and orientation patterns. Tomographic data allowed 3D visualization of the mineral phase as individual crystallites and/or aggregates that were correlated with atomic-resolution TEM images and corresponding diffraction patterns. Integration of STEM tomography with HRTEM and crystallographic data resulted in a model of 3D mineral morphology and its association with the organic matrix. RESULTS: To visualize and characterize the crystallites within the extracellular matrix, we recorded imaging data of the bone mineral in two orthogonal projections with respect to the arrays of mineralized collagen fibrils. Three motifs of mineral organization were observed: “filamentous” (longitudinal or in-plane) and “lacy” (out-of-plane) motifs, which have been reported previously, and a third “rosette” motif comprising hexagonal crystals. Tomographic reconstructions showed that these three motifs were projections of the same 3D assembly. Our data revealed that needle-shaped, curved nanocrystals merge laterally to form platelets, which further organize into stacks of roughly parallel platelets separated by gaps of approximately 2 nanometers. These stacks of platelets, single platelets, and single acicular crystals coalesce into larger polycrystalline aggregates exceeding the lateral dimensions of the collagen fibrils, and the aggregates span adjacent fibrils as continuous, cross-fibrillar mineralization. CONCLUSION: Our findings can be described by a model of mineral and collagen assembly in which the mineral organization is hierarchical at the nanoscale. First, the data reveal that mineral particles are neither exclusively needle- nor platelet-shaped, but indeed are a combination of both, because curved acicular elements merge laterally to form slightly twisted plates. This can only be detected when the organic extracellular matrix is preserved in the sample. Second, the mineral particles are neither exclusively intrafibrillar nor extrafibrillar, but rather form a continuous cross-fibrillar phase where curved and merging crystals splay beyond the typical dimensions of a single collagen fibril. Third, in the organization of the mineral phase of bone, a helical pattern can be identified. This 3D observation, integrated with previous studies of bone hierarchy and structure, illustrates that bone (as a material, as a tissue, and as an organ) follows a fractal-like organization that is self-affine. The assembly of bone components into nested, helix-like patterns helps to explain the paradoxical combination of enhanced stiffness and toughness of bone and results in an expansion of the previously known hierarchical structure of bone to at least 12 levels

Three-dimensional coherent X-ray diffraction imaging of a ceramic nanofoam: determination of structural deformation mechanisms

Ultra-low density polymers, metals, and ceramic nanofoams are valued for their high strength-to-weight ratio, high surface area and insulating properties ascribed to their structural geometry. We obtain the labrynthine internal structure of a tantalum oxide nanofoam by X-ray diffractive imaging. Finite element analysis from the structure reveals mechanical properties consistent with bulk samples and with a diffusion limited cluster aggregation model, while excess mass on the nodes discounts the dangling fragments hypothesis of percolation theory.Comment: 8 pages, 5 figures, 30 reference

Three-dimensional coherent X-ray diffraction imaging of a ceramic nanofoam: determination of structural deformation mechanisms

Ultra-low density polymers, metals, and ceramic nanofoams are valued for their high strength-to-weight ratio, high surface area and insulating properties ascribed to their structural geometry. We obtain the labrynthine internal structure of a tantalum oxide nanofoam by X-ray diffractive imaging. Finite element analysis from the structure reveals mechanical properties consistent with bulk samples and with a diffusion limited cluster aggregation model, while excess mass on the nodes discounts the dangling fragments hypothesis of percolation theory.Comment: 8 pages, 5 figures, 30 reference

Quantifying the hydroxyapatite orientation near the ossification front in a piglet femoral condyle using X-ray diffraction tensor tomography

While a detailed knowledge of the hierarchical structure and morphology of the extracellular matrix is considered crucial for understanding the physiological and mechanical properties of bone and cartilage, the orientation of collagen fibres and carbonated hydroxyapatite (HA) crystallites remains a debated topic. Conventional microscopy techniques for orientational imaging require destructive sample sectioning, which both precludes further studies of the intact sample and potentially changes the microstructure. In this work, we use X-ray diffraction tensor tomography to image non-destructively in 3D the HA orientation in a medial femoral condyle of a piglet. By exploiting the anisotropic HA diffraction signal, 3D maps showing systematic local variations of the HA crystallite orientation in the growing subchondral bone and in the adjacent mineralized growth cartilage are obtained. Orientation maps of HA crystallites over a large field of view (~ 3 7 3 7 3 mm3) close to the ossification (bone-growth) front are compared with high-resolution X-ray propagation phase-contrast computed tomography images. The HA crystallites are found to predominantly orient with their crystallite c-axis directed towards the ossification front. Distinct patterns of HA preferred orientation are found in the vicinity of cartilage canals protruding from the subchondral bone. The demonstrated ability of retrieving 3D orientation maps of bone-cartilage structures is expected to give a better understanding of the physiological properties of bones, including their propensity for bone-cartilage diseases

Functional lung imaging with synchrotron radiation : Methods and preclinical applications

Many lung disease processes are characterized by structural and functional heterogeneity that is not directly appreciable with traditional physiological measurements. Experimental methods and lung function modeling to study regional lung function are crucial for better understanding of disease mechanisms and for targeting treatment. Synchrotron radiation offers useful properties to this end: coherence, utilized in phase-contrast imaging, and high flux and a wide energy spectrum which allow the selection of very narrow energy bands of radiation, thus allowing imaging at very specific energies. K-edge subtraction imaging (KES) has thus been developed at synchrotrons for both human and small animal imaging. The unique properties of synchrotron radiation extend X-ray computed tomography (CT) capabilities to quantitatively assess lung morphology, and also to map regional lung ventilation, perfusion, inflammation and biomechanical properties, with microscopic spatial resolution. Four-dimensional imaging, allows the investigation of the dynamics of regional lung functional parameters simultaneously with structural deformation of the lung as a function of time. This review summarizes synchrotron radiation imaging methods and overviews examples of its application in the study of disease mechanisms in preclinical animal models, as well as the potential for clinical translation both through the knowledge gained using these techniques and transfer of imaging technology to laboratory X-ray sources.Peer reviewe

Assessing the Accuracy of Soot Nanoparticle Morphology Measurements Using Three- Dimensional Electron Tomography

Morphology plays an important role in determining behaviour and impact of soot nanoparticles, including effect on human health, atmospheric optical properties, contribution to engine wear, and role in marine ecology. However, its nanoscopic size has limited the ability to directly measure useful morphological parameters such as surface area and effective volume. Recently, 3D morphology characterization of soot nanoparticles via electron tomography has been the subject of several introductory studies. So-called '3D-TEM' has been posited as an improvement over traditional 2D-TEM characterization due to the elimination of the error-inducing information gap that exists between 3-dimensional soot structures and 2-dimensional TEM projections. Little follow-up work has been performed due to difficulties with developing methodologies into robust high-throughput techniques. Recent work by the authors has exhibited significant improvements in efficiency, though as yet due consideration has not been given to assessing fidelity of the technique. This is vital to confirm significant and tangible improvements in soot-characterization accuracy that will establish 3D-TEM as a legitimate tool. Synthetic ground-truth data was developed to closely mimic real soot structures and the 3D-TEM volume-reconstruction process. A variety of procedures were tested to assess the magnitude and nuances of deviations from ground-truth values. Results showed average Z-elongation due to the 'missing-wedge' at 3.5% for the previously developed optimized procedure. Mean deviations from ground-truth in volume and surface area were 2.0% and-0.1% respectively. Results indicate highly accurate 3D-reconstruction can be achieved with an optimized procedure that can bridge the gap to permit high-throughput 3D morphology characterization of soot

Advances in shape measurement in the digital world

The importance of particle shape in terms of its effects on the behaviour of powders and other particulate systems has long been recognised, but particle shape information has been rather difficult to obtain and use until fairly recently, unlike its better-known counterpart, particle size. However, advances in computing power and 3D image acquisition and analysis techniques have resulted in major progress being made in the measurement, description and application of particle shape information in recent years. Because we are now in a digital era, it is fitting that many of these advanced techniques are based on digital technology. This review article aims to trace the development of these new techniques, highlight their contributions to both academic and practical applications, and present a perspective for future developments

Development of algorithms and methods for three-dimensional image analysis and biomedical applications

2010/2011Tomographic imaging is both the science and the tool to explore the internal structure of objects. The mission is to use images to characterize the static and/or dynamic properties of the imaged object in order to further integrate these properties into principles, laws or theories. Among the recent trends in tomographic imaging, three- dimensional (3D) methods are gaining preference and there is the quest for overcoming the bare qualitative observation towards the extraction of quantitative parameters directly from the acquired images. To this aim, Computed Tomography (CT) and Magnetic Resonance Imaging (MRI), as well as the related micro-scale techniques (μ-CT and μ-MRI), are promising tools for all the fields of science in which non-destructive tests are required. In order to support the interpretation of the images produced by these techniques, there is a growing demand of reliable image analysis methods for the specific 3D domain. The aim of this thesis is to present approaches for effective and efficient three-dimensional image analysis with special emphasis on porous media analysis. State-of-the art as well as innovative tools are included in a special software and hardware solution named Pore3D, developed in a collaboration with the Italian 3rd generation synchrotron laboratory Elettra (Basovizza - Trieste, Italy). Algorithms and methods for the characterization of different kinds of porous media are described. The key steps of image segmentation and skeletonization of the segmented pore space are also discussed in depth. Three different clinical and biomedical applications of quantitative analysis of tomographic images are presented. The reported applications have in common the characterization of the micro-architecture of trabecular bone. The trabecular (or cancellous) bone is a 3D mesh- work of bony trabeculae and void spaces containing the bone marrow. It can then be thought of as a porous medium with an interconnected porous space. To be more specific, the first application aims at characterizing a structure (a tissue engineering scaffold) that has to mimic the architecture of trabecular bone. The relevant features of porosity, pore- and throat-size distributions, connectivity and structural anisotropy indexes are automatically extracted from μ-CT images. The second application is based on ex vivo experiments carried out on femurs and lumbar spines of mice affected by microgravity conditions. Wild type and transgenic mice were hosted in the International Space Station (ISS) for 3 months and the observed bone loss due to the near-zero gravity was quantified by means of synchrotron radiation μ-CT image analysis. Finally, the results of an in vivo study on the risk of fracture in osteoporotic subjects is reported. The study is based on texture analysis of high resolution clinical magnetic resonance (MR) images.XXIV Ciclo198

Synchrotron Radiation Micro-CT Imaging of Bone Tissue

International audienc

Mapping the 3D orientation of nanocrystals and nanostructures in human bone: Indications of novel structural features

Bone is built from collagen fibrils and biomineral nanoparticles. In humans, they are organized in lamellar twisting patterns on the microscale. It has been a central tenet that the biomineral nanoparticles are co-aligned with the bone nanostructure. Here, we reconstruct the three-dimensional orientation in human lamellar bone of both the nanoscale features and the biomineral crystal lattice from small-angle x-ray scattering and wide-angle x-ray scattering, respectively. While most of the investigated regions show well-aligned nanostructure and crystal structure, consistent with current bone models, we report a localized difference in orientation distribution between the nanostructure and the biomineral crystals in specific bands. Our results show a robust and systematic, but localized, variation in the alignment of the two signals, which can be interpreted as either an additional mineral fraction in bone, a preferentially aligned extrafibrillar fraction, or the result of transverse stacking of mineral particles over several fibrils