Search

Significance Axons, the brain’s communication cables, have been described as cylinders since their discovery in 1860. Their structure is linked to how fast they conduct signals and is thus indicative of brain health and function. Here, we demonstrate an interplay between the micromorphology of axons and other extra-axonal structures, showing that axons are noncylindrical and exhibit environment-dependent diameter and trajectory variations. The nonspecificity in diameter, and thus conduction velocity, challenges the current knowledge of how axons communicate signals. Diffusion magnetic resonance imaging can be used to measure axon diameter in the living brain in order to explore the brain network and detect potential biomarkers of disease, but we show here that the observed complex morphologies of axons bias these measurements., Axonal conduction velocity, which ensures efficient function of the brain network, is related to axon diameter. Noninvasive, in vivo axon diameter estimates can be made with diffusion magnetic resonance imaging, but the technique requires three-dimensional (3D) validation. Here, high-resolution, 3D synchrotron X-ray nano-holotomography images of white matter samples from the corpus callosum of a monkey brain reveal that blood vessels, cells, and vacuoles affect axonal diameter and trajectory. Within single axons, we find that the variation in diameter and conduction velocity correlates with the mean diameter, contesting the value of precise diameter determination in larger axons. These complex 3D axon morphologies drive previously reported 2D trends in axon diameter and g-ratio. Furthermore, we find that these morphologies bias the estimates of axon diameter with diffusion magnetic resonance imaging and, ultimately, impact the investigation and formulation of the axon structure–function relationship.

There is a growing interest in developing 3D microscopy for the exploration of thick biological tissues. Recently, 3D X-ray nanocomputerised tomography has proven to be a suitable technique for imaging the bone lacunocanalicular network. This interconnected structure is hosting the osteocytes which play a major role in maintaining bone quality through remodelling processes. 3D images have the potential to reveal the architecture of cellular networks, but their quantitative analysis remains a challenge due to the density and complexity of nanometre sized structures and the need to handle and process large datasets, for example, 20483 voxels corresponding to 32 GB per individual image in our case. In this work, we propose an efficient image processing approach for the segmentation of the network and the extraction of characteristic parameters describing the 3D structure. These parameters include the density of lacunae, the porosity of lacunae and canaliculi, and morphological features of lacunae (volume, surface area, lengths, anisotropy etc.). We also introduce additional parameters describing the local environment of each lacuna and its canaliculi. The method is applied to analyse eight human femoral cortical bone samples imaged by magnified X-ray phase nanotomography with a voxel size of 120 nm, which was found to be a good compromise to resolve canaliculi while keeping a sufficiently large field of view of 246 μm in 3D. The analysis was performed on a total of 2077 lacunae showing an average length, width and depth of 17.1 μm × 9.2 μm × 4.4 μm, with an average number of 58.2 canaliculi per lacuna and a total lacuno-canalicular porosity of 1.12%. The reported descriptive parameters provide information on the 3D organisation of the lacuno-canalicular network in human bones.

Bone is an intriguingly complex material. It combines high strength, toughness and lightweight via an elaborate hierarchical structure. This structure results from a biologically driven self-assembly and self-organisation, and leads to different deformation mechanisms along the length scales. Characterising multiscale bone mechanics is fundamental to better understand these mechanisms including changes due to bone-related diseases. It also guides us in the design of new bio-inspired materials. A key-gap in understanding bone’s behaviour exists for its fundamental mechanical unit, the mineralised collagen fibre, a composite of organic collagen molecules and inorganic mineral nanocrystals. Here, we report an experimentally informed statistical elasto-plastic model to explain the fibre behaviour including the nanoscale interplay and load transfer with its main mechanical components. We utilise data from synchrotron nanoscale imaging, and combined micropillar compression and synchrotron X-ray scattering to develop the model. We see that a 10-15% micro- and nanomechanical heterogeneity in mechanical properties is essential to promote the ductile microscale behaviour preventing an abrupt overall failure even when individual fibrils have failed. We see that mineral particles take up 45% of strain compared to collagen molecules while interfibrillar shearing seems to enable the ductile post-yield behaviour. Our results suggest that a change in mineralisation and fibril-to-matrix interaction leads to different mechanical properties among mineralised tissues. Our model operates at crystalline-, molecular- and continuum-levels and sheds light on the micro- and nanoscale deformation of fibril-matrix reinforced composites.

Attributingin vivoneurophysiology to the brains’ ultrastructure requires a large field of view containing contextual anatomy. Electron microscopy (EM) is the gold standard technique to identify ultrastructure, yet acquiring volumes containing full mammalian neural circuits is challenging and time consuming using EM. Here, we show that synchrotron X-ray computed tomography (SXRT) provides rapid imaging of EM-prepared tissue volumes of several cubic millimetres. Resolution was sufficient for distinguishing cell bodies as well as for tracing apical dendrites in olfactory bulb and hippocampus, for up to 350 μm. Correlating EM with SXRT allowed us to associate dendritic spines on pyramidal cell apical dendrites in the stratum radiatum to their corresponding soma locations. Superficial pyramidal neurons had larger spine apparatus density compared to deeper ones, implying differential synaptic plasticity for superficial and deeper cells. Finally, we show that X-ray tomography and volume EM can be reliably correlated to priorin vivoimaging. Thus, combining functional measurements with multiscale X-ray microscopy and volume EM establishes a correlative workflow that enables functional and structural investigation of subcellular features in the context of cellular morphologies, tissues and ultimately whole organs.

Recently, increasing attention has been given to the study of osteocytes, the cells that are thought to play an important role in bone remodeling and in the mechanisms of bone fragility. The interconnected osteocyte system is deeply embedded inside the mineralized bone matrix and lies within a closely fitted porosity known as the lacuno-canalicular network. However, quantitative data on human samples remain scarce, mostly measured in 2D, and there are gaps to be filled in terms of spatial resolution. In this work, we present data on femoral samples from female donors imaged with isotropic 3D spatial resolution by magnified X-ray phase nano computerized-tomography. We report quantitative results on the 3D structure of canaliculi in human femoral bone imaged with a voxel size of 30 nm. We found that the lacuno-canalicular porosity occupies on average 1.45% of the total tissue volume, the ratio of the canalicular versus lacunar porosity is about 37.7%, and the primary number of canaliculi stemming from each lacuna is 79 on average. The examination of this number at different distances from the surface of the lacunae demonstrates branching in the canaliculi network. We analyzed the impact of spatial resolution on quantification by comparing parameters extracted from the same samples imaged with 120 nm and 30 nm voxel sizes. To avoid any bias related to the analysis region, the volumes at 120 nm and 30 nm were registered and cropped to the same field of view. Our results show that the measurements at 120 and 30 nm are strongly correlated in our data set but that the highest spatial resolution provides more accurate information on the canaliculi network and its branching properties.

A deeper knowledge of the architecture of the peripheral nerve with three-dimensional (3D) imaging of the nerve tissue at the sub-cellular scale may contribute to unravel the pathophysiology of neuropathy. Here we demonstrate the feasibility of X-ray phase contrast holographic nanotomography to enable 3D imaging of nerves at high resolution, while covering a relatively large tissue volume. We show various subcomponents of human peripheral nerves in biopsies from patients with type 1 and 2 diabetes and in a healthy subject. Together with well-organized, parallel myelinated nerve fibres we show regenerative clusters with twisted nerve fibres, a sprouted axon from a node of Ranvier and other specific details. A novel 3D construction (with movie created) of a node of Ranvier with end segment of a degenerated axon and sprout of a regenerated one is captured. Many of these architectural elements are not described in the literature. Thus, X-ray phase contrast holographic nanotomography enables identifying specific morphological structures in 3D in peripheral nerve biopsies from a healthy subject and from patients with type 1 and 2 diabetes. Funding Agencies|EU InterReg project MAX4ESSFUN [KU-008]; Swedish Research CouncilSwedish Research Council [E0605401, E0605402]; Lund University

The osteocyte lacuno-canalicular network (LCN) is essential for bone remodeling because osteocytes regulate cell recruitment. This has been proposed to occur through liquid-flow-induced shear forces in the canaliculi. Models of the LCN have thus far assumed that it contains canaliculi connecting the osteocyte lacunae. However, here, we reveal that enlarged spaces occur at places where several canaliculi cross; we name these spaces canalicular junctions. We characterize them in detail within mice cortical bone using synchrotron nanotomography at two length scales, with 50 and 130 nm voxel size, and show that canalicular junctions occur at a density similar to that of osteocyte lacunae and that canalicular junctions tend to cluster. Through confocal laser scanning microscopy, we show that canalicular junctions are widespread as we have observed them in cortical bone from several species, even though the number density of the canalicular junctions was not universal. Fluid flow simulations of a simple model system with and without a canalicular junction clearly show that liquid mass transport and flow velocities are altered by the presence of canalicular junctions. We suggest that these canalicular junctions may play an important role in osteocyte communication and possibly also in canalicular fluid flow. Therefore, we believe that they constitute an important component in the bone osteocyte network.

Elucidating the structure of neuronal networks provides a foundation for understanding how the nervous system processes information to generate behavior. Despite technological breakthroughs in visible light and electron microscopy, imaging dense nanometer-scale neuronal structures over millimeter-scale tissue volumes remains a challenge. Here, we demonstrate that X-ray holographic nano-tomography is capable of imaging large tissue volumes with sufficient resolution to disentangle dense neuronal circuitry in Drosophila melanogaster and mammalian central and peripheral nervous tissue. Furthermore, we show that automatic segmentation using convolutional neural networks enables rapid extraction of neuronal morphologies from these volumetric datasets. The technique we present allows rapid data collection and analysis of multiple specimens, and can be used correlatively with light microscopy and electron microscopy on the same samples. Thus, X-ray holographic nano-tomography provides a new avenue for discoveries in neuroscience and life sciences in general.

International audience; We have recently developed a series of tamoxifen-like metallocifens of the group-8 metals (Fe, Ru and Os) with strong antiproliferative activity on the triple negative breast cancer cells (MDA-MB-231). This property, not observed for the organic analog 4-hydroxytamoxifen, has been associated with the unique redox behavior of metallocenic moieties, readily affording reactive quinone methides in cancer cells. To shed light on the mechanism of action of these molecules, synchrotron radiation X-ray fluorescence (SR-XRF) nanoimaging studies were performed on cells exposed to osmocenyltamoxifen (Oc-OH-Tam) to disclose its intracellular distribution using osmium as an intrinsic reporter. High resolution mapping of the lipophilic Oc-OH-Tam in cells, revealed its preferential accumulation in the endomembrane system encompassing endoplasmic reticulum,nuclear envelope and vesicular structures. This is consistent with the ability of the amino nitrogen chain of the compounds to be protonated at physiological pH and responsible for electrostatic interactions between Oc-OH-Tam and membranes. We propose a comprehensive scenario that provides new insight into the cellular behavior and activation of Oc-OH-Tam and advances the understanding of its mechanism of action.

The roles of osteocytes in bone homeostasis have garnered increasing attention since it has been realized that osteocytes communicate with other organs. It has long been debated whether and/or to which degree osteocytes can break down the bone matrix surrounding them in a process called osteocytic osteolysis. Osteocytic osteolysis has been indicated to be induced by a number of skeletal challenges including lactation in CD1 and C57BL/6 mice, whereas immobilization-induced osteocytic osteolysis is still a matter of controversy. Motivated by the wish to understand this process better, we studied osteocyte lacunae in lactating NMRI mice, which is a widely used outbred mouse strain. Surprisingly, no trace of osteocytic osteolysis could be detected in tibial or femoral cortical bone either by 3D investigation by synchrotron nanotomography, by studies of lacunar cross-sectional areas using scanning electron microscopy, or by light microscopy. These results lead us to conclude that osteocytic osteolysis does not occur in NMRI mice as a response to lactation, in turn suggesting that osteocytic osteolysis may not play a generic role in mobilizing calcium during lactation.

Alzheimer's disease (AD), the most common form of dementia, is a progressive neurodegenerative disorder associated with aberrant production of beta-amyloid (Aβ) peptide depositing in brain as amyloid plaques. While animal models allow investigation of disease progression and therapeutic efficacy, technology to fully dissect the pathological mechanisms of this complex disease at cellular and vascular levels is lacking. X-ray phase contrast tomography (XPCT) is an advanced non-destructive 3D multi-scale direct imaging from the cell through to the whole brain, with exceptional spatial and contrast resolution. We exploit XPCT to simultaneously analyse disease-relevant vascular and neuronal networks in AD mouse brain, without sectioning and staining. The findings clearly show the different typologies and internal structures of Aβ plaques, together with their interaction with patho/physiological cellular and neuro-vascular microenvironment. XPCT enables for the first time a detailed visualization of amyloid-angiopathy at capillary level, which is impossible to achieve with other approaches. XPCT emerges as added-value technology to explore AD mouse brain as a whole, preserving tissue chemistry and structure, enabling the comparison of physiological vs. pathological states at the level of crucial disease targets. In-vivo translation will permit to monitor emerging therapeutic approaches and possibly shed new light on pathological mechanisms of neurodegenerative diseases.

Background The evolution of cartilage degeneration is still not fully understood, partly due to its thinness, low radio-opacity and therefore lack of adequately resolving imaging techniques. X-ray phase-contrast imaging (X-PCI) offers increased sensitivity with respect to standard radiography and CT allowing an enhanced visibility of adjoining, low density structures with an almost histological image resolution. This study examined the feasibility of X-PCI for high-resolution (sub-) micrometer analysis of different stages in tissue degeneration of human cartilage samples and compare it to histology and transmission electron microscopy. Methods Ten 10%-formalin preserved healthy and moderately degenerated osteochondral samples, post-mortem extracted from human knee joints, were examined using four different X-PCI tomographic set-ups using synchrotron radiation the European Synchrotron Radiation Facility (France) and the Swiss Light Source (Switzerland). Volumetric datasets were acquired with voxel sizes between 0.7 × 0.7 × 0.7 and 0.1 × 0.1 × 0.1 µm3. Data were reconstructed by a filtered back-projection algorithm, post-processed by ImageJ, the WEKA machine learning pixel classification tool and VGStudio max. For correlation, osteochondral samples were processed for histology and transmission electron microscopy. Results X-PCI provides a three-dimensional visualization of healthy and moderately degenerated cartilage samples down to a (sub-)cellular level with good correlation to histologic and transmission electron microscopy images. X-PCI is able to resolve the three layers and the architectural organization of cartilage including changes in chondrocyte cell morphology, chondrocyte subgroup distribution and (re-)organization as well as its subtle matrix structures. Conclusions X-PCI captures comprehensive cartilage tissue transformation in its environment and might serve as a tissue-preserving, staining-free and volumetric virtual histology tool for examining and chronicling cartilage behavior in basic research/laboratory experiments of cartilage disease evolution.

International audience

In bone remodeling, maturation of the newly formed osteonal tissue is associated with a rapid primary increase followed by a slower secondary increase of mineralization. This requires supply and precipitation of mineral into the bone matrix. Mineral delivery can occur only from the extracellular fluid via interfaces such as the Haversian system and the osteocyte pore network. We hypothesized that in mineralization, mineral exchange is achieved by the diffusion of mineral from the lacunar-canalicular network (LCN) to the bone matrix, resulting in a gradual change in tissue mineralization with respect to the distance from the pore-matrix interface. We expected to observe alterations in the mass density distribution with tissue age. We further hypothesized that mineral exchange occurs not only at the lacunar but also at the canalicular boundaries. The aim of this study was, therefore, to investigate the spatial distribution of mass density in the perilacunar and pericanalicular bone matrix and to explore how these densities are influenced by tissue aging. This is achieved by analyzing human jawbone specimens originating from four healthy donors and four treated with high-dosage bisphosphonate using synchrotron radiation phase-contrast nano-CT with a 50-nm voxel size. Our results provide the first experimental evidence that mass density in the direct vicinity of both lacunae (p

There have been great efforts on the nanoscale 3D probing of brain tissues to image subcellular morphologies. However, limitations in terms of tissue coverage, anisotropic resolution, stain dependence, and complex sample preparation all hinder achieving a better understanding of the human brain functioning in the subcellular context. Herein, X-ray nanoholotomography is introduced as an emerging synchrotron radiation-based technology for large-scale, label-free, direct imaging with isotropic voxel sizes down to 25 nm, exhibiting a spatial resolution down to 88 nm. The procedure is nondestructive as it does not require physical slicing. Hence, it allows subsequent imaging by complementary techniques, including histology. The feasibility of this 3D imaging approach is demonstrated on human cerebellum and neocortex specimens derived from paraffin-embedded tissue blocks. The obtained results are compared to hematoxylin and eosin stained histological sections and showcase the ability for rapid hierarchical neuroimaging and automatic rebuilding of the neuronal architecture at the level of a single cell nucleolus. The findings indicate that nanoholotomography can complement microscopy not only by large isotropic volumetric data but also by morphological details on the sub-100 nm level, addressing many of the present challenges in brain tissue characterization and probably becoming an important tool in nanoanatomy.

Bone properties at all length scales have a major impact on the fracture risk in disease such as osteoporosis. However, quantitative 3D data on bone tissue at the cellular scale are still rare. Here we propose to use magnified X-ray phase nano-CT to quantify bone ultra-structure in human bone, on the new setup developed on the beamline ID16A at the ESRF, Grenoble. Obtaining 3D images requires the application of phase retrieval prior to tomographic reconstruction. Phase retrieval is an ill-posed problem for which various approaches have been developed. Since image quality has a strong impact on the further quantification of bone tissue, our aim here is to evaluate different phase retrieval methods for imaging bone samples at the cellular scale. Samples from femurs of female donors were scanned using magnified phase nano-CT at voxel sizes of 120 and 30 nm with an energy of 33 keV. Four CT scans at varying sample-to-detector distances were acquired for each sample. We evaluated three phase retrieval methods adapted to these conditions: Paganin’s method at single distance, Paganin’s method extended to multiple distances, and the contrast transfer function (CTF) approach for pure phase objects. These methods were used as initialization to an iterative refinement step. Our results based on visual and quantitative assessment show that the use of several distances (as opposed to single one) clearly improves image quality and the two multi-distance phase retrieval methods give similar results. First results on the segmentation of osteocyte lacunae and canaliculi from such images are presented.

The degenerative effects of multiple sclerosis at the level of the vascular and neuronal networks in the central nervous system are currently the object of intensive investigation. Preclinical studies have demonstrated the efficacy of mesenchymal stem cell (MSC) therapy in experimental autoimmune encephalomyelitis (EAE), the animal model for multiple sclerosis, but the neuropathology of specific lesions in EAE and the effects of MSC treatment are under debate. Because conventional imaging techniques entail protocols that alter the tissues, limiting the reliability of the results, we have used non-invasive X-ray phase-contrast tomography to obtain an unprecedented direct 3D characterization of EAE lesions at micro-to-nano scales, with simultaneous imaging of the vascular and neuronal networks. We reveal EAE-mediated alterations down to the capillary network. Our findings shed light on how the disease and MSC treatment affect the tissues, and promote X-ray phase-contrast tomography as a powerful tool for studying neurovascular diseases and monitoring advanced therapies.

International audience; Micro-computed tomography (micro-CT)—a version of X-ray CT operating at high spatial resolution—has had a considerable success for the investigation of trabecular bone micro-architecture. Currently, there is a lot of interest in exploiting CT techniques at even higher spatial resolutions to assess bone tissue at the cellular scale. After recalling the basic principles of micro-CT, we review the different existing system, based on either standard X-ray tubes or synchrotron sources. Then, we present recent applications of micro- and nano-CT for the analysis of osteocyte lacunae and the lacunar-canalicular network. We also address the question of the quantification of bone ultrastructure to go beyond the sole visualization.

The architectural properties of the osteocyte cell network provide a valuable basis for understanding the mechanisms of bone remodelling, mineral homeostasis, ageing and pathologies. Recent advance ...

Purpose: The fundamental role of the osteocyte cell network in regulating the bone remodeling has become evident in the last years. This has raised the necessity to explore this complex three-dimensional interconnected structure, but the existing investigation methods cannot provide an adequate assessment. The authors propose to use parallel beam synchrotron radiation computed tomography at the nanoscale to image in three dimensions the osteocyte lacunocanalicular network. To this aim, the authors study the feasibility of this technique and present an optimized imaging protocol suited for the bone cell network. Moreover, they demonstrate the multifaceted information provided by this method. Methods: The high brilliance of synchrotron radiation combined with state of art detectors permits reaching nanoscale spatial resolution. With a nominal pixel size of 280 nm, the parallel beam computed tomography setup at the ID19 experimental station of the ESRF is capable of imaging the bone lacunocanalicular network, considering that the reported diameter of canaliculi is in the range 300–600 nm. However, the actual resolution is limited by the detector and by the radiationdose causing sample damage during the scan. The authors sought to overcome these limitations by optimizing the imaging setup and the acquisition parameters in order to minimize the necessary radiationdose to create the images and to improve the spatial resolution of the detector. Results: The authors achieved imaging of the osteocyte cell network in human bone. Due to the optimization of the imaging setup and acquisition parameters, they obtained simultaneously a radiationdose reduction and an increase of the signal to noise ratio in the images. This permitted the authors to generate the first three-dimensional images of the lacunocanalicular network in an area covering several osteons, the fundamental functional units in the bone cortex. The method enables assessment of both architectural parameters of the microporosity and of mineralization degree in the bone matrix. The authors found that the cell network is dense and connected inside osteonal tissue. Conversely, the cell lacunae are sparse, unorganized, and disconnected in interstitial tissue. Conclusions: The authors show that synchrotron radiation computed tomography is a feasible technique to assess the lacunocanalicular network in three dimensions. This is possible due to an optimal imaging setup in which the detector plays an important role. The authors could establish two valid setups, based on two different insertion devices. These results give access to new information on the bone cell network architecture, covering a number of cells two orders of magnitude greater than existing techniques. This enables biomedical studies on series of samples, paving the way to better understanding of bone fragility and to new treatments for bone diseases.

International audience; Osteocytes are hypothesized to regulate bone remodeling guided by both biological and mechanical stimuli. Morphology of the lacunar-canalicular network of osteocytes has been hypothesized to be strongly related to the level of mechanical loading and to various bone diseases. Finite element modeling could help to better understand the mechanosensation process by predicting the physiological strain environment. The aims of this study were to (i) quantify the lacunar-canalicular morphology in the cortex of the human femur; (ii) predict the in situ local deformations around and in osteocytes by means of case-specific finite element models; and (iii) investigate the potential relationship between morphology and deformations. Human femoral cortical bone samples were imaged using synchrotron X-ray phase nano-tomography with 50 nm voxel size. Rectangular volumes of interest were selected to contain single osteocyte lacunae and the surrounding matrix. Lacunar-canalicular morphology was quantified and the cell geometry was artificially reconstructed based on a priori assumptions. Finite element models of the volumes of interest were generated, containing the extracellular matrix, osteocyte and peri-cellular matrix, and subjected to uniaxial compression. The morphological analysis revealed that canalicular number was dictated by lacunar size, that the spacing of canaliculi fell within a narrow range, suggesting that these pores are well distributed throughout the bone matrix and indicated the trend that lacunae at the outer osteon boundary were less elongated than others. No apparent relationship was found between the morphological parameters and the predicted strains. The globally applied strain was amplified locally by factors up to 10 and up to 70 in the extracellular matrix and the in cells, respectively. Cell deformations were localized mainly at the body-dendrite junctions, with magnitudes reaching the in vitro stimulatory threshold reported for osteocytes.

International audience; In the context of bone diseases research, recent works have highlighted the crucial role of the osteocyte system. This system, hosted in the lacuno-canalicular network (LCN), plays a key role in the bone remodeling process. However, few data are available on the LCN due to the limitations of current microscopy techniques, and have mainly only been obtained from 2D histology sections. Here we present, for the first time, an automatic method to quantify the LCN in 3D from synchrotron radiation micro-tomography images. After segmentation of the LCN, two binary images are generated, one of lacunae (hosting the cell body) and one of canaliculi (small channels linking the lacunae). The binary image of lacunae is labeled, and for each object, lacunar descriptors are extracted after calculating the second order moments and the intrinsic volumes. Furthermore, we propose a specific method to quantify the ramification of canaliculi around each lacuna. To this aim, a signature of the numbers of canaliculi at different distances from the lacunar surface is estimated through the calculation of topological parameters. The proposed method was applied to the 3D SR micro-CT image of a human femoral mid-diaphysis bone sample. Statistical results are reported on 399 lacunae and their surrounding canaliculi.

International audience; Recent works highlighted the crucial role of the osteocyte system in bone fragility. The number of canaliculi of osteocyte lacuna (Lc.NCa) is an important parameter that reflects the functionality of bone tissue, but rarely reported due to the limitations of current microscopy techniques, and only assessed from 2D histology sections. Previously, we showed the Synchrotron Radiation nanotomography (SR-nanoCT) is a promising technique to image the 3D lacunar-canalicular network. Here we present, for the first time, an automatic method to quantify the connectivity of bone cells in 3D. After segmentation, our method first separates and labels each lacuna in the network. Then, by creating a bounding surface around lacuna, the Lc.NCa is calculated through estimating 3D topological parameters. The proposed method was successfully applied to a 3D SR-nanoCT image of cortical femoral bone. Statistical results on 165 lacunae are reported, showing a mean of 51, which is consistent with the literature.

International audience; The osteocyte cell network in bone tissue is thought to orchestrate tissue adaptation and remodeling, thus holding responsibility for tissue quality. Previously, this structure has been studied mainly in 2D and its architecture and functions are not fully elucidated. The assessment of the osteocyte system is prerequisite for deeper understanding of bone remodeling and for advances in management of bone diseases. Our goal is to enable 3D isotropic imaging of bone at cellular level and to develop algorithms for quantitative image analysis of the cell network. We recently demonstrated accurate 3D imaging of this cell structure with synchrotron radiation tomography at submicrometric scale. Due to the limited spatial resolution of the imaging system and the constraints in terms of radiation dose, the images suffer from low signal to noise ratio and the detection of the cell dendrites is challenging. Here we detail a method for enhancement of the osteocyte network in human bone from 3D microtomography images. The approach combines Hessian-based 3D line enhancement and bilateral filtering. Our method enables extraction of the interconnected cells from noisy images, preserving the integrity of the cells and of their slender dendrites. Qualitative and quantitative results are presented

We investigate the three-dimensional (3-D) organization of mineralized collagen fibrils in human cortical bone based on synchrotron X-ray phase nano-tomography images. In lamellar bone the collagen fibrils are assumed to have a plywood-like arrangement, but due to experimental limitations the 3-D fibril structure has only been deduced from section surfaces so far and the findings have been controversial. Breakthroughs in synchrotron tomographic imaging have given access to direct 3-D information on the bone structure at the nanoscale level. Using an autocorrelation-based orientation measure we confirm that the fibrils are unidirectional in quasi-planes of sub-lamellae and find two specific dominant patterns, oscillating and twisted plywoods coexisting in a single osteon. Both patterns exhibit smooth orientation changes between adjacent quasi-planes. Moreover, we find that the periodic changes in collagen fibril orientation are independent of fluctuations in local mass density. These data improve our understanding of the lamellar arrangement in bone and allow more detailed investigations of structure-function relationships at this scale, providing templates for bio-inspired materials. The presented methodology can be applied to non-destructive 3-D characterization of the sub-micron scale structure of other natural and artificial mineralized biomaterials.

International audience; During the last decade, X-ray micro Computerized Tomography (CT) has become a conventional technique for the three-dimensional (3D) investigation of trabecular bone micro-architecture. Coupling micro-CT to synchrotron sources possesses significant advantages in terms of image quality and gives access to information on bone mineralization which is an important factor of bone quality. We present an overview of the investigation of bone using Synchrotron Radiation (SR) CT from the micro to the nano scale. We introduce two synchrotron CT systems developed at the ESRF based on SR parallel-beam micro-CT and magnified phase CT respectively, achieving down to submicrometric and nanometric spatial resolution. In the latter, by using phase retrieval prior to tomographic reconstruction, the system provides maps of the 3D refractive index distribution. Parallel-beam SR micro-CT has extensively been used for the analysis of trabecular or cortical bone in human or small animals with spatial resolution in the range [3-10] μm. However, the characterization of the bone properties at the cellular scale is also of major interest. At the micrometric scale, the shape, density and morphology of osteocyte lacunae can be studied on statistically representative volumes. At the nanometric scale, unprecedented 3D displays of the canaliculi network have been obtained on fields of views including a large number of interconnected osteocyte lacunae. Finally SR magnified phase CT provides a detailed analysis of the lacuno-canalicular network and in addition information on the organization of the collagen fibers. These findings open new perspectives for three-dimensional quantitative assessment of bone tissue at the cellular scale.

International audience; Bone strength and failure are increasingly thought to be due to ultrastructural properties, such as the morphology of thelacuno-canalicular network, the collagen fiber orientation and the mineralization on the nanoscale. However, theseproperties have not been studied in 3D so far. Here we report the investigation of the human bone ultrastructure with X-rayphase nanotomography, which now provides the required sensitivity, spatial resolution and field of view. The 3Dorganization of the lacuno-canalicular network is studied in detail over several cells in osteonal and interstitial tissue.Nanoscale density variations are revealed and show that the cement line separating these tissues is hypermineralized.Finally, we show that the collagen fibers are organized as a twisted plywood structure in 3D

International audience; Imaging of the trabecular bone network has made significant advances in the last decade thanks to the outstanding development of 3D X-ray micro-CT. Compared to standard histomorphometry, this technique provides non-destructively 3D images of the trabecular bone permitting a so-called model-independent quantification of this network. Today, the assessment of the bone Lacuno-Canalicular Network (LCN) is a new challenge at the cellular scale. The LCN forms a communication network interconnecting the osteocytes, the bone cells embedded in the mineralized matrix. It plays a major role in mechanotransduction with important implications on bone remodeling and finally bone strength. However, methods for the 3D assessment of the LCN are lacking. In a recent work, we have shown the feasibility of imaging the LCN in 3D thanks to Synchrotron Radiation nano-CT (voxel size 280 nm). These first images of the LCN in 3D open new challenges in image processing to segment and quantify the LCN. After recalling the principle of image acquisition, we present our first approaches for enhancing the contrast of canaliculi and segmenting the LCN. Finally, to increase the connectivity of the network, we consider a new approach based on 3D geodesic voting, offering promising perspectives. Results on experimental 3D images of the 3D LCN in human femoral bone are presented.

The investigation of cellular aspects of bone tissue is generally performed from 2D microscopic techniques. Recent advances in Synchrotron Radiation Micro-CT allow to image biological samples at a sub-micrometric scale. In this paper, we propose to study the feasibility of this technique to study the bone lacuno-canalicular system, consisting in a complex network of thin canaliculi (diameter ∼ 500 nm) joining osteocytes. Due to the limited spatial resolution of the imaging system compared to the structure size and the constraints in term of dose, the images have a low signal to noise ratio and the detection of canaliculi is challenging. We propose a 3D non-linear enhancement technique based on Hessian analysis. The method is first evaluated on synthetic images, then we show, for the first time, a 3D rendering of the canalicular network based on experimental data.

International audience; Osteonecrosis of the jaw, in association with bisphosphonates (BRONJ) used for treating osteoporosis or cancer, is a severe and most often irreversible side effect whose underlying pathophysiological mechanisms remain largely unknown. Osteocytes are involved in bone remodeling and mineralization where they orchestrate the delicate equilibrium between osteoclast and osteoblast activity and through the active process called osteocytic osteolysis. Here, we hypothesized that (i) changes of the mineralized tissue matrix play a substantial role in the pathogenesis of BRONJ, and (ii) the osteocyte lacunar morphology is altered in BRONJ. Synchrotron µCT with phase contrast is an appropriate tool for assessing both the 3D morphology of the osteocyte lacunae and the bone matrix mass density. Here, we used this technique to investigate the mass density distribution and 3D osteocyte lacunar properties at the sub-micrometer scale in human bone samples from the jaw, femur and tibia. First, we compared healthy human jaw bone to human tibia and femur in order to assess the specific differences and address potential explanations of why the jaw bone is exclusively targeted by the necrosis as a side effect of BP treatment. Second, we investigated the differences between BRONJ and control jaw bone samples to detect potential differences which could aid an improved understanding of the course of BRONJ. We found that the apparent mass density of jaw bone was significantly smaller compared to that of tibia, consistent with a higher bone turnover in the jaw bone. The variance of the lacunar volume distribution was significantly different depending on the anatomical site. The comparison between BRONJ and control jaw specimens revealed no significant increase in mineralization after BP. We found a significant decrease in osteocyte-lacunar density in the BRONJ group compared to the control jaw. Interestingly, the osteocyte-lacunar volume distribution was not altered after BP treatment.

Imaging neuronal networks provides a foundation for understanding the nervous system, but resolving dense nanometer-scale structures over large volumes remains challenging for light microscopy (LM) and electron microscopy (EM). Here we show that X-ray holographic nano-tomography (XNH) can image millimeter-scale volumes with sub-100-nm resolution, enabling reconstruction of dense wiring in Drosophila melanogaster and mouse nervous tissue. We performed correlative XNH and EM to reconstruct hundreds of cortical pyramidal cells and show that more superficial cells receive stronger synaptic inhibition on their apical dendrites. By combining multiple XNH scans, we imaged an adult Drosophila leg with sufficient resolution to comprehensively catalog mechanosensory neurons and trace individual motor axons from muscles to the central nervous system. To accelerate neuronal reconstructions, we trained a convolutional neural network to automatically segment neurons from XNH volumes. Thus, XNH bridges a key gap between LM and EM, providing a new avenue for neural circuit discovery. Kuan, Phelps, et al. used synchrotron X-ray imaging and deep learning to map dense neuronal wiring in fly and mouse tissue, enabling examination of individual cells and connectivity in circuits governing motor control and perceptual decision-making.