Advanced Synchroton radiation X-ray micro- and nano-tomography for bone, vascularisation and soft tissue analysis

National audienc

3D micro-computed tomography of trabecular and cortical bone architecture with application to a rat model of immobilisation osteoporosis

Bone mass and microarchitecture are the main determinants of bone strength. Three-dimensional micro-computed tomogrpahy has the potential to examine complete bones of small laboratory animals with very high resolution in a non-invasive way. In the presented work, the proximal part of the tibiae of hindlimb unloaded and control rats were measured with 3D MicroCT, and the secondary spongiosa of the scanned region was evaluated using direct evaluation techniques that do not require model assumptions. For determination of the complete bone status, the cortex of the tibiae was evaluated and characterised by its thickness. It is shown that with the proposed anatomically conforming volume of interest (VOI), up to an eight-fold volume increase can be evaluated compared to cubic or spherical VOIs. A pronounced trabecular bone loss of −50% is seen after 23 days of tail suspension. With the new evaluation techniques, it is shown that most of this bone loss is caused by the thinning of trabeculae, and to a lesser extent by a decrease in their number. What changes most radically is the structure type: the remaining bone is more rod-like than the control group's bone. Cortical bone decreases less than trabecular bone, with only −18% after 23 day

Adaptive filtering for enhancement of the osteocyte cell network in 3D microtomography images

International audienceThe 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 presente

Propagation based X-ray phase microtomography of multi-material objects for simultaneous bone and soft tissue visualisation

International audienceWe present a method for phase microtomography based on X-ray propagation based phase contrast imaging for multi-material objects. Previously, homogeneous composition assumptions have been in the Radon domain to overcome low sensitivity in the low frequency range. Here, we introduce a prior in the object domain based on multiple, but known, materials in the sample. This is achieved by first reconstructing a tomographic attenuation scan, introducing the prior by segmentation, and finally forward projecting this initial object estimate to yield a priori phase maps. The method is applied to the imaging of a mouse knee where analysis of both soft and hard tissue is of interest, and is shown to perform better than previously proposed methods

Structure and quantification of microvascularisation within mouse long bones: what and how should we measure?

International audienceBone marrow vascularisation is involved in both remodeling and hematopoïesis. Challenged mouse models often require imaging and quantitative assessment of blood vessels and bone cell activities for a better understanding of the role of the vascular system. In this study we compared images of mouse hind limb long bone vascularisation after infusion of either barium sulfate or lead chromate-loaded silicon. The images were then analyzed through histology as well as low-resolution and synchrotron-radiation microtomography. We show that barium sulfate infusion provides the best vessel images and furthermore, that it is compatible with staining procedures used in bone histomorphometry and CD31 immunohistochemistry. Bone marrow vascularisation displays large structural and spatial distribution heterogeneity, including large lobular clusters of sinusoids and an unexpectedly substantial amount of capillaries in the adipocytes-rich distal third of the tibia. For an unbiased assessment of bone vascular development/changes, these features must be taken into account. We describe the conditions under which the quantification of microvascularisation on histological sections of barium-infused long bones is reproducible, as applied to seven-month-old male C57/Bl6J and mixed CD1/129Sv/J mice, and we propose a nomenclature for the histological parameters measured. Finally, we validate our technique by studying the effect of ovariectomy on mouse tibial vascular density

Cell cycling determines integrin-mediated adhesion in osteoblastic ROS 17/2.8 cells exposed to space-related conditions.

Six days of microgravity (Bion10 mission) induced dramatic shape changes in ROS 17/2.8 osteoblasts (7). During the Foton 11 and 12 space flights, we studied the kinetics (0-4 days) of ROS 17/2.8 morphology and adhesion, the relationships between adhesion and cell cycle progression after 4 days in space, and osteoblastic growth and activity after 6 days in space. Quantitative analysis of high-resolution adhesion [focal adhesion area imaged by total interference reflection fluorescent microscopy (TIRFM)] and integrin-dependent adhesion (imaged on confocal microscope by vinculin and phosphotyrosine staining) as well as cell cycle phase classification [Ki-67 staining, S-G2, mitotic cells and G1 (postmitotic cells)] were performed using programs validated in parabolic flight and clinostat. We observed disorganization of the cytoskeleton associated with disassembling of vinculin spots and phosphorylated proteins within focal contacts with no major change in TIRFM adhesion after 2 and 4 days of microgravity. Postmitotic cells, alone, accounted for the differences observed in the whole population. They are characterized by immature peripheral contacts with complete loss of central spots and decreased spreading. Osteocalcin, P1CP and alkaline phosphatase, and proliferation were similar in flight cells and 1 g centrifuge and ground controls after 6 days. In conclusion, microgravity substantially affected osteoblastic integrin-mediated cell adhesion. ROS17/2.8 cells responded differently, whether or not they were cycling by reorganizing adhesion plaque topography or morphology. In ROS 17/2.8, this reorganization did not impair osteoblastic phenotype

Use of TALEN for a double gene extinction on two closely linked loci

International audienc