Cécile Olivier

Synchrotron Radiation Micro-CT at the Micrometer Scale for the Analysis of the Three-Dimensional Morphology of Microcracks in Human Trabecular Bone

Bone quality is an important concept to explain bone fragility in addition to bone mass. Among bone quality factors, microdamage which appears in daily life is thought to have a marked impact on bone strength and plays a major role in the repair process. The starting point for all studies designed to further our understanding of how bone microdamage initiate or dissipate energy, or to investigate the impact of age, gender or disease, remains reliable observation and measurement of microdamage. In this study, 3D Synchrotron Radiation (SR) micro-CT at the micrometric scale was coupled to image analysis for the three-dimensional characterization of bone microdamage in human trabecular bone specimens taken from femoral heads. Specimens were imaged by 3D SR micro-CT with a voxel size of 1.4 µm. A new tailored 3D image analysis technique was developed to segment and quantify microcracks. Microcracks from human trabecular bone were observed in different tomographic sections as well as from 3D renderings. New 3D quantitative measurements on the microcrack density and morphology are reported on five specimens. The 3D microcrack density was found between 3.1 and 9.4/mm3 corresponding to a 2D density between 0.55 and 0.76 /mm2. The microcrack length and width measured in 3D on five selected microcrack ranged respectively from 164 µm to 209 µm and 100 µm to 120 µm. This is the first time that various microcracks in unloaded human trabecular bone - from the simplest linear crack to more complex cross-hatch cracks - have been examined and quantified by 3D imaging at this scale. The suspected complex morphology of microcracks is here considerably more evident than in the 2D observations. In conclusion, this technique opens new perspective for the 3D investigation of microcracks and the impact of age, disease or treatment.

Surface delivery of tunable doses of BMP-2 from an adaptable polymeric scaffold induces volumetric bone regeneration

The rapid and effective bone regeneration of large non-healing defects remains challenging. Bioactive proteins, such as bone morphogenetic protein (BMP)-2, are proved their osteoinductivity, but their clinical use is currently limited to collagen as biomaterial. Being able to deliver BMP-2 from any other biomaterial would broaden its clinical use. This work presents a novel means for repairing a critical size volumetric bone femoral defect in the rat by combining a osteoinductive surface coating (2D) to a polymeric scaffold (3D hollow tube) made of commercially-available PLGA. Using a polyelectrolyte film as BMP-2 carrier, we tune the amount of BMP-2 loaded in and released from the polyelectrolyte film coating over a large extent by controlling the film crosslinking level and initial concentration of BMP-2 in solution. Using microcomputed tomography and quantitative analysis of the regenerated bone growth kinetics, we show that the amount of newly formed bone and kinetics can be modulated: an effective and fast repair was obtained in 1-2 weeks in the best conditions, including complete defect bridging, formation of vascularized and mineralized bone tissue. Histological staining and high-resolution computed tomography revealed the presence of bone regeneration inside and around the tube with spatially distinct organization for trabecular-like and cortical bones. The amount of cortical bone and its thickness increased with the BMP-2 dose. In view of the recent developments in additive manufacturing techniques, this surface-coating technology may be applied in combination with various types of polymeric or metallic scaffolds to offer new perspectives of bone regeneration in personalized medicine.

Alterations of mass density and 3D osteocyte lacunar properties in bisphosphonate-related osteonecrotic human jaw bone, a synchrotron µCT study.

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.

Computer vision tools to optimize reconstruction parameters in x-ray in-line phase tomography

In this article, a set of three computer vision tools, including scale invariant feature transform (SIFT), a measure of focus, and a measure based on tractography are demonstrated to be useful in replacing the eye of the expert in the optimization of the reconstruction parameters in x-ray in-line phase tomography. We demonstrate how these computer vision tools can be used to inject priors on the shape and scale of the object to be reconstructed. This is illustrated with the Paganin single intensity image phase retrieval algorithm in heterogeneous soft tissues of biomedical interest, where the selection of the reconstruction parameters was previously made from visual inspection or physical assumptions on the composition of the sample.

Information-based analysis of X-ray in-line phase tomography with application to the detection of iron oxide nanoparticles in the brain

The study analyzes noise in X-ray in-line phase tomography in a biomedical context. The impact of noise on detection of iron oxide nanoparticles in mouse brain is assessed. The part of the noise due to the imaging system and the part due to biology are quantitatively expressed in a Neyman Pearson detection strategy with two models of noise. This represents a practical extension of previous work on noise in phase-contrast X-ray imaging which focused on the theoretical expression of the signal-to-noise ratio in mono-dimensional phantoms, taking account of the statistical noise of the imaging system only. We also report the impact of the phase retrieval step on detection performance. Taken together, this constitutes a general methodology of practical interest for quantitative extraction of information from X-ray in-line phase tomography, and is also relevant to assessment of contrast agents with a blob-like signature in high resolution imaging.

Characterizing microcrack orientation distribution functions in osteonal bone samples

Prefailure microdamage in bone tissue is considered to be the most detrimental factor in defining its strength and toughness with respect to age and disease. To understand the influence of microcracks on bone mechanics it is necessary to assess their morphology and three‐dimensional distribution. This requirement reaches beyond classic histology and stereology, and methods to obtain such information are currently missing. Therefore, the aim of the study was to develop a methodology that allows to characterize three‐dimensional microcrack distributions in bulk bone samples.

A new quantitative approach for estimating bone cell connections from nano-CT images

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.

Label-free imaging of bone multiscale porosity and interfaces using third-harmonic generation microscopy

Interfaces provide the structural basis of essential bone functions. In the hierarchical structure of bone tissue, heterogeneities such as porosity or boundaries are found at scales ranging from nanometers to millimeters, all of which contributing to macroscopic properties. To date, however, the complexity or limitations of currently used imaging methods restrict our understanding of this functional integration. Here we address this issue using label-free third-harmonic generation (THG) microscopy. We find that the porous lacuno-canalicular network (LCN), revealing the geometry of osteocytes in the bone matrix, can be directly visualized in 3D with submicron precision over millimetric fields of view compatible with histology. THG also reveals interfaces delineating volumes formed at successive remodeling stages. Finally, we show that the structure of the LCN can be analyzed in relation with that of the extracellular matrix and larger-scale structures by simultaneously recording THG and second-harmonic generation (SHG) signals relating to the collagen organization.

Synchrotron radiation CT from the micro to nanoscale for the investigation of bone tissue

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.

Cortical bone elasticity measured by resonant ultrasound spectroscopy is not altered by defatting and synchrotron X-ray imaging

In the study of mechanical properties of human bone, specimens may be defatted before experiments to prevent contamination and the risk of infections. High energy synchrotron radiation micro-computed tomography (SR-μCT) is a popular technique to study bone microstructure. However, little is known about the effects of defatting or irradiation during SR-μCT imaging on different elastic coefficients including shear and longitudinal moduli in different anatomical directions. In this work, these effects are evaluated on a set of 24 samples using resonant ultrasound spectroscopy (RUS), which allows one to accurately measure the complete set of elastic coefficients of cortical bone non destructively. The results show that defatting with diethylether and methanol and irradiation up to 2.5kGy has no detectable effect on any of the elastic coefficients of human cortical bone.