Hard X-ray imaging and tomography at the Biomedical Imaging and Therapy beamlines of Canadian Light Source.

The Biomedical Imaging and Therapy facility of the Canadian Light Source comprises two beamlines, which together cover a wide X-ray energy range from 13 keV up to 140 keV. The beamlines were designed with a focus on synchrotron applications in preclinical imaging and veterinary science as well as microbeam radiation therapy. While these remain a major part of the activities of both beamlines, a number of recent upgrades have enhanced the versatility and performance of the beamlines, particularly for high-resolution microtomography experiments. As a result, the user community has been quickly expanding to include researchers in advanced materials, batteries, fuel cells, agriculture, and environmental studies. This article summarizes the beam properties, describes the endstations together with the detector pool, and presents several application cases of the various X-ray imaging techniques available to users.

MRI overestimates articular cartilage thickness and volume compared to synchrotron radiation phase-contrast imaging.

Accurate evaluation of morphological changes in articular cartilage are necessary for early detection of osteoarthritis (OA). 3T magnetic resonance imaging (MRI) has highly sensitive contrast resolution and is widely used clinically to detect OA. However, synchrotron radiation phase-contrast imaging computed tomography (SR-PCI) can also provide contrast to tissue interfaces that do not have sufficient absorption differences, with the added benefit of very high spatial resolution. Here, MRI was compared with SR-PCI for quantitative evaluation of human articular cartilage. Medial tibial condyles were harvested from non-OA donors and from OA patients receiving knee replacement surgery. Both imaging methods revealed that average cartilage thickness and cartilage volume were significantly reduced in the OA group, compared to the non-OA group. When comparing modalities, the superior resolution of SR-PCI enabled more precise mapping of the cartilage surface relative to MRI. As a result, MRI showed significantly higher average cartilage thickness and cartilage volume, compared to SR-PCI. These data highlight the potential for high-resolution imaging of articular cartilage using SR-PCI as a solution for early OA diagnosis. Recognizing current limitations of using a synchrotron for clinical imaging, we discuss its nascent utility for preclinical models, particularly longitudinal studies of live animal models of OA.

Low-density tissue scaffold imaging by synchrotron radiation propagation-based imaging computed tomography with helical acquisition mode.

Visualization of low-density tissue scaffolds made from hydrogels is important yet challenging in tissue engineering and regenerative medicine (TERM). For this, synchrotron radiation propagation-based imaging computed tomography (SR-PBI-CT) has great potential, but is limited due to the ring artifacts commonly observed in SR-PBI-CT images. To address this issue, this study focuses on the integration of SR-PBI-CT and helical acquisition mode (i.e. SR-PBI-HCT) to visualize hydrogel scaffolds. The influence of key imaging parameters on the image quality of hydrogel scaffolds was investigated, including the helical pitch (p), photon energy (E) and the number of acquisition projections per rotation/revolution (Np), and, on this basis, those parameters were optimized to improve image quality and to reduce noise level and artifacts. The results illustrate that SR-PBI-HCT imaging shows impressive advantages in avoiding ring artifacts with p = 1.5, E = 30 keV and Np = 500 for the visualization of hydrogel scaffolds in vitro. Furthermore, the results also demonstrate that hydrogel scaffolds can be visualized using SR-PBI-HCT with good contrast while at a low radiation dose, i.e. 342 mGy (voxel size of 26 µm, suitable for in vivo imaging). This paper presents a systematic study on hydrogel scaffold imaging using SR-PBI-HCT and the results reveal that SR-PBI-HCT is a powerful tool for visualizing and characterizing low-density scaffolds with a high image quality in vitro. This work represents a significant advance toward the non-invasive in vivo visualization and characterization of hydrogel scaffolds at a suitable radiation dose.

Conversion of Pulse Protein Foam-Templated Oleogels into Oleofoams for Improved Baking Application.

The food industry has long been searching for an efficient replacement for saturated-fatty-acid-rich fats for baking applications. Although oleogels have been considered a potential alternative for saturated and trans fats, their success in food application has been poor. The present study explored the use of oleofoams obtained by whipping the pulse protein foam-templated oleogels for cake baking. Oleogels were prepared at room temperature by adding canola oil containing high-melting monoglyceride (MAG) or candelilla wax (CW) to the freeze-dried pea or faba bean protein-stabilized foams. Oleogels were then whipped to create the oleofoams; however, only the oleogels containing MAG could form oleofoams. CW-oleogel could not form any oleofoam. The most stable oleofoams with the highest overrun, stability, and storage modulus were obtained from 3% MAG+pulse protein foam-templated oleogels. The MAG plus protein foam-templated oleogels showed smaller and more packed air bubbles than MAG-only oleofoam, which was ascribed to the protein's ability to stabilize air bubbles and provide a network in the continuous oil phase to restrict air bubble movement. A novel batter preparation method for oleofoam was developed to increase air bubble incorporation. The X-ray microtomography images of the cakes showed a non-homogeneous distribution of larger air bubbles in the oleofoam cake compared to the shortening cake although their total porosity was not much different. The oleofoam cakes made with the new method yielded similar hardness and chewiness compared to the shortening cakes. By improving rheology and increasing air incorporation in the batter, high-quality cakes can be obtained with MAG-containing oleofoams made from pulse protein foam-templated oleogels.

Activation of Focal Adhesion Kinase Restores Simulated Microgravity-Induced Inhibition of Osteoblast Differentiation via Wnt/Β-Catenin Pathway.

Simulated microgravity (SMG) inhibits osteoblast differentiation (OBD) and induces bone loss via the inhibition of the Wnt/ß-catenin pathway. However, the mechanism by which SMG alters the Wnt/ß-catenin pathway is unknown. We previously demonstrated that SMG altered the focal adhesion kinase (FAK)-regulated mTORC1, AMPK and ERK1/2 pathways, leading to the inhibition of tumor cell proliferation/metastasis and promoting cell apoptosis. To examine whether FAK similarly mediates SMG-dependent changes to Wnt/ß-catenin in osteoblasts, we characterized mouse MC3T3-E1 cells cultured under clinostat-modeled SMG (µg) conditions. Compared to cells cultured under ground (1 g) conditions, SMG reduces focal adhesions, alters cytoskeleton structures, and down-regulates FAK, Wnt/ß-catenin and Wnt/ß-catenin-regulated molecules. Consequently, protein-2 (BMP2), type-1 collagen (COL1), alkaline-phosphatase activity and matrix mineralization are all inhibited. In the mouse hindlimb unloading (HU) model, SMG-affected tibial trabecular bone loss is significantly reduced, according to histological and micro-computed tomography analyses. Interestingly, the FAK activator, cytotoxic necrotizing factor-1 (CNF1), significantly suppresses all of the SMG-induced alterations in MC3T3-E1 cells and the HU model. Therefore, our data demonstrate the critical role of FAK in the SMG-induced inhibition of OBD and bone loss via the Wnt/ß-catenin pathway, offering FAK signaling as a new therapeutic target not only for astronauts at risk of OBD inhibition and bone loss, but also osteoporotic patients.

Insights into biogenic and diagenetic lead exposure in experimentally altered modern and archaeological bone: Synchrotron radiation X-ray fluorescence imaging.

Bones represent a valuable biological archive of environmental lead (Pb) exposure for modern and archaeological populations. Synchrotron radiation X-ray fluorescence imaging (SR-XFI) generates maps of Pb in bone on a microstructural scale, potentially providing insights into an individual's history of Pb exposure and, in the context of archaeological bone, the biogenic or diagenetic nature of its uptake. The aims of this study were to (1) examine biogenic spatial patterns for Pb from bone samples of modern cadavers compared with patterns observed archaeologically, and (2) test the hypothesis that there are spatial differences in the distribution of Pb for diagenetic and biogenic modes of uptake in bone. To address these aims, this study used inductively coupled plasma-mass spectrometry (ICP-MS) and SR-XFI on unaltered and experimentally altered cadaveric bone samples (University of Saskatchewan, Saskatoon, SK) and archaeological bone samples from 18th to 19th century archaeological sites from Antigua and Lithuania. Bone concentrations of modern individuals are relatively low compared to those of archaeological individuals. SR-XFI results provide insights into modern Saskatchewan Pb exposure with some samples demonstrating a pattern of relatively low Pb exposure with higher levels of Pb exposure occurring in bone structures of a relatively older age that formed earlier in life, likely during the era of leaded gasoline (pre-1980s), and other samples demonstrating a pattern of fairly consistent, low-level exposure. Results support hypotheses for the spatial distribution of Pb corresponding to biogenic vs. diagenetic uptake. Diagenetic Pb is mainly confined to the periosteal surface of each sample with some enrichment of cracks and sub-periosteal canals. This may be useful in the future for differentiating diagenetic from biogenic Pb accumulation, analyzing environmental contamination, and informing sampling strategies in archaeological or fossil bone.

The physiopathology of osteoarthritis: Paleopathological implications of non-articular lesions from a modern surgical sample.

OBJECTIVES: This research focused on osteoarthritis (OA) lesions on modern patients to 1) identify consistently observed lesions not included within current paleopathological measures of OA, 2) assess the correspondence of bone and cartilage lesions with clinical OA diagnostic criteria, and 3) discuss the correspondence of bone lesions with sources of pain reported in clinical literature. MATERIALS: Tibial plateaus from 62 patients undergoing total knee replacement surgery due to OA were examined. METHODS: Plateaus were scored for several non-standard OA criteria, including non-articular and X-ray visible lesions and pre-maceration cartilage lesions, as well as articular surface criteria standard in paleopathology. RESULTS: Proliferative bone in the intercondylar region was present in 95 % of specimens, while areas of dense trabecular bone and lytic defects, both on the inferior side of the plateaus, were present in 98 % and 83 %, respectively. CONCLUSIONS: The inferior lytic defects may be physical evidence of bone marrow lesions (BML), a clinical OA indicator visible via MRI. Previous research has linked BML to pain, inflammation, and ligament pathology. The latter conditions have also been associated with intercondylar enthesophytes and third intercondylar tubercle of Parsons (TITP), both of which were observed in the intercondylar regions. SIGNIFICANCE: Several non-articular lesions not currently included in paleopathological measures of OA were consistently observed. SUGGESTIONS FOR FUTURE RESEARCH: A similar analysis of a control sample of non-OA tibial plateaus would better contextualize these results. LIMITATIONS: The sample's high average age (65.8 years) and severe OA stage may hamper generalizability to archaeological collections.

Mechanobiological osteocyte feedback drives mechanostat regulation of bone in a multiscale computational model.

Significant progress has been made to identify the cells and signaling molecules involved in the mechanobiological regulation of bone remodeling. It is now well accepted that osteocytes act as mechanosensory cells in bone expressing several signaling molecules such as nitric oxide (NO) and sclerostin (Scl) which are able to control bone remodeling responses. In this paper, we present a comprehensive multiscale computational model of bone remodeling which incorporates biochemical osteocyte feedback. The mechanostat theory is quantitatively incorporated into the model using mechanical feedback to control expression levels of NO and Scl. The catabolic signaling pathway RANK-RANKL-OPG is co-regulated via (continuous) PTH and NO, while the anabolic Wnt signaling pathway is described via competitive binding reactions between Wnt, Scl and the Wnt receptors LRP5/6. Using this novel model of bone remodeling, we investigate the effects of changes in the mechanical loading and hormonal environment on bone balance. Our numerical simulations show that we can calibrate the mechanostat anabolic and catabolic regulatory mechanisms so that they are mutually exclusive. This is consistent with previous models that use a Wolff-type law to regulate bone resorption and formation separately. Furthermore, mechanical feedback provides an effective mechanism to obtain physiological bone loss responses due to mechanical disuse and/or osteoporosis.

A wavelet gradient sparsity based algorithm for reconstruction of reduced-view tomography datasets obtained with a monochromatic synchrotron-based X-ray source.

High-resolution synchrotron computed tomography (CT) is very helpful in the diagnosis and monitor of chronic diseases including osteoporosis. Osteoporosis is characterized by low bone mass and cortical bone porosity best imaged with CT. Synchrotron CT requires a large number of angular projections to reconstruct images with high resolution for detailed and accurate diagnosis. However, this poses great risks and challenges for serial in-vivo human and animal imaging due to a large amount of X-ray radiation dose required that can damage living specimens. Also, longer scan times are associated with increased risk of specimen movement and motion artifact in the reconstructed images. We developed a wavelet-gradient sparsity based algorithm to be utilized as a synchrotron tomography reconstruction technique allowing accurate reconstruction of cortical bone porosity assessed for in-vivo preclinical study which significantly reduces the radiation dose and scan time required while maintaining satisfactory image resolution for diagnosis. The results of our study on a rat forelimb sample imaged in the Biomedical Imaging and Therapy Bending Magnet (BMIT-BM) beamline at the Canadian Light Source show that the proposed algorithm can produce satisfactory image quality with more than 50 percent X-ray dose reduction as indicated by both visual and quantitative-based performance.

Evaluating differential nuclear DNA yield rates and osteocyte numbers among human bone tissue types: A synchrotron radiation micro-CT approach.

Molecular human identification has conventionally focused on DNA sampling from dense, weight-bearing cortical bone tissue, typically from femora or tibiae. A comparison of skeletal elements from three contemporary individuals demonstrated that elements with high quantities of cancellous bone yielded nuclear DNA at the highest rates, suggesting that preferentially sampling cortical bone may be suboptimal (Mundorff & Davoren, 2014). Despite these findings, the reason for the differential DNA yields between cortical and cancellous bone tissues remains unknown. The primary goal of this work is to ascertain whether differences in bone microstructure can be used to explain differential nuclear DNA yield among bone tissue types observed by Mundorff and Davoren (2014), with a focus on osteocytes and the three-dimensional (3D) quantification of their associated lacunae. Osteocytes and other bone cells are recognized to house DNA in bone tissue, thus examining the density of their lacunae may explain why nuclear DNA yield rates differ among bone tissue types. Lacunae were visualized and quantified using synchrotron radiation-based micro-Computed Tomographic imaging (SR micro-CT). Volumes of interest (VOIs) from cortical and cancellous bone tissues (n=129) were comparatively analyzed from the three skeletons sampled for Mundorff and Davoren's (2014) study. Analyses tested the primary hypothesis that the abundance and density of osteocytes (inferred from their lacunar spaces) vary between cortical and cancellous bone tissue types. Results demonstrated that osteocyte lacunar abundance and density vary between cortical and cancellous bone tissue types, with cortical bone VOIs containing a higher lacunar abundance and density. We found that the osteocyte lacunar density values are independent of nuclear DNA yield, suggesting an alternative explanation for the higher nuclear DNA yields from bones with greater quantities of cancellous bone tissue. The use of SR micro-CT allowed for a scale of analysis that revealed a high range of variation in lacunar abundance in both tissue types. Moreover, high-resolution SR micro-CT imaging revealed potential soft tissue remnants within marrow spaces not visible macroscopically. It is hypothesized that soft tissue remnants observed among the trabeculae of skeletal elements with high quantities of cancellous bone tissue are responsible for the high nuclear DNA yields. These findings have significant implications for bone-sample selection for nuclear DNA analysis in a forensic context when skeletal remains are recovered from the ground surface.

Three-dimensional labeling of newly formed bone using synchrotron radiation barium K-edge subtraction imaging.

Bone is a dynamic tissue which exhibits complex patterns of growth as well as continuous internal turnover (i.e. remodeling). Tracking such changes can be challenging and thus a high resolution imaging-based tracer would provide a powerful new perspective on bone tissue dynamics. This is, particularly so if such a tracer can be detected in 3D. Previously, strontium has been demonstrated to be an effective tracer which can be detected by synchrotron-based dual energy K-edge subtraction (KES) imaging in either 2D or 3D. The use of strontium is, however, limited to very small sample thicknesses due to its low K-edge energy (16.105 keV) and thus is not suitable for in vivo application. Here we establish proof-of-principle for the use of barium as an alternative tracer with a higher K-edge energy (37.441 keV), albeit for ex vivo imaging at the moment, which enables application in larger specimens and has the potential to be developed for in vivo imaging of preclinical animal models. New bone formation within growing rats in 2D and 3D was demonstrated at the Biomedical Imaging and Therapy bending magnet (BMIT-BM) beamline of the Canadian Light Source synchrotron. Comparative x-ray fluorescence imaging confirmed those patterns of uptake detected by KES. This initial work provides a platform for the further development of this tracer and its exploration of applications for in vivo development.

Three-dimensional reconstruction of Haversian systems in human cortical bone using synchrotron radiation-based micro-CT: morphology and quantification of branching and transverse connections across age.

This study uses synchrotron radiation-based micro-computed tomography (CT) scans to reconstruct three-dimensional networks of Haversian systems in human cortical bone in order to observe and analyse interconnectivity of Haversian systems and the development of total Haversian networks across different ages. A better knowledge of how Haversian systems interact with each other is essential to improve understanding of remodeling mechanisms and bone maintenance; however, previous methodological approaches (e.g. serial sections) did not reveal enough detail to follow the specific morphology of Haversian branching, for example. Accordingly, the aim of the present study was to identify the morphological diversity of branching patterns and transverse connections, and to understand how they change with age. Two types of branching morphologies were identified: lateral branching, resulting in small osteon branches bifurcating off of larger Haversian canals; and dichotomous branching, the formation of two new osteonal branches from one. The reconstructions in this study also suggest that Haversian systems frequently target previously existing systems as a path for their course, resulting in a cross-sectional morphology frequently referred to as 'type II osteons'. Transverse connections were diverse in their course from linear to oblique to curvy. Quantitative assessment of age-related trends indicates that while in younger human individuals transverse connections were most common, in older individuals more evidence of connections resulting from Haversian systems growing inside previously existing systems was found. Despite these changes in morphological characteristics, a relatively constant degree of overall interconnectivity is maintained throughout life. Altogether, the present study reveals important details about Haversian systems and their relation to each other that can be used towards a better understanding of cortical bone remodeling as well as a more accurate interpretation of morphological variants of osteons in cross-sectional microscopy. Permitting visibility of reversal lines, synchrotron radiation-based micro-CT is a valuable tool for the reconstruction of Haversian systems, and future analyses have the potential to further improve understanding of various important aspects of bone growth, maintenance and health.

A sparsity-based iterative algorithm for reconstruction of micro-CT images from highly undersampled projection datasets obtained with a synchrotron X-ray source.

Synchrotron X-ray Micro Computed Tomography (Micro-CT) is an imaging technique which is increasingly used for non-invasive in vivo preclinical imaging. However, it often requires a large number of projections from many different angles to reconstruct high-quality images leading to significantly high radiation doses and long scan times. To utilize this imaging technique further for in vivo imaging, we need to design reconstruction algorithms that reduce the radiation dose and scan time without reduction of reconstructed image quality. This research is focused on using a combination of gradient-based Douglas-Rachford splitting and discrete wavelet packet shrinkage image denoising methods to design an algorithm for reconstruction of large-scale reduced-view synchrotron Micro-CT images with acceptable quality metrics. These quality metrics are computed by comparing the reconstructed images with a high-dose reference image reconstructed from 1800 equally spaced projections spanning 180°. Visual and quantitative-based performance assessment of a synthetic head phantom and a femoral cortical bone sample imaged in the biomedical imaging and therapy bending magnet beamline at the Canadian Light Source demonstrates that the proposed algorithm is superior to the existing reconstruction algorithms. Using the proposed reconstruction algorithm to reduce the number of projections in synchrotron Micro-CT is an effective way to reduce the overall radiation dose and scan time which improves in vivo imaging protocols.

3-D localization of non-radioactive strontium in osteoarthritic bone: Role in the dynamic labeling of bone pathological changes.

The study objective was to visualize regions of bone that undergo pathological mineralization and/or remodeling during pathogenesis of osteoarthritis, by employing non-radioactive strontium as a dynamic tracer of bone turnover. Post traumatic osteoarthritis was surgically induced in skeletally mature rats, followed by in vivo micro-CT imaging for 12 weeks to assess bone micro-structural changes. Rats either received strontium ranelate daily for the entire course of study or only last 10 days before euthanization. Distribution of strontium in bone was assessed in two and three dimensions, using electron probe micro-analysis (EPMA) and synchrotron dual energy K-edge subtraction micro-CT (SRµCT), respectively. Considerable early formation of osteophytes around the collateral ligament attachments and margins of articulating surfaces were observed, followed by subchondral sclerosis at the later stages. Accordingly, strontium was heavily incorporated by mineralizing osteophytes at 4, 8, and 12 weeks post-surgery, whereas subchondral bone only incorporated strontium between weeks 8-12.This study showed low dose stable strontium can effectively serve as a dynamic tracer of bone turnover to study pathological bone micro-structural changes, at resolution higher than nuclear medicine. Co-administration of strontium during therapeutic drug intervention may show enormous utility in assessing the efficacy of those compounds upon adaptive bone physiology.

Variation in osteocyte lacunar morphology and density in the human femur--a synchrotron radiation micro-CT study.

In recent years there has been growing interest in the spatial properties of osteocytes (including density and morphology) and how these potentially relate to adaptation, disease and aging. This interest has, in part, arisen from the availability of increasingly high-resolution 3D imaging modalities such as synchrotron radiation (SR) micro-CT. As resolution increases, field of view generally decreases. Thus, while increasingly detailed spatial information is obtained, it is unclear how representative this information is of the skeleton or even the isolated bone. The purpose of this research was to describe the variation in osteocyte lacunar density, morphology and orientation within the femur from a healthy young male human. Multiple anterior, posterior, medial and lateral blocks (2 mm × 2 mm) were prepared from the proximal femoral shaft and SR micro-CT imaged at the Advanced Photon Source. Average lacunar densities (± standard deviation) from the anterior, posterior, medial and lateral regions were 27,169 ± 1935, 26,3643 ± 1262, 37,521 ± 6416 and 33,972 ± 2513 lacunae per mm(3) of bone tissue, respectively. These values were significantly different between the medial and both the anterior and posterior regions (p<0.05). The density of the combined anterior and posterior regions was also significantly lower (p=0.001) than the density of the combined medial and lateral regions. Although no difference was found in predominant orientation, shape differences were found; with the combined anterior and posterior regions having more elongated (p=0.004) and flattened (p=0.045) lacunae, than those of the medial and lateral regions. This study reveals variation in osteocyte lacunar density and morphology within the cross-section of a single bone and that this variation can be considerable (up to 30% difference in density between regions). The underlying functional significance of the observed variation in lacunar density likely relates to localized variations in loading conditions as the pattern corresponds well with mechanical axes. Lower density and more elongate shapes being associated with the antero-posterior oriented neutral axis. Our findings demonstrate that the functional and pathological interpretations that are increasingly being drawn from high resolution imaging of osteocyte lacunae need to be better situated within the broader context of normal variation, including that which occurs even within a single skeletal element.

Degenerative knee joint disease in mice lacking 3'-phosphoadenosine 5'-phosphosulfate synthetase 2 (Papss2) activity: a putative model of human PAPSS2 deficiency-associated arthrosis.

OBJECTIVE: Murine brachymorphism (bm) results from an autosomal recessive mutation of the Papss2 gene that encodes 3'-phosphoadenosine 5'-phosphosulfate synthetase 2, one of the principal enzymes required for the sulfation of extracellular matrix molecules in cartilage and other tissues. A spondyloepimetaphyseal dysplasia has been identified in Pakistani kindred having a mutation of PAPSS2. In addition to skeletal malformations that include short stature evident at birth due to limb shortening, brachydactyly, and kyphoscoliosis, affected individuals demonstrate premature onset degenerative joint disease. We investigated whether loss of Papss2 activity would similarly lead to degenerative joint disease in mice. METHODS: Mice carrying the bm mutation on a C57BL/6 background were obtained from the Jackson Laboratory. Limbs were analyzed by micro-computed tomography (microCT) and histology. RESULTS: At 12 months of age both male and female bm mice exhibited severe degenerative knee joint disease, with cartilage damage being primarily evident in the patello-femoral and medial compartments. Control 12-14-month-old C57BL/6 mice, in contrast, only occasionally demonstrated minimal cartilage damage. muCT imaging of bm limbs revealed shortened diaphyses associated with flared metaphyses in the proximal elements of both fore and hind limbs. Additionally, the bm hind limbs demonstrated extensive structural alterations, characterized by distortion of the patello-femoral groove, and prominent bowing of both tibia and fibula. CONCLUSIONS: The bm mutant, which develops severe articular cartilage lesions of the knee joint by approximately 12 months of age, represents a novel example of murine degenerative joint disease, possibly representing a model of human PAPSS2 deficiency-associated arthrosis.