Monochromatic computed microtomography using laboratory and synchrotron sources and X-ray fluorescence analysis for comprehensive analysis of structural changes in bones

A combination of X-ray tomography at different wavelengths and X-ray fluorescence analysis was applied in the study of two types of bone tissue changes: prolonged presence in microgravity conditions and age-related bone growth. The proximal tail vertebrae of geckos were selected for investigation because they do not bear the supporting load in locomotion, which allows them to be considered as an independent indicator of gravitational influence. For the vertebrae of geckos no significant differences were revealed in the elemental composition of the flight samples and the synchronous control samples. In addition, the gecko bone tissue samples from the jaw apparatus, spine and shoulder girdle were measured. The dynamics of structural changes in the bone tissue growth was studied using samples of a human fetal hand. The hands of human fetuses of 11–15 weeks were studied. Autonomous zones of calcium accumulation were found not only in individual fingers but in each of the investigated phalanges. The results obtained are discussed

Multiparametric optical bioimaging reveals the fate of epoxy crosslinked biomeshes in the mouse subcutaneous implantation model

Biomeshes based on decellularized bovine pericardium (DBP) are widely used in reconstructive surgery due to their wide availability and the attractive biomechanical properties. However, their efficacy in clinical applications is often affected by the uncontrolled immunogenicity and proteolytic degradation. To address this issue, we present here in vivo multiparametric imaging analysis of epoxy crosslinked DBPs to reveal their fate after implantation. We first analyzed the structure of the crosslinked DBP using scanning electron microscopy and evaluated proteolytic stability and cytotoxicity. Next, using combination of fluorescence and hypoxia imaging, X-ray computed microtomography and histology techniques we studied the fate of DBPs after subcutaneous implantation in animals. Our approach revealed high resistance to biodegradation, gradual remodeling of a surrounding tissue forming the connective tissue capsule and calcification of crosslinked DBPs. These changes were concomitant to the development of hypoxia in the samples within 3 weeks after implantation and subsequent induction of angiogenesis and vascularization. Collectively, presented approach provides new insights on the transplantation of the epoxy crosslinked biomeshes, the risks associated with its applications in soft-tissue reconstruction and can be transferred to studies of other types of implants

X-ray computed tomography

X-ray computed tomography (CT) can reveal the internal details of objects in three dimensions non-destructively. In this Primer, we outline the basic principles of CT and describe the ways in which a CT scan can be acquired using X-ray tubes and synchrotron sources, including the different possible contrast modes that can be exploited. We explain the process of computationally reconstructing three-dimensional (3D) images from 2D radiographs and how to segment the 3D images for subsequent visualization and quantification. Whereas CT is widely used in medical and heavy industrial contexts at relatively low resolutions, here we focus on the application of higher resolution X-ray CT across science and engineering. We consider the application of X-ray CT to study subjects across the materials, metrology and manufacturing, engineering, food, biological, geological and palaeontological sciences. We examine how CT can be used to follow the structural evolution of materials in three dimensions in real time or in a time-lapse manner, for example to follow materials manufacturing or the in-service behaviour and degradation of manufactured components. Finally, we consider the potential for radiation damage and common sources of imaging artefacts, discuss reproducibility issues and consider future advances and opportunities

Multimodal Imaging of Silver Nanoclusters

Recent developments in Nanobiotechnology have given rise to a novel brand of fluorescent labels, fluorescent metal nanoclusters, e.g., gold and silver nanoclusters. Generally, high atomic number elements such as silver can attenuate more X-ray and consider as label in X-ray microtomography. Features such as ultra-small size, good biocompatibility, non-toxicity and photo-stability made nanoclusters more attractive as a fluorescent label than conventional fluorophores dye in biological imaging. The core concept of this thesis is to analyze silver nanoclusters as contrast agent by the multimodal imaging approaches of X-ray microtomography (MicroCT) and Optical Projection Tomography (OPT). To estimate the absorption and relation of X-ray and fluorescent signal by different concentrations of silver nanoclusters in samples. AgNCs-Agar with different concentrations of AgNCs, diluted with agar and water and filter paper coated with silver nanoclusters with different dipping time were studied in this work. The imaging implementation divided into three parts: 1. MicroCT imaging of samples (both AgNC-Agar and filter paper), 2. Optical imaging of AgNC-Agar samples by both fluorescent and bright-field modes. 3. MicroCT imaging of samples which were imaged by OPT first. Afterward, quantitative approach employed to both microCT and optical images to evaluate the relation between X-ray energy and light intensity with different concentrations of AgNCs to assess the amount of X-ray and light absorption by samples. Ideally, higher ratio of AgNCs revealed brighter microCT images due to more X-ray absorption. In sum, our results showed that the tested silver nanoclusters can be used as a label in both X-ray microtomography and fluorescent OPT since they show the contrast in X-ray and optical images. Moreover, depicted graphs demonstrate the linear correlation between data from images of both modalities and different amounts of silver material

X-ray and neutron μCT of biomedical samples: from image acquisition to quantification

Even though the validity of x-ray computed tomography in the analysis of biomedical samples is nowadays undisputed, the more recent imaging techniques and more advanced instruments (such as synchrotrons) are still relatively unknown to many medical doctors that could benefit from them.The doctoral work presented in this thesis joins a collective effort from the imaging community to demonstrate potential applications of advanced x-ray and neutron imaging methods to preclinical medical research, with the hope of contributing to reach a “critical mass” in the medical community and in the public opinion as well.Two main lines of work are detailed, one focused on the ex vivo evaluation of corrosion processes of magnesium-based biodegradable implants for osteosynthesis, the other dedicated to the assessment of neuropathy in human gastroenteric dysmotility. The aimed endpoint was to develop pipelines, from image acquisition all the way to data quantification, that could be used by other research groups with similar questions and may inspire future interdisciplinary collaborations between medicine, natural science and engineering.In the first line of work, we have attempted to employ synchrotron-radiation micro-computed tomography (µCT) coupled with in situ loading tests to assess the mechanical properties of the bone-implant interface (Paper I). We have revealed the crucial importance of the radiation dose deposited on the sample, and that the mechanical loading geometry should be accurately determined in the planning steps of the experiment. Moving away from the mechanical testing, we have also explored a novel three-dimensional analysis of the corrosion by-products of biodegradable implants by combining x-ray µCT, neutron µCT and x-ray fluorescence mapping (Papers IV and V). The second line of work has assessed the potential of x-ray phase-contrast µCT and nano-resolution holotomography as ways to perform virtual histology of unstained peripheral and autonomic neural tissue. In full-thickness biopsies of the myenteric nervous system, qualitative and potentially quantitative differences have been shown between controls and patients affected by gastrointestinal dysmotility (Paper II). In unstained skin biopsies, the methods have failed to visualise peripheral nerves, but we could identify structural changes in the connective tissue of some patients when compared to controls and other patients (Paper III)

Synchrotron-based characterization of mechanobiological effects on the nanoscale in musculoskeletal tissues

Collagen is the main organic building block of musculoskeletal tissues. Despite collagen being their smallest load bearing unit, these tissues differ significantly in mechanical function and properties. A major factor behind these differences is their hierarchical organization, from the collagen molecule up to the organ scale. It is thus of high importance to understand the characteristics of each level, as well as how they interact and relate to each other. With such knowledge, improved prevention and rehabilitation of musculoskeletal pathologies may be achieved.Both mineralized and soft collagenous tissues respond to their mechanical loading environment according to specific mechanobiological principles. During prenatal development, immobilization can cause dramatic effects on the developing skeleton, causing the newly formed bones to be smaller, deformed and more prone to fracture. But how immobilization affects the deposition, structure and composition of the developing bones is still unclear. In tendons, both insufficient and excessive mechanical loading increases the risk of injury. After rupture, reduced mechanical loading results in altered collagen structure and cell activity, thus influencing the mechanical properties of the healing tendon. How the loading environment affects the structure of intact and ruptured tendons is still debated.The work presented in this thesis aims to thoroughly characterize the mechanobiological effects on the mineralization process in developing bones as well as the collagen structure and multiscale mechanical response of intact and healing tendons. This is achieved through a multimodal approach including a range of high-resolution synchrotron- and lab-based techniques, in combination with mechanical testing.In the first part of the thesis, humeri from “muscle-less” embryonic mice and their healthy littermates at development stages from start of mineralization to shortly before birth were investigated. The multimodal approach revealed a highly localized spatial pattern of Zinc during normal development to sites of ongoing mineralization, accompanied by larger mineral particles. Healthy bones also showed signs of remodeling at later time points. In the absence of skeletal muscle, it was revealed that the developing bones exhibited a delayed but increased mineral deposition and growth, with no signs of remodeling.In the second part of the thesis, intact Achilles tendons from rats subjected to either full in vivo loading through free cage activity or unloading by Botox injections combined with cast immobilization were investigated. It was shown that the nanoscale fibrils in the Achilles tendon respond to the applied tissue loads and exhibit viscoelastic responses. It was revealed that in vivo unloading results in a more disorganized microstructure and an impaired viscoelastic response. Unloading also altered the nanoscale fibril mechanical response, possibly through alterations in the strain partitioning between hierarchical levels.In the third part of the thesis, Achilles tendons were transected and allowed to heal while subjected to either full in vivo loading, reduced loading through Botox injections or unloading. In vivo unloading during the early healing process resulted in a delayed and more disorganized collagen structure and a larger presence of adipose tissue. Unloading also delayed the remodeling of the stumps as well as callus maturation. Additionally, the nanoscale fibril mechanical response was altered, with unloaded tendons exhibiting a low degree of fibril recruitment as well as a decreased ability for fibril extension.The work in this thesis further illustrates the important role of the mechanical environment on the nanostructure of musculoskeletal tissues. It also highlights the power of combining high-resolution tissue characterization techniques into a multimodal and multiscale approach, allowing us to study the effects on several hierarchical length scales simultaneously and as a result be able to elucidate the intricate connection between hierarchical scales

Development of 3D-Printed Cartilage Constructs and Their Non-Invasive Assessment by Synchrotron-Based Inline-Phase Contrast Imaging Computed Tomography

One goal of cartilage tissue engineering (CTE) is to create constructs for regeneration of hyaline cartilage. Three-dimensional (3D)-printed cartilage constructs fabricated from polycaprolactone (PCL) and chondrocyte-impregnated alginate mimic the biphasic nature of articular cartilage and offers great promise for CTE applications. However, ensuring that these constructs provide biologically conducive environment and mechanical support for cellular activities and articular cartilage regeneration is still a challenge. That said, the regulatory pathway for medical device development requires validation of implants such as these through in vitro bench test and in vivo preclinical examination prior to their premarket approval. Furthermore, mechano-transduction and secretion of cartilage-specific ECM are influenced by mechanical stimuli directed at chondrocytes. Thus, ensuring that these cartilage constructs have mechanical properties similar to that of human articular cartilage is crucial to their success. Non-invasive imaging techniques are required for effective evaluation of progression of these cartilage constructs. However, current non-invasive techniques cannot decipher components of the cartilage constructs, nor their time-dependent structural changes, because they contain hydrophobic and hydrophilic biomaterials with different X-ray refractive indices. The aims of this thesis were to develop 3D-printed cartilage constructs that biologically and mechanically mimic human articular cartilage and to investigate synchrotron radiation inline phase contrast computed tomography (SR-inline-PCI-CT) as a non-invasive imaging technique to characterize components of these constructs and associated time-dependent structural changes. The first objective was to determine in vitro biological functionality of the cartilage constructs over a 42-day period with regards to cell viability and secretion of extracellular matrix by traditional invasive assays. In parallel, performance of SR-inline-PCI-CT for non-invasive visualization of components and associated structural changes within the constructs in vitro over a 42-day was examined. To achieve this objective, three sample-to-detector distances (SDDs): 0.25 m, 1 m and 3 m were investigated. Then, the optimal SDD with better phase contrast and edge enhancement fringes for characterization of the multiple refractive indices within the constructs was utilized to visualize their structural changes over a 42-day culture period. Like the first objective, the second objective was to examine in vivo biological functionality of the cartilage constructs by traditional invasive assays and utilize SR-inline-PCI-CT to non-invasively visualize components of the hybrid cartilage constructs over a 21-day period post-implantation in mice. The third objective was to modulate mechanical properties of PCL framework of the 3D-printed PCL-based cartilage constructs to mimic mechanical properties of human articular cartilage. To achieve this, effect of modulation of PCL's molecular weight (MW) and scaffold's pore geometric configurations: strand size (SZ), strand spacing (SS), and strand orientation (SO), on mechanical properties of 3D-printed PCL scaffolds were studied. Then, regression models showing the effect of SZ, SS, and SO on porosity, tensile moduli and compressive moduli of scaffold were developed. Compressive and tensile properties of these scaffolds were compared with those of human articular cartilage. Then, “modulated PCL scaffolds” with mechanical and biomimetic properties that better mimic human articular cartilage was identified and recommended for fabrication of PCL-based cartilage constructs. This thesis demonstrated effective in vitro and in vivo biological performance of the 3D-printed hybrid cartilage constructs studied and presented a significant advancement in CTE applications. To be precise, cell viability was at a minimum of 77 % and secretion of sulfated GAGs and Col2 increased progressively within cartilage constructs over a 42-day in vitro. Similarly, cell viability was consistently above 70 %, and secretion of sulfated GAGs and Col2 increased post-implantation of constructs in mice over a 21-day period. Furthermore, SR-inline-PCI-CT demonstrated phase contrast and edge-enhancement fringes effective for visualization of the different components and subtle variations within the biphasic cartilage constructs, and thus, offers great potential for their non-invasive and three-dimensional visualization. Lastly, this thesis presented a significant advancement towards development of PCL constructs with mechanical behavior that mimic that of human articular cartilage. The statistical regression models developed showed the effect of SZ, SS, and SO on porosity, tensile moduli and compressive moduli of scaffolds and recommended sets of parameters for fabrication of “modulated PCL scaffolds” with mechanical properties that better mimic mechanical behavior of human articular cartilage. These “modulated PCL scaffolds” could serve as a better framework and could guide more effective secretion of cartilage-specific ECM within PCL-based constructs for CTE applications

Recent developments in X-ray diffraction/scattering computed tomography for materials science

X-ray diffraction/scattering computed tomography (XDS-CT) methods are a non-destructive class of chemical imaging techniques that have the capacity to provide reconstructions of sample cross-sections with spatially resolved chemical information. While X-ray diffraction CT (XRD-CT) is the most well-established method, recent advances in instrumentation and data reconstruction have seen greater use of related techniques like small angle X-ray scattering CT and pair distribution function CT. Additionally, the adoption of machine learning techniques for tomographic reconstruction and data analysis are fundamentally disrupting how XDS-CT data is processed. The following narrative review highlights recent developments and applications of XDS-CT with a focus on studies in the last five years. This article is part of the theme issue 'Exploring the length scales, timescales and chemistry of challenging materials (Part 2)'