Morphological characterisation of unstained and intact tissue microarchitecture by x-ray computed micro- and nano-tomography

Characterisation and quantification of tissue structures is limited by sectioning-induced artefacts and by the difficulties of visualising and segmenting 3D volumes. Here we demonstrate that, even in the absence of X-ray contrast agents, X-ray computed microtomography (microCT) and nanotomography (nanoCT) can circumvent these problems by rapidly resolving compositionally discrete 3D tissue regions (such as the collagen-rich adventitia and elastin-rich lamellae in intact rat arteries) which in turn can be segmented due to their different X-ray opacities and morphologies. We then establish, using X-ray tomograms of both unpressurised and pressurised arteries that intra-luminal pressure not only increases lumen cross-sectional area and straightens medial elastic lamellae but also induces profound remodelling of the adventitial layer. Finally we apply microCT to another human organ (skin) to visualise the cell-rich epidermis and extracellular matrix-rich dermis and to show that conventional histological and immunohistochemical staining protocols are compatible with prior X-ray exposure. As a consequence we suggest that microCT could be combined with optical microscopy to characterise the 3D structure and composition of archival paraffin embedded biological materials and of mechanically stressed dynamic tissues such as the heart, lungs and tendons

Multiscale Quantitative Imaging of Human Femoral Heads Using X-ray Microtomography

PhDClinical diagnostic tools provide limited information on the underlying structural and mechanical properties of bone-tissue affected by degenerative and bone metabolic diseases. In-vivo bone failure studies provide limited information due to constraints such as X-ray dosage, cost and various other practicalities. In-vitro studies are thus required to enhance understanding of this phenomenon. The aims of this study were to use quantitative high-definition X-ray Micro-Tomography (XMT) to assess factors contributing to pathological and non-pathological bone failure and repair in relation to the mechanics of whole human femoral heads. XMT images of one normal and six pathological femoral heads were collected at 26 – 8.8 μm voxel resolution and evaluated to determine structural features; bone mineral concentration (BMC); and using image analysis, identify microcallus formations. In addition, in-vitro compression tests were carried out on specimens taken from regions with different anatomical loading. Bone quality was then related to the anatomical loading and BMC. Results from non-pathological tissue where used to establish a baseline for measurements of structural features. Microcallus formations where identified and used as markers to map the occurrence of bone damage. In osteoarthritic (OA) heads, the damage was found to be concentrated in localised clusters. Conversely, in the osteoporotic head damage was distributed homogeneously throughout the entire specimen. No significant difference in the BMC was observed, however there was a iii significant difference in the bone quality values between the non-pathological and pathological heads, and also between the pathologies. In-vitro mechanical testing revealed a difference in the mechanical properties of OA trabecular bone in relation to bone quality measurements but the samples exhibited no significant correlation to anatomical loading. X-ray Ultra Microscopy (XuM) at 200nm and 775nm voxel resolution was used to investigate the nano-morphology of individual trabeculae. The XuM images showed differences in bone structure and fewer osteocyte lacunae present close to fracture site. XuM also identified micro-cracks within trabeculae that were encased by microcallus formations. The application of novel quantitative high definition X-ray imaging to clinically relevant tissue at multiple length scales has provided new metrological data on the distribution of damage within pathological tissue. Insight into the vulnerability of diseased tissue to damage could ultimately lead to improved diagnosis from clinical radiographs

X-ray microtomography study of carious dentine and a comparison of its removal by three techniques

The wisdom on mechanical removal of carious dentine based on hardness has been challenged and chemo-mechanical technique has been proposed as a more conservative method. However, the extent and comparison of excessive removal of sound dentine and insufficient removal of carious dentine have not been studied. The aims of the present study are to use X-ray microtomography (XMT) to determine the mineral concentrations of sound and carious dentine, and the excavated dentine using a hand excavation (HE) technique, a hand excavation technique aided by Caries Detector Dye (CD) and a chemo-mechanical removal technique using Carisolv (CSS). Comparison of the three techniques with respect to the mineral concentrations of the excavated dentine caries and the volumes of sound dentine removed were investigated. It was aimed to identify the boundary between ―infected‖ and ―affected‖ using the X-ray linear attenuation coefficients (LAC) from the XMT results and the ultrastructural images obtained from the Back Scattered Electron (BSE) imaging and Atomic Force Microscopy (AFM). Thirty eight deciduous molars with open carious cavities were sectioned in half. One half of each molar had the carious tissue removed by HE and the other by CSS or hand excavation plus CD. XMT images were taken before and after caries removal. From the data set, an assessor, who was ‗blind‘ to which technique was used, used LAC histograms to assess the efficacy of the excavation techniques. The volumes of sound dentine removed by the 3 techniques were calculated and compared. Detailed analyses were carried out using XMT slices to investigate the mineral concentration of removed and residual dentine. Remineralisation experiments of residual dentine were performed 7 after caries removal. Backscattered electron (BSE) microscopy and atomic force microscopy (AFM) were used to investigate the ultrastructure of the carious dentine. The results showed that CSS was a better technique; conforming to the principles of minimally invasive dentistry. Volume analyses showed that both CSS and CD were effective in removing less sound dentine than conventional hand excavation. It was shown for the first time that the partially demineralised layer of dentine in a natural carious lesion, which was maintained by the CSS technique, had the potential to remineralise up to 80% of the mineral level for sound dentine. Combining XMT results with AFM and BSE images, ultrastructural changes were found at the boundary around a LAC value of 0.8 cm-1 which corresponded to a KHN of 7.66 kgmm-2. It was concluded that carious dentine removal up to a hardness level of 7.66 kgmm-2 could be recommended in order to preserve dentine that has potential to remineralise

Towards A 4-D Spatial and Temporal Model of Human Enamel Biomineralisation

PhDPrecise timings and spatial progression of human enamel biomineralisation are still largely unknown due to scarcity of developing human enamel specimens available for investigation. This information is crucial for optimising emerging biomimetic regenerative and reparative dentistry routes. Five developing permanent incisors were obtained from an archaeological source and used alongside mature contemporary teeth for comparison. X-ray microtomography (XMT), synchrotron X-ray diffraction (S-XRD) and quantitative back-scattered electrons (qBSE) imaging were used to investigate the mineral density distribution, the crystallites texture magnitude and orientation and the nanostructure of dental enamel at various developmental stages, respectively. XMT revealed that there was a bi-directional mineralisation "front" that starts at the cusp tip and at the enamel-dentine junction (EDJ) travelling cervically and peripherally until the relative mineral density is uniform in the fully mature tooth (2.75 g/cm3 ± 0.01 g/cm3). S-XRD revealed that within one probed region, two populations of crystallite orientations exist simultaneously with an angular separation of 20-50°, with one population being more dominant than the other by a factor of approximately 3:7. Furthermore, one population displayed a higher degree of crystallite texture than the other. These phenomena were observed in all stages of tooth development. The crystallites in both populations were oriented approximately perpendicular to the EDJ regardless of development stage, indicating initial preferred directions of crystallites persist from early through to full maturation. The direction and magnitude of organisation within two distinct populations of crystallites within the developing and mature enamel has not been quantified previously. qBSE analyses suggested that the two observed populations are most likely due to prism decussation and revealed that mineralisation of prism cores precedes that of prism boundaries. These results provide new insights towards building a quantitative spatio-temporal model of human enamel biomineralisation in order to inform emerging biomimetic reparative/regenerative dental technologies

A study of the influence of combined Glucosamine Sulfate and Chondroitin Sulfate systemic supplements on root resorption and tooth movement in rats

Orthodontic tooth movement is a periodontal ligament and alveolar bone phenomenon that involves microscopic and macroscopic changes in the periodontal ligament, alveolar bone and dental pulp. Root resorption is recognized as an unavoidable side effect of orthodontic tooth movement and numerous studies have been conducted in order to identify potential risk factors and possible methods to reduce or prevent this unwanted and often devastating side effect while maintaining or even improving the rate of tooth movement

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