I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[25602]
Open Access
Abstract: The bone-cartilage unit (BCU) is a universal feature in diarthrodial joints, which is mechanically-graded and subjected to shear and compressive strains. Changes in the BCU have been linked to osteoarthritis (OA) progression. Here we report existence of a physiological internal strain gradient (pre-strain) across the BCU at the ultrastructural scale of the extracellular matrix (ECM) constituents, specifically the collagen fibril. We use X-ray scattering that probes changes in the axial periodicity of fibril-level D-stagger of tropocollagen molecules in the matrix fibrils, as a measure of microscopic pre-strain. We find that mineralized collagen nanofibrils in the calcified plate are in tensile pre-strain relative to the underlying trabecular bone. This behaviour contrasts with the previously accepted notion that fibrillar pre-strain (or D-stagger) in collagenous tissues always reduces with mineralization, via reduced hydration and associated swelling pressure. Within the calcified part of the BCU, a finer-scale gradient in pre-strain (0.6% increase over ~50μm) is observed. The increased fibrillar pre-strain is linked to prior research reporting large tissue-level residual strains under compression. The findings may have biomechanical adaptative significance: higher in-built molecular level resilience/damage resistance to physiological compression, and disruption of the molecular-level pre-strains during remodelling of the bone-cartilage interface may be potential factors in osteoarthritis-based degeneration.
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Sep 2022
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I13-2-Diamond Manchester Imaging
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Diamond Proposal Number(s):
[10315, 16497, 22575]
Open Access
Abstract: Musculoskeletal tissues are complex hierarchical materials where mechanical response is linked to structural and material properties at different dimensional levels. Therefore, high-resolution three-dimensional tomography is very useful for assessing tissue properties at different scales. In particular, Synchrotron Radiation micro-Computed Tomography (SR-microCT) has been used in several applications to analyze the structure of bone and biomaterials. In the past decade the development of digital volume correlation (DVC) algorithms applied to SR-microCT images and its combination with in situ mechanical testing (four-dimensional imaging) have allowed researchers to visualise, for the first time, the deformation of bone tissue and its interaction with biomaterials under different loading scenarios. However, there are several experimental challenges that make these measurements difficult and at high risk of failure. Challenges relate to sample preparation, imaging parameters, loading setup, accumulated tissue damage for multiple tomographic acquisitions, reconstruction methods and data processing. Considering that access to SR-microCT facilities is usually associated with bidding processes and long waiting times, the failure of these experiments could notably slow down the advancement of this research area and reduce its impact. Many of the experimental failures can be avoided with increased experience in performing the tests and better guidelines for preparation and execution of these complex experiments; publication of negative results could help interested researchers to avoid recurring mistakes. Therefore, the goal of this article is to highlight the potential and pitfalls in the design and execution of in situ SR-microCT experiments, involving multiple scans, of musculoskeletal tissues for the assessment of their structural and/or mechanical properties. The advice and guidelines that follow should improve the success rate of this type of experiment, allowing the community to reach higher impact more efficiently.
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May 2022
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I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[18524]
Open Access
Abstract: Fibrotic scarring is prevalent in a range of collagenous tissue disorders. Understanding the role of matrix biophysics in contributing to fibrotic progression is important to develop therapies, as well as to elucidate biological mechanisms. Here, we demonstrate how microfocus small-angle X-ray scattering (SAXS), with in situ mechanics and correlative imaging, can provide quantitative and position-resolved information on the fibrotic matrix nanostructure and its mechanical properties. We use as an example the case of keloid scarring in skin. SAXS mapping reveals heterogeneous gradients in collagen fibrillar concentration, fibril pre-strain (variations in D-period) and a new interfibrillar component likely linked to proteoglycans, indicating evidence of a complex 3D structure at the nanoscale. Furthermore, we demonstrate a proof-of-principle for a diffraction-contrast correlative imaging technique, incorporating, for the first time, DIC and SAXS, and providing an initial estimate for measuring spatially resolved fibrillar-level strain and reorientation in such heterogeneous tissues. By application of the method, we quantify (at the microscale) fibrillar reorientations, increases in fibrillar D-period variance, and increases in mean D-period under macroscopic tissue strains of ~20%. Our results open the opportunity of using synchrotron X-ray nanomechanical imaging as a quantitative tool to probe structure–function relations in keloid and other fibrotic disorders in situ.
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Mar 2022
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I13-2-Diamond Manchester Imaging
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Diamond Proposal Number(s):
[22575]
Abstract: Machine learning methods have the potential to transform imaging techniques and analysis for healthcare applications with automation, making diagnostics and treatment more accurate and efficient, as well as to provide mechanistic insights into tissue deformation and fracture in physiological and pathological conditions. Here we report an exploratory investigation for the classification and prediction of mechanical states of cortical and trabecular bone tissue using convolutional neural networks (CNNs), residual neural networks (ResNet), and transfer learning applied to a novel dataset derived from high-resolution synchrotron-radiation micro-computed tomography (SR-microCT) images acquired in uniaxial continuous compression in situ. We present the systematic optimization of CNN architectures for classification of this dataset, visualization of class-defining features detected by the CNNs using gradient class activation maps (Grad-CAMs), comparison of CNN performance with ResNet and transfer learning models, and perhaps most critically, the challenges that arose from applying machine learning methods to an experimentally-derived dataset for the first time. With optimized CNN architectures, we obtained trained models that classified novel images between failed and pristine classes with over 98% accuracy for cortical bone and over 90% accuracy for trabecular bone. Harnessing a pre-trained ResNet with transfer learning, we further achieved over 98% accuracy on the cortical dataset, and 99% on the trabecular dataset. This demonstrates that powerful classifiers for high-resolution SR-microCT images can be developed even with few unique training samples and invites further development through the inclusion of more data and training methods to move towards novel, fundamental, and machine learning-driven insights into microstructural states and properties of bone.
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Aug 2021
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I13-2-Diamond Manchester Imaging
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Diamond Proposal Number(s):
[22575, 20132]
Abstract: Digital volume correlation (DVC) in combination with high-resolution micro-computed tomography (microCT) imaging and in situ mechanical testing is gaining popularity for quantifying 3D full-field strains in bone and biomaterials. However, traditional in situ time-lapsed (i.e., interrupted) mechanical testing cannot fully capture the dynamic strain mechanisms in viscoelastic biological materials. The aim of this study was to investigate the time-resolved deformation of bone structures and analogues via continuous in situ synchrotron-radiation microCT (SR-microCT) compression and DVC to gain a better insight into their structure-function relationships. Fast SR-microCT imaging enabled the deformation behaviour to be captured with high temporal and spatial resolution. Time-resolved DVC highlighted the relationship between local strains and damage initiation and progression in the different biostructures undergoing plastic deformation, bending and/or buckling of their main microstructural elements. The results showed that SR-microCT continuous mechanical testing complemented and enhanced the information obtained from time-lapsed testing, which may underestimate the 3D strain magnitudes as a result of the stress relaxation occurring in between steps before image acquisition in porous biomaterials. Altogether, the findings of this study highlight the importance of time-resolved in situ experiments to fully characterise the time-dependent mechanical behaviour of biological tissues and biomaterials and to further explore their micromechanics under physiologically relevant conditions.
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Jun 2021
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I13-2-Diamond Manchester Imaging
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Diamond Proposal Number(s):
[22575]
Open Access
Abstract: As a composite material, the mechanical properties of bone are highly dependent on its hierarchical organisation, thus, macroscopic mechanical properties are dictated by local phenomena, such as microdamage resulting from repetitive cyclic loading of daily activities. Such microdamage is associated with plastic deformation and appears as a gradual accumulation of residual strains. The aim of this study is to investigate local residual strains in cortical bone tissue following compressive cyclic loading, using in situ X-ray computed tomography (XCT) and digital volume correlation (DVC) to provide a deeper insight on the three-dimensional (3D) relationship between residual strain accumulation, cortical bone microstructure and failure patterns. Through a progressive in situ XCT loading-unloading scheme, localisation of local residual strains was observed in highly compressed regions. In addition, a multi-scale in situ XCT cyclic test highlighted the differences on residual strain distribution at the microscale and tissue level, where high strains were observed in regions with the thinnest vascular canals and predicted the failure location following overloading. Finally, through a continuous in situ XCT compression test of cycled specimens, the full-field strain evolution and failure pattern indicated the reduced ability of bone to plastically deform after damage accumulation due to high number of cyclic loads. Altogether, the novel experimental methods employed in this study, combining high-resolution in situ XCT mechanics and DVC, showed a great potential to investigate 3D full-field residual strain development under repetitive loading and its complex interaction with bone microstructure, microdamage and fracture.
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Oct 2020
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I13-2-Diamond Manchester Imaging
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Diamond Proposal Number(s):
[14080]
Abstract: Osteoregenerative biomaterials for the treatment of bone defects are under much development, with the aim of favouring osteointegration up to complete bone regeneration. A detailed investigation of bone-biomaterial integration is vital to understanding and predicting the ability of such materials to promote bone formation, preventing further bone damage and supporting load-bearing regions. This study aims to characterise the ex vivo micromechanics and microdamage evolution of bone-biomaterial systems at the tissue level, combining high resolution synchrotron micro-computed tomography, in situ mechanics and digital volume correlation. Results showed that the main microfailure events were localised close to or within the newly formed bone tissue, in proximity to the bone-biomaterial interface. The apparent nominal compressive load applied to the composite structures resulted in a complex loading scenario, mainly due to the higher heterogeneity but also to the different biomaterial degradation mechanisms. The full-field strain distribution allowed characterisation of microdamage initiation and progression. The findings reported in this study provide a deeper insight into bone-biomaterial integration and micromechanics in relation to the osteoregeneration achieved in vivo, for a variety of biomaterials. This could ultimately be used to improve bone tissue regeneration strategies.
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Apr 2019
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I13-2-Diamond Manchester Imaging
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Diamond Proposal Number(s):
[16497]
Open Access
Abstract: Digital volume correlation (DVC), combined with in situ synchrotron microcomputed tomography (SR-microCT) mechanics, allows for 3D full-field strain measurement in bone at the tissue level. However, long exposures to SR radiation are known to induce bone damage, and reliable experimental protocols able to preserve tissue properties are still lacking. This study aims to propose a proof-of-concept methodology to retain bone tissue integrity, based on residual strain determination using DVC, by decreasing the environmental temperature during in situ SR-microCT testing. Compact and trabecular bone specimens underwent five consecutive full tomographic data collections either at room temperature or 0 °C. Lowering the temperature seemed to reduce microdamage in trabecular bone but had minimal effect on compact bone. A consistent temperature gradient was measured at each exposure period, and its prolonged effect over time may induce localised collagen denaturation and subsequent damage. DVC provided useful information on irradiation-induced microcrack initiation and propagation. Future work is necessary to apply these findings to in situ SR-microCT mechanical tests, and to establish protocols aiming to minimise the SR irradiation-induced damage of bone.
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Nov 2018
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I13-2-Diamond Manchester Imaging
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Diamond Proposal Number(s):
[14080]
Abstract: The use of synchrotron radiation micro-computed tomography (SR-microCT) is becoming increasingly popular for studying the relationship between microstructure and bone mechanics subjected to in situ mechanical testing. However, it is well known that the effect of SR X-ray radiation can considerably alter the mechanical properties of bone tissue. Digital volume correlation (DVC) has been extensively used to compute full-field strain distributions in bone specimens subjected to step-wise mechanical loading, but tissue damage from sequential SR-microCT scans has not been previously addressed. Therefore, the aim of this study is to examine the influence of SR irradiation-induced microdamage on the apparent elastic properties of trabecular bone using DVC applied to in situ SR-microCT tomograms obtained with different exposure times. Results showed how DVC was able to identify high local strain levels (>10,000 µε) corresponding to visible microcracks at high irradiation doses (~230 kGy), despite the apparent elastic properties remained unaltered. Microcracks were not detected and bone plasticity was preserved for low irradiation doses (~33 kGy), although image quality and consequently, DVC performance were reduced. DVC results suggested some local deterioration of tissue that might have resulted from mechanical strain concentration further enhanced by some level of local irradiation even for low accumulated dose.
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Aug 2018
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I13-2-Diamond Manchester Imaging
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Diamond Proposal Number(s):
[14080]
Abstract: A micromechanical characterization of biomaterials for bone tissue engineering is essential to understand the quality of the newly regenerated bone, enabling the improvement of tissue regeneration strategies. A combination of microcomputed tomography in conjunction with in situ mechanical testing and digital volume correlation (DVC) has become a powerful technique to investigate the internal deformation of bone structure at a range of dimensional scales. However, in order to obtain accurate three‐dimensional strain measurement at tissue level, high‐resolution images must be acquired, and displacement/strain measurement uncertainties evaluated. The aim of this study was to optimize imaging parameters, image postprocessing and DVC settings to enhance computation based on ‘zero‐strain’ repeated high‐resolution synchrotron microCT scans of trabecular bone and bone‐biomaterial systems. Low exposures to SR X‐ray radiation were required to minimize irradiation‐induced tissue damage, resulting in the need of advanced three‐dimensional filters on the reconstructed images to reduce DVC‐measured strain errors. Furthermore, the computation of strain values only in the hard phase (i.e. bone, biomaterial) allowed the exclusion of large artefacts localized in the bone marrow. This study demonstrated the suitability of a local DVC approach based on synchrotron microCT images to investigate the micromechanics of trabecular bone and bone‐biomaterial composites at tissue level with a standard deviation of the errors in the region of 100 microstrain after a thorough optimization of DVC computation.
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Jul 2018
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