Tomo-SAXS: Imaging full-field molecular-to-macroscale biophysics of fibrous tissues

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Biological tissues - e.g. joints, arteries, ligaments - operate in a dynamic mechanical environment. Examples include the frictionless sliding of joints and the periodic stress waves in blood vessels. The body's response to these forces is mediated by a hierarchy of biophysical processes from the smallest (molecular) to the largest (organ) level. These processes - e.g. sliding of collagen fibrils at the nanoscale or shearing of fibre-bundles at the microscale - are very challenging experimentally to measure in situ. This is important because biophysics of the extracellular matrix at these small length-scales crucially affects cell and tissue growth and mediates progression of multiple noncommunicable disorders (e.g. osteoarthritis and abnormal wound healing). However, the state of the art in analysing such processes largely relies on imaging without direct mechanical quantification at the sub-micron scales or measuring mechanics of individual molecules ex situ. In this regard, X-ray illumination of an organ can build up a 3D map of the collagen fibre bundles in the matrix (tomography or CT) with micron-level resolution (size of a human hair). At a hundred times smaller size, these same X-rays can interact with the molecules making up the fibres via interference, building up a picture like a diffraction grating (small angle scattering or SAXS). When a brilliant X-ray beam (like the kind available at synchrotrons) is available, these methods can be used to study load-induced biophysical changes dynamically. If the information from these two techniques - CT and SAXS - could be combined, we would have an unprecedented molecular-to-macroscale visualisation of tissue biophysics. Here, we bring together expertise in X-ray imaging and synchrotron techniques to develop a path-breaking new technique - TomoSAXS - which will image the multiscale biophysics of tissues, integrating phase-contrast CT with SAXS into a single platform. By using the information from each method as input into the other in a synergistic manner, we will develop advanced reconstruction algorithms to generate full-field 3D images of molecular to macroscale soft tissue structure. These advances in analysis will be coupled with hardware development of a unique mechanical rig which can be used for simultaneous CT- and SAXS imaging on the same tissue or organ. Because the SAXS signal from fibrous tissues is a highly complex 3D anisotropic pattern, we will develop the technique on simpler model systems before progressing to real tissues and organs. Starting with reconstituted collagen biomaterials, we will advance to organs like the intervertebral disc, which is crucially important for posture and preventing back pain. The intervertebral disc is a highly ordered collagenous tissue, with strong signal contrast in CT- and SAXS, and is well-suited to establish the method on. After establishment of the technique, we will demonstrate its application and utility by i) carrying out training workshops for bioengineers and biomedical scientists on using the technique effectively and ii) engaging with the modelling community to incorporate the new insights from TomoSAXS in the next generation of predictive models. The load- or stimuli-induced changes in micro- and nanostructure visualised in 3D volume maps of tissue will enable a step-change in realism, prediction and analysis of tissue health and disease. Examples include detection of localised supramolecular changes in the tissue matrix at early stages in disease and degeneration, defining structural biomarkers in conditions like osteoarthritis, and testing the effectiveness of drugs in repairing or regenerating tissue in situ. By establishing the method at the UK's national synchrotron, we will make this unique technique available to the UK bioengineering and biomedical community as well as internationally.