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

生物组织-例如关节、动脉、韧带-在动态机械环境中操作。例子包括关节的无摩擦滑动和血管中的周期性应力波。身体对这些力量的反应是由一系列生物物理过程介导的，从最小的（分子）到最大的（器官）水平。这些过程-例如，胶原纤维在纳米级的滑动或纤维束在微米级的剪切-在实验上非常具有挑战性，难以原位测量。这是重要的，因为细胞外基质在这些小长度尺度上的生物物理学对细胞和组织生长具有关键性影响，并介导多种非传染性疾病（例如骨关节炎和异常伤口愈合）的进展。然而，分析这些过程的现有技术在很大程度上依赖于成像，而没有在亚微米尺度下的直接机械定量或非原位测量单个分子的力学。 在这方面，器官的X射线照射可以建立具有微米级分辨率（人类头发的大小）的基质中胶原纤维束的3D图（断层扫描或CT）。在小一百倍的尺寸下，这些相同的X射线可以通过干涉与构成纤维的分子相互作用，形成像衍射光栅（小角散射或SAXS）一样的图像。当有明亮的X射线束（如同步加速器中可用的那种）时，这些方法可以用来动态地研究负载引起的生物物理变化。如果来自这两种技术的信息- CT和SAXS -可以结合起来，我们将有一个前所未有的组织生物物理学的分子到宏观尺度的可视化。在这里，我们汇集了X射线成像和同步加速器技术的专业知识，开发了一种开创性的新技术- TomoSAXS -该技术将对组织的多尺度生物物理进行成像，将相位对比CT与SAXS集成到一个平台中。通过使用每种方法的信息作为输入到其他协同方式，我们将开发先进的重建算法，以生成分子宏观软组织结构的全场3D图像。这些分析方面的进展将与独特机械装置的硬件开发相结合，该装置可用于同一组织或器官的同时CT和SAXS成像。 因为来自纤维组织的SAXS信号是高度复杂的3D各向异性图案，所以我们将在进行到真实的组织和器官之前在更简单的模型系统上开发该技术。从重组胶原蛋白生物材料开始，我们将推进到椎间盘等器官，这对姿势和预防背痛至关重要。椎间盘是高度有序的胶原组织，在CT和SAXS中具有强信号对比，并且非常适合于建立该方法。我们将通过i）为生物工程师和生物医学科学家举办关于有效使用该技术的培训讲习班和ii）与建模社区合作，将TomoSAXS的新见解纳入下一代预测模型。在组织的3D体积图中可视化的微结构和纳米结构中的负载或刺激诱导的变化将使组织健康和疾病的真实性、预测和分析发生阶跃变化。例如，在疾病和退化的早期阶段检测组织基质中的局部超分子变化，定义骨关节炎等疾病的结构生物标志物，以及测试药物在原位修复或再生组织中的有效性。通过在英国国家同步加速器上建立这种方法，我们将使这种独特的技术可用于英国生物工程和生物医学界以及国际上。

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.