Image quality and scan time optimisation for in situ phase contrast x-ray tomography of the intervertebral disc.

In-line phase contrast synchrotron tomography combined with in situ mechanical loading enables the characterisation of soft tissue micromechanics via digital volume correlation (DVC) within whole organs. Optimising scan time is important for reducing radiation dose from multiple scans and to limit sample movement during acquisition. Also, although contrasted edges provided by in-line phase contrast tomography of soft tissues are useful for DVC, the effect of phase contrast imaging on its accuracy has yet to be investigated. Due to limited time at synchrotron facilities, scan parameters are often decided during imaging and their effect on DVC accuracy is not fully understood. Here, we used previously published data of intervertebral disc phase contrast tomography to evaluate the influence of i) fibrous image texture, ii) number of projections, iii) tomographic reconstruction method, and iv) phase contrast propagation distance on DVC results. A greater understanding of how image texture influences optimal DVC tracking was obtained by visualising objective function mapping, enabling tracking inaccuracies to be identified. When reducing the number of projections, DVC was minimally affected by image high frequency noise but with a compromise in accuracy. Iterative reconstruction methods improved image signal-to-noise and consequently significantly lowered DVC displacement uncertainty. Propagation distance was shown to affect DVC accuracy. Consistent DVC results were achieved within a propagation distance range which provided contrast to the smallest scale features, where; too short a distance provided insufficient features to track, whereas too long led to edge effect inconsistencies, particularly at greater deformations. Although limited to a single sample type and image setup, this study provides general guidelines for future investigations when optimising image quality and scan times for in situ phase contrast x-ray tomography of fibrous connective tissues.

Regional variations in discrete collagen fibre mechanics within intact intervertebral disc resolved using synchrotron computed tomography and digital volume correlation.

Many soft tissues, such as the intervertebral disc (IVD), have a hierarchical fibrous composite structure which suffers from regional damage. We hypothesise that these tissue regions have distinct, inherent fibre structure and structural response upon loading. Here we used synchrotron computed tomography (sCT) to resolve collagen fibre bundles (∼5µm width) in 3D throughout an intact native rat lumbar IVD under increasing compressive load. Using intact samples meant that tissue boundaries (such as endplate-disc or nucleus-annulus) and residual strain were preserved; this is vital for characterising both the inherent structure and structural changes upon loading in tissue regions functioning in a near-native environment. Nano-scale displacement measurements along >10,000 individual fibres were tracked, and fibre orientation, curvature and strain changes were compared between the posterior-lateral region and the anterior region. These methods can be widely applied to other soft tissues, to identify fibre structures which cause tissue regions to be more susceptible to injury and degeneration. Our results demonstrate for the first time that highly-localised changes in fibre orientation, curvature and strain indicate differences in regional strain transfer and mechanical function (e.g. tissue compliance). This included decreased fibre reorientation at higher loads, specific tissue morphology which reduced capacity for flexibility and high strain at the disc-endplate boundary. STATEMENT OF SIGNIFICANCE: The analyses presented here are applicable to many collagenous soft tissues which suffer from regional damage. We aimed to investigate regional intervertebral disc (IVD) structural and functional differences by characterising collagen fibre architecture and linking specific fibre- and tissue-level deformation behaviours. Synchrotron CT provided the first demonstration of tracking discrete fibres in 3D within an intact IVD. Detailed analysis of regions was performed using over 200k points, spaced every 8 µm along 10k individual fibres. Such comprehensive structural characterisation is significant in informing future computational models. Morphological indicators of tissue compliance (change in fibre curvature and orientation) and fibre strain measurements revealed localised and regional differences in tissue behaviour.

Phase-contrast 3D tomography of HeLa cells grown in PLLA polymer electrospun scaffolds using synchrotron X-rays.

Advanced imaging is useful for understanding the three-dimensional (3D) growth of cells. X-ray tomography serves as a powerful noninvasive, nondestructive technique that can fulfill these purposes by providing information about cell growth within 3D platforms. There are a limited number of studies taking advantage of synchrotron X-rays, which provides a large field of view and suitable resolution to image cells within specific biomaterials. In this study, X-ray synchrotron radiation microtomography at Diamond Light Source and advanced image processing were used to investigate cellular infiltration of HeLa cells within poly L-lactide (PLLA) scaffolds. This study demonstrates that synchrotron X-rays using phase contrast is a useful method to understand the 3D growth of cells in PLLA electrospun scaffolds. Two different fiber diameter (2 and 4 µm) scaffolds with different pore sizes, grown over 2, 5 and 8 days in vitro, were examined for infiltration and cell connectivity. After performing visualization by segmentation of the cells from the fibers, the results clearly show deeper cell growth and higher cellular interconnectivity in the 4 µm fiber diameter scaffold. This indicates the potential for using such 3D technology to study cell-scaffold interactions for future medical use.

Synchrotron tomography of intervertebral disc deformation quantified by digital volume correlation reveals microstructural influence on strain patterns.

The intervertebral disc (IVD) has a complex and multiscale extracellular matrix structure which provides unique mechanical properties to withstand physiological loading. Low back pain has been linked to degeneration of the disc but reparative treatments are not currently available. Characterising the disc's 3D microstructure and its response in a physiologically relevant loading environment is required to improve understanding of degeneration and to develop new reparative treatments. In this study, techniques for imaging the native IVD, measuring internal deformation and mapping volumetric strain were applied to an in situ compressed ex vivo rat lumbar spine segment. Synchrotron X-ray micro-tomography (synchrotron CT) was used to resolve IVD structures at microscale resolution. These image data enabled 3D quantification of collagen bundle orientation and measurement of local displacement in the annulus fibrosus between sequential scans using digital volume correlation (DVC). The volumetric strain mapped from synchrotron CT provided a detailed insight into the micromechanics of native IVD tissue. The DVC findings showed that there was no slipping at lamella boundaries, and local strain patterns were of a similar distribution to the previously reported elastic network with some heterogeneous areas and maximum strain direction aligned with bundle orientation, suggesting bundle stretching and sliding. This method has the potential to bridge the gap between measures of macro-mechanical properties and the local 3D micro-mechanical environment experienced by cells. This is the first evaluation of strain at the micro scale level in the intact IVD and provides a quantitative framework for future IVD degeneration mechanics studies and testing of tissue engineered IVD replacements. STATEMENT OF SIGNIFICANCE: Synchrotron in-line phase contrast X-ray tomography provided the first visualisation of native intact intervertebral disc microstructural deformation in 3D. For two annulus fibrosus volumes of interest, collagen bundle orientation was quantified and local displacement mapped as strain. Direct evidence of microstructural influence on strain patterns could be seen such as no slipping at lamellae boundaries and maximum strain direction aligned with collagen bundle orientation. Although disc elastic structures were not directly observed, the strain patterns had a similar distribution to the previously reported elastic network. This study presents technical advances and is a basis for future X-ray microscopy, structural quantification and digital volume correlation strain analysis of soft tissue.

A review of techniques for visualising soft tissue microstructure deformation and quantifying strain Ex Vivo.

Many biological tissues have a complex hierarchical structure allowing them to function under demanding physiological loading conditions. Structural changes caused by ageing or disease can lead to loss of mechanical function. Therefore, it is necessary to characterise tissue structure to understand normal tissue function and the progression of disease. Ideally intact native tissues should be imaged in 3D and under physiological loading conditions. The current published in situ imaging methodologies demonstrate a compromise between imaging limitations and maintaining the samples native mechanical function. This review gives an overview of in situ imaging techniques used to visualise microstructural deformation of soft tissue, including three case studies of different tissues (tendon, intervertebral disc and artery). Some of the imaging techniques restricted analysis to observational mechanics or discrete strain measurement from invasive markers. Full-field local surface strain measurement has been achieved using digital image correlation. Volumetric strain fields have successfully been quantified from in situ X-ray microtomography (micro-CT) studies of bone using digital volume correlation but not in soft tissue due to low X-ray transmission contrast. With the latest developments in micro-CT showing in-line phase contrast capability to resolve native soft tissue microstructure, there is potential for future soft tissue mechanics research where 3D local strain can be quantified. These methods will provide information on the local 3D micromechanical environment experienced by cells in healthy, aged and diseased tissues. It is hoped that future applications of in situ imaging techniques will impact positively on the design and testing of potential tissue replacements or regenerative therapies. LAY DESCRIPTION: The soft tissues in our bodies, such as tendons, intervertebral discs and arteries, have evolved to have complicated structures which deform and bear load during normal function. Small changes in these structures can occur with age and disease which then leads to loss of function. Therefore, it is important to image tissue microstructure in 3D and under functional conditions. This paper gives an overview of imaging techniques used to record the deformation of soft tissue microstructures. Commonly there are compromises between obtaining the best imaging result and retaining the samples native structure and function. For example, invasive markers and dissecting samples damages the tissues natural structure, and staining or clearing (making the tissue more transparent) can distort tissue structure. Structural deformation has been quantified from 2D imaging techniques (digital image correlation) to create surface strain maps which help identify local tissue mechanics. When extended to 3D (digital volume correlation), deformation measurement has been limited to bone samples using X-ray micro-CT. Recently it has been possible to image the 3D structure of soft tissue using X-ray micro-CT meaning that there is potential for internal soft tissue mechanics to be mapped in 3D. Future application of micro-CT and digital volume correlation will be important for soft tissue mechanics studies particularly to understand normal function, progression of disease and in the design of tissue replacements.

Visualising the 3D microstructure of stained and native intervertebral discs using X-ray microtomography.

Intervertebral disc degeneration (IVDD) is linked to low back pain. Microstructural changes during degeneration have previously been imaged using 2D sectioning techniques and 3D methods which are limited to small specimens and prone to inducing artefacts from sample preparation. This study explores micro computed X-ray tomography (microCT) methods with the aim of resolving IVD 3D microstructure whilst minimising sample preparation artefacts. Low X-ray absorption contrast in non-mineralised tissue can be enhanced using staining and phase contrast techniques. A step-wise approach, including comparing three stains, was used to develop microCT for bovine tail IVD using laboratory and synchrotron sources. Staining successfully contrasted collagenous structures; however not all regions were stained and the procedure induced macroscopic structural changes. Phase contrast microCT of chemically fixed yet unstained samples resolved the nucleus pulposus, annulus fibrosus and constituent lamellae, and finer structures including collagen bundles and cross-bridges. Using the same imaging methods native tissue scans were of slightly lower contrast but free from sample processing artefacts. In the future these methods may be used to characterise structural remodelling in soft (non-calcified) tissues and to conduct in situ studies of native loaded tissues and constructs to characterise their 3D mechanical properties.