Tendon Biomechanics : Characterizing hierarchical loading and mineralization during healing through advanced synchrotron techniques

Over the past 50 years, the number of tendon ruptures has been increasing. Ruptures may be traumatic, or as a response to long term tendinopathy including e.g. inflammation, altered structure and composition of the tendon. Tendons are primarily made up of water and load bearing collagen molecules that hierarchically assemble into fibrils, aggregate into fibres, that combine into fascicles and eventually make up the entire tendon. Their assembly is crucial for the tissue's mechanical properties. Tendon’s response to loading is governed by the structural rearrangement of collagen, which has been investigated at many length scales independently, but rarely simultaneously. Furthermore, after rupture, the tendon healing process is long and complex, and the tissue never fully regains the intact mechanical properties. One challenge during the healing process is formation of heterotopic ossifications, which result in pain and discomfort. However, the understanding of how and why these heterotopic ossifications form is very limited. Part I of the work presented in this thesis aims to thoroughly investigate the hierarchical response of tendons during loading to better understand the strain partitioning in tendons. In part II of the work, it aims to characterize heterotopic ossifications and the interaction between the mineralized and surrounding healing collagen matrix in tendons which is crucial for providing further prevention and rehabilitation methods. These aims are addressed by using a multimodal approach combining synchrotron- and lab-based tissue characterization techniques probing the tissue with high spatio-temporal resolution while characterizing the mechanical, structural, and compositional properties across samples and loading protocols. More specifically, in part I the combination of X-ray scattering (nano-scale), digital image correlation (meso-scale), and in situ mechanical loading (whole organ) provides a comprehensive hierarchical method to investigate the strain partitioning across length scales. In part II, 2D X-ray scattering, and fluorescence techniques are used to understand the structural and compositional nano-scale characteristics of heterotopic ossifications. Further, cutting edge 3D scattering and fluorescence techniques in combination with tomography are applied to explore the heterogeneity of heterotopic ossifications within the healing tendon matrix.In part I, a mechanical setup is validated through a multimodal approach on rat Achilles tendons and further expanded with bovine tendons. A clear strain partitioning across length scales was found, and a simultaneous strain rate dependence on the hierarchical response where two different fibril adaptations were captured: stretching at lower strain rates and sliding at higher strain rates. Furthermore, time-dependent viscoelastic response was observed during stress-relaxation where a different relaxation behaviour was observed at the tissue level, while the fibrils behaved in a similar manner. The multimodal approach captured heterogenous strain distributions in the tendon across all loading conditions demonstrating that load is not uniformly distributed across the tendon. In part II, healing rat Achilles tendons with established heterotopic ossifications were characterized in both 2D and 3D to elucidate the structural and compositional variability within and between samples. Imaging of heterotopic ossification deposits showed more porous morphology than bone. The multimodal approach revealed a localized spatial pattern of Zinc and Iron at the boundaries of the heterotopic ossification-deposits, with hydroxyapatite crystallites varying between needle-shaped (early healing time points) and towards wider oval-shaped that were found in bone. Collagen d-spacing from nano-scale fibrils were higher within heterotopic ossifications compared to the surrounding healing collagen matrix. The observed heterogeneity in the 2D investigation was further probed in 3D where no obvious directionality for further growth of the heterotopic ossification deposits was seen. In conclusion, the work in this thesis further illustrates the importance of multimodal investigations to provide more comprehensive data on complex hierarchical materials such as tendon tissue. This work highlights the power of high spatio-temporal X-ray techniques and the importance of further developing methods that allow for simultaneous applications of multiple techniques to understand the tissue strain partitioning in response to loading, and tissue characterization as a tool for understanding the mechanisms behind heterotopic ossification in soft tendon tissue.