Quantification of 3D microstructures in Achilles tendons during in situ loading reveals anisotropic fiber response

While the number of studies investigating Achilles tendon pathologies has grown exponentially, more research is needed to gain a better understanding of the complex relation between its hierarchical structure, mechanical response, and failure. At the microscale, collagen fibers are, with some degree of dispersion, primarily aligned along the principal loading direction. However, during tension, rearrangements and reorientations of these fibers are believed to occur. As 3D micro-movements are hard to capture, the precise nature of this fiber reorganization remains unknown. This study aimed to visualize and quantify the intricate fiber changes occurring within rat Achilles tendons under tension. Rat tendons were in situ loaded with concurrent synchrotron phase contrast microCT imaging. The results are heterogenous and show that collagen fibers' response to loading is nonuniform and depends on anatomical orientation. Furthermore, damage propagation could be visualized, revealing that in the presence of heterotopic ossification, damage proceeds within the ossified deposits rather than at the interface between hard and soft tissues. Our approach could effectively capture the microstructural changes occurring during loading and shows promise in understanding the relation between microstructure and mechanical response for ex-vivo Achilles tendons and other biological tissues. STATEMENT OF SIGNIFICANCE: Achilles tendons endure high mechanical loads during daily motion and physical activities. Understanding the structural and mechanical responses of Achilles tendons to such loads is vital for elucidating their function in health and pathology. We have combined the use of synchrotron phase contrast microCT with in situ mechanical loading to contribute to a better understanding of the relation between microstructural response and organ scale mechanical properties. The proposed methodology will be valuable for future research into the interplay between structure, mechanics, and pathology of tendons, and for the development of more effective strategies to preserve tendon function and possibly mitigating musculoskeletal disorders.