In Situ Characterization Reveals an Impaired Fibril Response to Loading Following Unloading during Early Achilles Tendon Healing

The Achilles tendon is the most frequently injured tendon in humans. Despite extensive research to understand its healing, there are still no clear rehabilitation guidelines to ensure full recovery and prevent reruptures. This could partly be due to limited understanding of how loading during tendon healing affects the multiscale mechanical response and in particular the nanoscale. We combined synchrotron small-angle X-ray scattering mapping with <i>in situ</i> tensile stress relaxation of early healing (1-, 2-, and 3 weeks) rat Achilles tendons subjected to either full activity or immobilization. Initially, <i>in vivo</i> unloading resulted in a similar collagen fibril structure and spatial distribution, but then, as healing progressed, the unloaded group exhibited more alterations in tissue distribution. While no clear differences were observed at the tissue scale, unloading clearly altered the nanoscale mechanical response already after 1 week of healing. Unloading led to an impaired nanoscale response, characterized by higher spatial variations, less fibril recruitment, and capacity for elongation. This indicates that the nanoscale of healing tendons is more susceptible to changes in the loading environment compared to the tissue scale. These insights contribute to a better understanding of the effects of <i>in vivo</i> loading on multiple length scales of the healing Achilles tendon.