Synchrotron-based characterization of mechanobiological effects on the nanoscale in musculoskeletal tissues

Collagen is the main organic building block of musculoskeletal tissues. Despite collagen being their smallest load bearing unit, these tissues differ significantly in mechanical function and properties. A major factor behind these differences is their hierarchical organization, from the collagen molecule up to the organ scale. It is thus of high importance to understand the characteristics of each level, as well as how they interact and relate to each other. With such knowledge, improved prevention and rehabilitation of musculoskeletal pathologies may be achieved.Both mineralized and soft collagenous tissues respond to their mechanical loading environment according to specific mechanobiological principles. During prenatal development, immobilization can cause dramatic effects on the developing skeleton, causing the newly formed bones to be smaller, deformed and more prone to fracture. But how immobilization affects the deposition, structure and composition of the developing bones is still unclear. In tendons, both insufficient and excessive mechanical loading increases the risk of injury. After rupture, reduced mechanical loading results in altered collagen structure and cell activity, thus influencing the mechanical properties of the healing tendon. How the loading environment affects the structure of intact and ruptured tendons is still debated.The work presented in this thesis aims to thoroughly characterize the mechanobiological effects on the mineralization process in developing bones as well as the collagen structure and multiscale mechanical response of intact and healing tendons. This is achieved through a multimodal approach including a range of high-resolution synchrotron- and lab-based techniques, in combination with mechanical testing.In the first part of the thesis, humeri from “muscle-less” embryonic mice and their healthy littermates at development stages from start of mineralization to shortly before birth were investigated. The multimodal approach revealed a highly localized spatial pattern of Zinc during normal development to sites of ongoing mineralization, accompanied by larger mineral particles. Healthy bones also showed signs of remodeling at later time points. In the absence of skeletal muscle, it was revealed that the developing bones exhibited a delayed but increased mineral deposition and growth, with no signs of remodeling.In the second part of the thesis, intact Achilles tendons from rats subjected to either full in vivo loading through free cage activity or unloading by Botox injections combined with cast immobilization were investigated. It was shown that the nanoscale fibrils in the Achilles tendon respond to the applied tissue loads and exhibit viscoelastic responses. It was revealed that in vivo unloading results in a more disorganized microstructure and an impaired viscoelastic response. Unloading also altered the nanoscale fibril mechanical response, possibly through alterations in the strain partitioning between hierarchical levels.In the third part of the thesis, Achilles tendons were transected and allowed to heal while subjected to either full in vivo loading, reduced loading through Botox injections or unloading. In vivo unloading during the early healing process resulted in a delayed and more disorganized collagen structure and a larger presence of adipose tissue. Unloading also delayed the remodeling of the stumps as well as callus maturation. Additionally, the nanoscale fibril mechanical response was altered, with unloaded tendons exhibiting a low degree of fibril recruitment as well as a decreased ability for fibril extension.The work in this thesis further illustrates the important role of the mechanical environment on the nanostructure of musculoskeletal tissues. It also highlights the power of combining high-resolution tissue characterization techniques into a multimodal and multiscale approach, allowing us to study the effects on several hierarchical length scales simultaneously and as a result be able to elucidate the intricate connection between hierarchical scales