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

Analysis of skeleton in a mouse model of Rett syndrome

Rett Syndrome (RTT) is an X-linked genetic disorder and a major cause of intellectual disability in girls. Mutations in the methyl-CpG binding protein 2 (MECP2) gene, are the primary cause of the disorder. Despite the dominant neurological phenotypes that characterise RTT, MECP2 is expressed ubiquitously throughout the body and a number of peripheral phenotypes such as growth retardation (reduced height and weight), skeletal deformities (scoliosis/kyphosis), reduced bone mass and low energy fractures are also common yet under-reported clinical features of the disorder. In order to explore whether MeCP2 protein deficiency results in altered structural and functional properties of bone and to test the potential reversibility of any such defects, I have conducted series of histological, imaging and biomechanical tests of bone using an accurate genetic (functional knockout) mouse model of RTT. Initial experiments using a GFP reporter mouse line demonstrated the presence of MeCP2 in bone cells and the effective silencing on the gene in functional knockout mice. Different aspects of the study were conducted in different types of bone tissues that were especially suited for individual assays. For instance, biomechanical three point bending tests were conducted in long bone (femur) whilst trabecular geometry measures were measured in spinal vertebrae. Both hemizygous Mecp2stop/y male mice in which Mecp2 is silenced in all cells and female Mecp2stop/+ mice in which Mecp2 is silenced in ~50% of cells as a consequence of random X-chromosome inactivation (XCI), revealed, lighter and smaller long bones and significant reductions in cortical bone mechanical properties (~ 39.5% reduction in stiffness, 31% reduction in ultimate load and 37% reduction in Young’s modulus respectively in Mecp2stop/y male mice; %) and material properties (microhardess reduced 12.3% in Mecp2stop/y male mice and 14% inMecp2stop/+ female mice) as compared to age wild type control mice. Micro structural analysis conducted using µCT also revealed a significant reduction in cortical (54% reduction in cortical thickness, 30% in bone volume, 20% in total area, and 38% in marrow area) and trabecular (~30% in trabecular thickness) bone parameters as compared to age matched wild-type controls MeCP2-deficent mice. Histological analysis using Sirius red staining as a marker of collagen revealed a ~25% reduction in collagen content in MeCP2 deficient mice as compared to age matched wild type controls. In experiments designed to establish the potential for reversal of MeCP2-related deficits, unsilencing of Mecp2 in adult mice by tamoxifen-induced and cre-mediated excision of a stop cassette located at the endogenous Mecp2 locus (male; Mecp2stop/y, CreER and female; Mecp2+/stop, CreER), resulted in a restoration of biomechanical properties towards the wild-type levels. Specifically, Male Mecp2stop/y, CreER mice displayed improvement in mechanical properties (stiffness 40%, ultimate load 10%, young’s modulus 61% and micro hardness 12%) and structural bone parameter (trabecular thickness 80%) as compared to Mecp2stop/y male mice. Female Mecp2+/stop, CreER, displayed a significant improvement (19%) in microhardess measures as compared to Mecp2 deficient mice. Overall, the results of my studies show that MeCP2-deficiency results in overt, but potentially reversible, alterations in the biomechanical integrity of bone and highlights the importance of targeting skeletal phenotypes in considering the development of pharmacological and gene-based therapies for Rett Syndrom