Bones beyond osteocytes

Bone, a mineralized nano biocomposite, is composed of an intricate arrangement of collagen fibers and carbonated hydroxyapatite crystals. This hierarchical material, observed across all vertebrate species, including bony fish, confers remarkable mechanical properties through tight interactions between the organic and inorganic phases. The organic collagen matrix interacts with non-collagenous proteins, such as proteoglycans (PGs), that attract water to the bone matrix and modulate tissue-level mechanical properties. Water within the open porosity of bone, spanning the micro to nanometer length scales, facilitates nutrient transport and influences the mechanical properties of bone tissue. Although bony fish possess a material composition similar to mammalian bone, anosteocytic bones, prevalent among advanced teleost fishes, challenge conventional concepts of bone biology by thriving without osteocytes and the associated lacuno-canalicular network (LCN). Despite the absence of an LCN, anosteocytic bones can adapt to external loading conditions. To elucidate the functional differences in bone matrix with and without osteocytes, this thesis compares the vertebrae of similarly-sized fishbones: medaka (Oryzias latipes, with anosteocytic bone) and zebrafish (Danio rerio, with osteocytic bone), using advanced techniques including synchrotron nanotomography, neutron tomography, X-Ray Fluorescence (XRF), and X-Ray Diffraction (XRD). Neutron imaging, sensitive to hydrogen, reveals differences in water permeability between the two species. Medaka bones, despite lacking an LCN, exhibit higher water diffusion through nanoscale porosity, contrasting with the more confined water flow in the LCN of zebrafish bones. The bone matrix of zebrafish appears to possess a mechanism for modulating water permeability, potentially enhancing the mechanosensation of external loads by osteocytes. The lower PG content in medaka bone, corroborated by histological staining and Raman spectroscopy, suggests that PGs may play a role as water permeability modulators within the bone matrix. Additionally, high-resolution imaging techniques reveal how zebrafish bone exhibits a network of voids that is absent in medaka bone. Importantly, the ultrastructure of both species reveals a similar elemental composition and nanocrystals orientation, with hydroxyapatite crystals aligned with collagen fibrils along the principal bone axis. Medaka and zebrafish bones show comparable average d-spacing, however, variations in the c-lattice parameter across bones hint to the presence of residual strain within bones of both species. Bones may have a built-in compressive residual strain mechanism to enhance fracture toughness. XRD analysis indicates higher residual strain in anosteocytic medaka bone, potentially entailing enhanced mechanical properties in bones lacking a biologically active LCN. This thesis deepens our understanding of bone nanostructure, water permeability, and mechanics in both osteocytic and anosteocytic bones, highlighting the role of PGs and residual strain in maintaining bone integrity. These findings have broader implications for bone health and resilience.