Connecting the dots: exploring the relationship between the arrangement of cortical porosity and the development of sex-specific whole bone phenotypes in cystine string protein alpha deficient mice

Abstract

Neurodegenerative diseases are characterised by synaptic loss and cognitive decline but are also typified by debilitating systemic comorbidities such as high prevalence of bone fracture. However, causative studies linking neurodegenerative diseases to bone fragility frequently omit the influence of bone porosity - a key contributor to bone strength - due to the size of pores within bone (frequently < 5 µm diameter) and their inaccessibility (within mineralised matrix). Bone porosity is comprised of a network of intracortical canals which house the bone vasculature and the mechanosensitive lacuna-canalicular network (LCN). Herein, this thesis aims to assess the morphology and organisation of porosity in a murine model of early synaptic loss - the cystine string protein alpha null mouse (CSPα-/-) - to understand how synaptic loss may alter bone phenotype. This will permit the characterization of links between CSPα-/- bone phenotypes on the whole bone scale, to the underpinning changes to the morphology, spatial arrangements and uniquely, on the cellular scale by visualising osteocytes within the LCN and relating osteocytes to their local mineral environment.

This thesis describes four primary objectives: (i) Firstly, to characterize the development of bone phenotypes in CSPα-/- mice at multiple skeletal sites using micro computed tomography. Minor alterations to the endocranial anatomy of CSPα-/- mice and no changes to tibial trabecular morphology were observed. However, both male and female mice possess tibial cortical thinning and exclusively female CSPα-/- mice possess a deficit in tissue mineral density, establishing the tibial cortical bone as a site of interest. Subsequently, immunohistochemical approaches determined that CSPα is expressed by the bone vasculature, suggesting that in CSPα-/- mice intracortical canal structure or function may be affected. (ii) Synchrotron radiation computed tomography (SR CT) was thereby harnessed to study and establish the morphological and spatial arrangements of tibial cortical porosity in skeletally mature 13-month-old male WT mice. The tibiofibular junction (TFJ) was selected as a landmark site as it contains solely cortical bone, with image analysis tools developed to assess the morphology and spatial organization of pores, in addition to an alignment technique to separate the TFJ into quadrants. Together, these tools identified the posterior TFJ as a region possessing a distinct microstructural profile, with abundant intracortical canals surrounded by large canal-associated osteocyte lacunae. Importantly, conditional removal of a key angiogenic mitogen involved in angiogenic-osteogenic coupling – vascular endothelial growth factor (VEGF) in osteocalcin expressing cells (OcnVEGFKO) in mice – removed this population of large, canal-associated osteocyte lacunae in a sex specific manner. Study of OcnVEGFKO microstructure provided evidence that this unique posterior microstructural arrangement was not an anomaly, but rather, a deliberate coupling of abundant intracortical canals to large osteocyte lacunae to fulfil a specific function. (iii) However, when applied to CSPα-/- mice these tools revealed no differences in the arrangement of cortical porosity between WT and CSPα-/- mice, while microstructural heterogeneity was again observed. (iv) Finally, utilization of propagation-based enhanced phase contrast SR CT permitted the visualization of the cellular components lying within cortical pores - vasculature within intracortical canals and osteocytes within lacunae - which was used to assess their cellular structure and function. A convoluted neural network was developed to segment soft tissues from within cortical pores, revealing that canal-associated osteocytes were smaller in both male and female CSPα-/- mice, specifically within the posterior TFJ. Further, assessment of the local mineral surrounding posterior canal-associated osteocytes found that WT osteocytes possessed a region of low mineral density surrounding each lacuna, which extended to ~ 1 µm. However, exclusively female CSPα-/- mice possessed an extension of this hypo-dense region, with the hypo-mineral region surrounding canal-associated osteocytes extending to ~ 2 µm from the lacunae.

Collectively, this thesis describes a sex-specific mineral deficit in female CSPα-/- mice, exclusively at the tibia. SR CT established the existence of region-specific arrangements of posterior osteocyte lacunae surrounding intracortical canals in WT mice, which is sex-specific in its facilitation by VEGF. Lastly, the value in assessing osteocyte subsets is highlighted through the identification of posterior, canal-associated osteocyte populations at the TFJ that contribute to the sex-specific deficit to bone mineral observed in female CSPα-/- mice. This finding provides potential functionality to this unique microstructural arrangement in CSPα-/- bone through the description of a region-specific association of low-density bone mineral surrounding canal-associated posterior osteocytes in female CSPα-/- mice. Together, it is hoped that this thesis highlights the importance and complexity of regional microstructural heterogeneity, linking microscale changes to bone porosity to altered murine bone macrostructure. Looking forward, it is hoped the continued study and understanding of these large, vasculature-associated osteocyte subpopulations could further our understanding of how changes to bone porosity could underpin the increased susceptibility to bone fracture of individuals living with neurodegenerative diseases.

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Identifiers

ORCID for Jacob Trend: orcid.org/0000-0003-0394-7665

ORCID for Katrin Deinhardt: orcid.org/0000-0002-6473-5298

ORCID for Philipp Schneider: orcid.org/0000-0001-7499-3576

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