Of mice and women: utilising high-resolution computed tomography for extrapolation of murine and human sexual dimorphism in cortical bone microstructure and fracture risk

Abstract

Introduction: Men and women exhibit skeletal differences throughout life, exacerbated by age. Cortical porosity comprising vascular canals and osteocyte lacunae has been reported to be sexually dimorphic in adult mice. Heterogeneity of blood vessel types in bone is described, and spatial heterogeneity in cortical porosity reported to exist in tibiae of female mice which is influenced by age. Since the arrangement of vascular canals and osteocyte lacunae within the cortex is a key determinant of bone strength and mechanosensitivity in both mice and humans, improved understanding of sex-specific regulatory mechanisms driving bone microstructure is required. As such, this thesis aims to investigate whether sexually dimorphic mechanisms affect spatial heterogeneity and mechanoadaptation of cortical porosity, and contribute to sex differences in fracture risk with specific focus on military recruits.
Methods: Computational tools were developed to enable 3D reconstruction, regionalisation, and spatial mapping of vascular canals in the transverse bone cortex of mice and humans as well as osteocyte lacunae in mice. Ex vivo high-resolution micro-computed tomography scans (CT, 0.60-1.70 μm resolution) were taken of tibiofibular junctions of male and female pre-pubertal (4 weeks old, n = 3 per sex) and post-pubertal (16 weeks, n = 5 per sex) wildtype (WT) mice, four-core genotype (FCG) mice with uncoupling of chromosomal and gonadal sex (16 weeks old, n = 2 per genotype), and male WT mice (12 weeks old, n = 4) subjected to 2 weeks of servo-hydraulic loading. Additionally, in vivo high-resolution peripheral quantitative CT scans (61 μm resolution) were taken of the tibial diaphysis of men (n = 5) and women (n = 6) prior to and during 44 weeks of British military training. This also included early scans of women (n = 5) who sustained bone stress injuries (BSIs) and were subsequently removed from the study.
Results: Following cross-sectional 3D analysis, no differences were observed in cortical porosity of male and female WT tibiae pre-puberty. Post-puberty, male mice exhibited more lacunae than female mice, with no significant differences in lacuna size. This area was then regionalised into anterior, medial, lateral, and posterior quadrants. It was revealed that females exhibited more canals than males pre-puberty, which localised to the posterior region. The post-pubertal differences in lacunae density in males were predominantly localised to the anterior region. Analysis of FCG mice showed sex differences predominantly localised to anterior and lateral regions, influenced both by chromosomal and gonadal sex. In tibiae of military recruits, cross-sectional analysis also revealed no sex differences in canal density or size, while regionalisation highlighted more canals in men than women predominantly localised to the anterior region. Prior to training and BSI, there were also more canals in injured than non-injured women predominantly localised to the posterior region. Regionalisation effects were evident in response to the mechanical loading too. Following servo-hydraulic loading, no cross-sectional differences in canals or lacunae were observed in male mice. Regionalisation however revealed a decreased canal density in the lateral region and increased lacuna density in the posterior region, with no difference in size. Following military training, a decreased canal density in men localised to the anterior region (from week 1 to 28), which was not observed in women. These instead exhibited a decreased canal size in the anterior region (from week 1 to 28) followed by an increase in the lateral region (from week 28 to 44), which was not evident in women who suffered from BSIs.
Conclusion: Sexual dimorphism in murine and human cortical porosity localises to specific tibial quadrants. In mice, these sex differences appear sensitive to age, sex chromosomes, sex hormones, and mechanoadaptation. Sex differences in cortical porosity also translate to humans, with regional heterogeneity linked to mechanoadaptation and bone stress injury. Regional heterogeneity in cortical porosity described across species likely associates with variation in mechanical strain experienced in mice and humans. Better understanding of sexually dimorphic mechanisms underlying bone microstructure could allow for more effective mitigation of sex-specific fracture risk in the military, potentially involving hormone manipulation and modified exercise regimes.

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Identifiers

ORCID for Lysanne Veerle Michels: orcid.org/0009-0008-6071-2567

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

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