Exploring longitudinal alterations in cortical bone architecture driven by SQSTM1 mutation as a determinant of fracture risk

Abstract

Femoral fractures are a major impediment in Paget’s Disease of Bone (PDB), the second most common bone disease. Little is known about cortical bone geometry in PDB; novel diagnostic tools and therapeutic strategies are urgently required. Herein, the objective of this thesis was to examine whether alterations in longitudinal cortical bone architecture underlie femoral fracture risk. 40% of familial cases of PDB are underpinned by sequestosome-1 (SQSTM1) mutation; A murine model exhibiting a proline to leucine mutation at codon 394 (P394L) of SQSTM1 was used to study 8-month old wildtype (WT) and homozygote (P394L+/+) mice. The main thesis aims were to i) characterise the gross anatomical features of the of P394L+/+ animals using lab-based microCT (12.7 µm), ii) determine underlying microstructural changes within the bone cortex of P394L+/+ femora using synchrotron radiation computed tomography (SR CT, 0.65 µm), iii) elucidate macrostructural changes of P394L+/+ femora using lab-based microCT (17.76 µm), iv) examine mechano-adaptation in P394L+/+ femora combining ex vivo mechanical testing and finite element analysis (FEA) and v) interrogate novel microstructure-based biomarkers for mechanical failure in P394L+/+ femora. Lab based microCT established sexual dimorphism in P394L+/+ skull geometry and identified geometrical alterations in male P394L+/+ skulls. Longitudinal femoral alterations were assessed according to percentage bone length with 0-50% and 50-100% covering the proximal and distal region respectively. Two primary components of cortical bone microstructure comprising of i) osteocyte lacunae and ii) intracortical canals were assessed using SR CT, with a greater accumulation of microstructural changes found in the distal region of both male and female P394L+/+ femora. In the distal region of male P394L+/+ femora, lacunar number density was significantly reduced (p=0.05) and there was a significant increase in canal diameter vs WT (p=0.0135). In the distal region of female P394L+/+ femora, lacunar circumference and lacunar volume were significantly decreased (p=0.0276 and p=0.0022 respectively), while small canal volume density was significantly increased compared to female WT (p<0.0001). Lab-based microCT showed a reduction in cross sectional area (CSA) in male P394L+/+ femora within the distal region compared to male WT (p=0.0182). In female P394L+/+ femora, CSA, second polar moment of area (J) and cortical thickness were decreased in the distal region, while ellipticity was increased in both the proximal and distal region when compared to female WT (p<0.05). In parallel, contralateral 8-month old femora were subjected to mechanical testing to assess the relationship between P394L+/+ driven alterations to cortical bone and fracture site. Four-point bending tests revealed a lesser force required to break female P394L+/+ femora (36.6 N) versus female WT (42.8 N, p=0.0257) with P394L+/+ exhibiting fracture at 65.8% and 48.3% of the bone length respectively, meaning P394L+/+ femora fracture within the distal region. No significant differences to the mechanical properties of male P394L+/+ femora were observed. Furthermore, FEA experiments revealed no significant differences in maximal stress between WT and P394L+/+ femora in males and females. Finally, SR CT analyses revealed disruption in osteocyte lacunar orientation and lacunar morphology in the distal, fracture prone, region of female P394L+/+ femora. Overall, microCT and SR CT in tandem with mechanical testing and FEA has revealed that geometrical alterations in bone macrostructure and microstructure associate directly with regions of bone prone to fracture. Geometrical alterations could therefore provide a novel clinical biomarker for fracture prediction in degenerative bone diseases such as PDB.

More information

Identifiers

Catalogue record

Export record