Ultrastructural Properties in Cortical Bone Vary Greatly in Two Inbred Strains of Mice as Assessed by Synchrotron Light Based Micro- and Nano-CT

Abstract

Nondestructive SR-based microCT and nano-CT methods have been designed for 3D quantification and morphometric analysis of ultrastructural phenotypes within murine cortical bone, namely the canal network and the osteocyte lacunar system. Results in two different mouse strains, C57BL/6J-Ghrhr(lit)/J and C3.B6-Ghrhr(lit)/J, showed that the cannular and lacunar morphometry and their bone mechanics were fundamentally different. To describe the different aspects of bone quality, we followed a hierarchical approach and assessed bone tissue properties in different regimens of spatial resolution, beginning at the organ level and going down to cellular dimensions. For these purposes, we developed different synchrotron radiation (SR)-based CT methods to assess ultrastructural phenotypes of murine bone. The femoral mid-diaphyses of 12 C57BL/6J-Ghrhr(lit)/J (B6-lit/lit) and 12 homozygous mutants C3.B6-Ghrhr(lit)/J (C3.B6-lit/lit) were measured with global SR microCT and local SR nano-CT (nCT) at nominal resolutions ranging from 3.5 microm to 700 nm, respectively. For volumetric quantification, morphometric indices were determined for the cortical bone, the canal network, and the osteocyte lacunar system using negative imaging. Moreover, the biomechanics of B6-lit/lit and C3.B6-lit/lit mice was determined by three-point bending. The femoral mid-diaphysis of C3.B6-lit/lit was larger compared with B6-lit/lit mice. On an ultrastructural level, the cannular indices for C3.B6-lit/lit were generally bigger in comparison with B6-lit/lit mice. Accordingly, we derived and showed a scaling rule, saying that overall cannular indices scaled with bone size, whereas indices describing basic elements of cannular and lacunar morphometry did not. Although in C3.B6-lit/lit, the mean canal volume was larger than in B6-lit/lit, canal number density was proportionally smaller in C3.B6-lit/lit, so that lacuna volume density was found to be constant and therefore independent of mouse strain and sex. The mechanical properties in C3.B6-lit/lit were generally improved compared with B6-lit/lit specimens. For C3.B6-lit/lit, we observed a sex specificity of the mechanical parameters, which could not be explained by bone morphometry on an organ level. However, there is evidence that for C3.B6-lit/lit, the larger cortical bone mass is counterbalanced or even outweighed by the larger canal network in the female mice. We established a strategy to subdivide murine intracortical porosity into ultrastructural phenotypes, namely the canal network and the osteocyte lacunar system. Nondestructive global and local SR-based CT methods have been designed for 3D quantification and subsequent morphometric analysis of these phenotypes. Results in the two different mouse strains C57BL/6J-Ghrhr(lit)/J and C3.B6-Ghrhr(lit)/J showed that the cannular and lacunar morphometry and the biomechanical properties were fundamentally different.