Nondestructive SR-based μCT 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-Ghrhrlit/J and C3.B6-Ghrhrlit/J, showed that the cannular and lacunar morphometry and their bone mechanics were fundamentally different. Introduction: 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. Materials and Methods: The femoral mid-diaphyses of 12 C57BL/6J-Ghrhrlit/J (B6-lit/lit) and 12 homozygous mutants C3.B6-Ghrhrlit/J (C3.B6-lit/lit) were measured with global SR μCT and local SR nano-CT (nCT) at nominal resolutions ranging from 3.5 μm 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. Results: 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. Conclusions: 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-Ghrhrlit/J and C3.B6-Ghrhrlit/J showed that the cannular and lacunar morphometry and the biomechanical properties were fundamentally different.

Advanced glycation end-products (AGE) contribute to age-related connective tissue damage and functional deficit. The documented association between AGE formation on collagens and the correlated progressive stiffening of tissues has widely been presumed causative, despite the lack of mechanistic understanding. The present study investigates precisely how AGEs affect mechanical function of the collagen fibril – the supramolecular functional load-bearing unit within most tissues. We employed synchrotron small-angle X-ray scattering (SAXS) and carefully controlled mechanical testing after introducing AGEs in explants of rat-tail tendon using the metabolite methylglyoxal (MGO). Mass spectrometry and collagen fluorescence verified substantial formation of AGEs by the treatment. Associated mechanical changes of the tissue (increased stiffness and failure strength, decreased stress relaxation) were consistent with reports from the literature. SAXS analysis revealed clear changes in molecular deformation within MGO treated fibrils. Underlying the associated increase in tissue strength, we infer from the data that MGO modified collagen fibrils supported higher loads to failure by maintaining an intact quarter-staggered conformation to nearly twice the level of fibril strain in controls. This apparent increase in fibril failure resistance was characterized by reduced side-by-side sliding of collagen molecules within fibrils, reflecting lateral molecular interconnectivity by AGEs. Surprisingly, no change in maximum fibril modulus (2.5 GPa) accompanied the changes in fibril failure behavior, strongly contradicting the widespread assumption that tissue stiffening in ageing and diabetes is directly related to AGE increased fibril stiffness. We conclude that AGEs can alter physiologically relevant failure behavior of collagen fibrils, but that tissue level changes in stiffness likely occur at higher levels of tissue architecture.