Recent data have shown that predicting bone strength can be greatly improved by including microarchitectural
parameters in the analysis. Moreover, bone ultrastructure has been implicated as an important contributor to bone
strength. We therefore hypothesized that a better understanding of phenotypes linked to bone ultrastructure will provide
new insight in the assessment of bone quality and its contribution to bone strength and fracture risk. Therefore, we first
developed an experimental design to assess quantitatively ultrastructural murine bone tissue properties non-invasively in
three dimensions by using synchrotron radiation-based (SR) computed tomography (CT) methods with resolutions on the
order of one micrometer and below. New morphometric indices were introduced to quantify ultrastructural phenotypes of
murine cortical bone assessed by our SR CT-based setup, namely the canal network and the osteocyte lacunar system.
These ultrastructural phenotypes were then successfully studied in two genetically distinct mouse strains. Finally, we
provided strong evidence for a significant influence of the canal network on murine bone mechanics. In the long run, we
believe that the morphometric analysis of the ultrastructural phenotypes and the study of bone phenotypes at different
hierarchy levels, in conjunction with bone mechanics, will provide new insights in the assessment of bone quality.
To describe the different aspects of bone quality, we follow a hierarchical approach and assess bone tissue properties in different regimes of spatial resolution, beginning at the organ level and going down to cellular dimensions. For these purposes we developed different synchrotron radiation (SR) based computed-tomography (CT) methods to assess murine bone ultrastructure. In a first step, a tubular system and the osteocyte lacunar system within murine cortical bone have been established as novel ultrastructural quantitative traits. Results in two mouse strains showed that morphometry of these quantitative traits was dependent on strain and partially on gender, and that their scaling behavior with bone size was fundamentally different. In a second step, we explored bone competence on an ultrastructural level and related our findings to the two ultrastructural quantitative traits introduced before. We showed that SR CT imaging is a powerful tool to investigate the initiation and propagation of microcracks, which may alter bone quality and may lead to increased fracture risk by means of microdamage accumulation. In summary, investigation of ultrastructural bone tissue properties will eventually lead to a better understanding of bone quality and its relative contribution to bone competence.
Biomechanical testing is the gold standard to determine bone competence, and has been used extensively. Direct mechanical testing provides detailed information on overall bone mechanical and material properties, but fails in revealing local properties such as local deformations and strains or quantification of fracture progression. Therefore, we incorporated several imaging methods in our mechanical setups in order to get a better insight into bone deformation and failure characteristics. Our aim was to develop an integrative approach for hierarchical investigation of bone, working at different scales of resolution ranging from the whole bone to its ultrastructure. At a macroscopic level, we used high-resolution and high-speed cameras which drastically increased the amount of information obtained from a biomechanical bone test. The new image data proved especially important when dealing with very small bones such as the murine femur. Here the feedback of the camera in the process of aligning and positioning the samples is indispensable for reproducibility. In addition, global failure behavior and fracture initiation can now be visualized with high temporal resolution. At a microscopic level, bone microstructure, i.e. trabecular architecture and cortical porosity, are known to influence bone strength and failure mechanisms significantly. For this reason, we developed an image-guided failure assessment technique, also referred to as functional microimaging, allowing direct time-lapsed 3D visualization and computation of local displacements and strains for better quantification of fracture initiation and progression at the microscopic level. While the resolution of typical desktop micro-computed tomography is around a few micrometers, highly brilliant X-rays from synchrotron radiation permit to explore the nanometer world. This allowed, for the first time, to uncover fully nondestructively the 3D ultrastructure of bone including vascular and cellular structures and to investigate their role in development of bone microcracks in an unprecedented resolution. We conclude that functional microimaging, i.e. the combination of biomechanical testing with non-destructive 3D imaging and visualization are extremely valuable in studying bone failure mechanisms. Functional investigation of microcrack initiation and propagation will lead to a better understanding of the relative contribution of bone mass and bone quality to bone competence.
In current biological and biomedical research, quantitative endpoints have become an important factor of success. Classically, such endpoints were investigated with 2D imaging, which is usually destructive and the 3D character of tissue gets lost. 3D imaging has gained in importance as a tool for both, qualitative and quantitative assessment of biological systems. In this context synchrotron radiation based tomography has become a very effective tool for opaque 3D tissue systems. Cell cultures and adherent scaffolds are visualized in 3D in a hydrated environment, even uncovering the shape of individual cells. Advanced morphometry allows to characterize the differences between the cell cultures of two distinct phenotypes. Moreover, a new device is presented enabling the 3D investigation of trabecular bone under mechanical load in a time-lapsed fashion. Using the highly brilliant X-rays from a synchrotron radiation source, bone microcracks and an indication for un-cracked ligament bridging are uncovered. 3D microcrack analysis proves that the classification of microcracks from 2D images is ambiguous. Fatigued bone was found to fail in burst-like fashion, whereas non-fatigued bone exhibited a distinct failure band. Additionally, a higher increase in microcrack volume was detected in fatigued in comparison to non-fatigued bone. The developed technologies prove to be very effective tools for advanced 3D imaging of both hard and soft tissue.