3D scanning SAXS: A novel method for the assessment of bone ultrastructure orientation

The arrangement and orientationof the ultrastructureplays an important role for the mechanical properties of inhomogeneous and anisotropic materials, such as polymers, wood, or bone. However, there is a lack of techniques to spatially resolve and quantify the material's ultrastructure orientationin a macroscopic context. In this study, a new method is presented, which allows deriving the ultrastructural3D orientationin a quantitative and spatially resolved manner. The proposed 3D scanning small-angle X-ray scattering (3D sSAXS) method was demonstrated on a thin trabecular bone specimen of a human vertebra. A micro-focus X-ray beam from a synchrotron radiation sourcewas used to raster scanthe sample for different rotation angles. Furthermore, a mathematical framework was developed, validated and employed to describe the relation between the SAXS data for the different rotation angles and the local 3D orientationand degree of orientation(DO) of the bone ultrastructure. The resulting local 3D orientationwas visualized by a 3D orientation mapusing vector fields. Finally, by applying the proposed 3D scanning SAXS method on consecutive bone sections, a 3D map of the local orientationof a complete trabecular element could be reconstructed for the first time. The obtained 3D orientation mapprovided information on the bone ultrastructureorganization and revealed links between trabecular bone microarchitectureand local bone ultrastructure. More specifically, we observed that trabecular bone ultrastructureis organized in orientationdomains of tens of micrometers in size. In addition, it was observed that domains with a high DO were more likely to be found near the surface of the trabecular structure, and domains with lower DO (or transition zones) were located in-between the domains with high DO. The method reproducibility was validated by comparing the results obtained when scanning the sample under different sample tilt angles. 3D orientation mapssuch as the ones created using 3D scanning SAXS will help to quantify and understand structure–function relationships between bone ultrastructureand bone mechanics. Beyond that, the proposed method can also be used in other research fields such as material sciences, with the aim to locally determine the 3D orientationof material components.