X-RAY NANOPROBE

84 ESRF

tenet, until now, has been that the mineral particles are aligned with the collagen fibrils. Yet, the microstructural properties of bone mineral depend on their position within the hierarchy of human bone structure [1]. Such observations prompted the hypothesis that an investigation of the 3D orientation of both bone nanostructure and HA nanocrystal orientation was important for unravelling the full structure of bone.

The direct imaging of all the constituents is not possible due to the very diverse size scales of the structures present in bone. It was necessary to resort to small-angle scattering tensor tomography (SASTT) [2], however, SASTT only allows for mapping of the 3D orientation of bone nanostructure. Therefore, the technique was extended to cover crystallographic information from wide-angle X-ray scattering (WAXS). Through this combination, it is possible to link information from the Ångström to the nanoscale and to use these contrast mechanisms for a tomographic imaging technique with µm-spatial resolution. Using the high-brilliance nanofocus beams at beamline ID13, and with help from the Partnership of Soft Condensed Matter, a setup was devised to carry out the experiment on

human bone (Figure 67). This experiment can easily be applied to other preferentially oriented samples.

The new combined SAXS/WAXS tensor tomography technique gives access to both SAXS (Figure 68b) and WAXS (Figure 68c) orientation tensors in 3D at a spatial resolution governed by the beam size and/or the scanning step size. Due to the concurrent data acquisition of the two signals on one detector, it is possible to compare them directly and judge the co-orientation in every voxel very easily (Figure 68d). This capability makes it possible to detect a localised difference in alignment between the crystallographic and nanostructural orientation of the mineral particles in human lamellar bone. This orientation difference is localised in specific regions between lamellae of the bone, a region with less-organised mineralised collagen fibres. The localised orientation differences can be interpreted in various ways. They could originate from the presence of two distinct sets of mineral, within and outside of the collagen fibres, respectively. Alternatively, the two signals may stem from mineral within the collagen fibres. However, as the collagen fibres are made up of fibril bundles, there are zones where the fibrils overlap and zones with holes. Fibrils are slightly tilted with respect to the collagen fibre, so the orientation difference could come from mineral that sits in either of these zones. Finally, the observations could be coupled to a breakdown of the usual assumption of a radio-symmetric fibre symmetry in the mineral.

This study shows a hitherto undetected potential means of mechanical adaptation in human bone. It emphasises the importance of considering orientation as an additional factor to the interplay of collagen and the forming mineral, influencing the fundamental understanding of the micromechanical properties of bone. This will ultimately help in predicting and treating bone disease.

Fig. 67: Experimental setup and the alignment of nano- and crystal structure. a) The setup comprised a µ-focused synchrotron X-ray beam and a sample that is raster scanned along the x and y direction for various rotations (α) and tilts (β) to assemble a tensor tomography dataset. In each point, both SAXS (nanostructure) and WAXS (biomineral diffraction) is measured. b) The orientation difference of the SAXS signal (green dashed line) and the hydroxyapatite (002) reflection (red

dashed line) is clearly visible in the raw data.

Fig. 68: a) High-resolution absorption tomogram with a cut through the middle of the sample and the corresponding reconstructed SAXS (b) and WAXS (c) tensor. Colour code is degree of orientation. d) Comparison of the SAXS and WAXS orientation, with the orientation difference as colour code with 1 (red) corresponding to co-alignment of the fibrils in SAXS and the mineral c-axis in WAXS and 0 (blue) for maximum orientation difference.