Bone has remarkable mechanical properties resulting in a unique ability to withstand mechanical loading. It is a hierarchically structured composite material made up primarily of type-I collagen and hydroxyapatite (HAp) mineral. The combination of these constituents and their complex structural organisation, from the molecular level to the whole organ, result in a tissue with an exceptional capacity to withstand fracture. Degenerative disorders and trauma reduce bone strength and fracture resistance. Thus, a better understanding of how bone structure and composition is affected is vital for improving and developing new treatments.
Both collagen and mineral change in composition and structure during maturation and growth of the bone, as well as during healing after an injury and as a result of some degenerative disorders such as osteoporosis. Small-angle X-ray scattering (SAXS) has been used to look at the mineral particles in bone, but for collagen, the lower interaction with X-rays, as compared to between X-rays and mineral, result in a low scattering signal overshadowed by the mineral scattering when SAXS is used to probe the collagen phase in bone. Collagen structure, in terms of meridional and axial spacing of the molecules, has been studied using neutron diffraction, but the complementarity of small-angle neutron (SANS) and X-ray scattering has not previously been investigated.
In this study, we systematically compared SANS and SAXS data from the same specimens. Since the interaction between the two probes and matter are not the same, the modalities yield different scattering contrasts. Hence, our aim was to elucidate the possibility of obtaining additional structural information compared to a single-contrast experiment.
Sections of compact bone from the thigh bone of cow, pig, and sheep were measured with SANS at the D11 beamline at the Institut Laue-Langevin (ILL) in Grenoble, France, and later with SAXS at the cSAXS beamline at the Paul Scherrer Institute (PSI) in Villigen, Switzerland (Figure 1). The measurements covered a q-range of 0.00046-0.36 Å-1 for SANS and 0.0048-0.109 Å-1 for SAXS. Data reduction, calibration to absolute intensity, and subsequent angular integration over 2π as well as parallel and perpendicular to the collagen fibre orientation were done using the softwares LAMP, GRASP, and Matlab. In addition, micro-computed X-ray tomography (micro-CT) measurements were carried out to determine tissue mineral density, porosity, and orientation of the microstructures.
The integrated scattering intensities from SANS and SAXS were compared to find overall and q-range dependent differences between the techniques, as well as differences in anisotropy as a function of q. The anisotropy was evaluated in terms of an order parameter, S, quantifying the orientational order of the mineral plates assuming uniaxial symmetry.
Figure 1. SANS and SAXS measurements on the same specimens show striking resemblance in the overlapping q-range.
Our findings show that SANS and SAXS patterns from compact bone are essentially identical, apart from a contribution of incoherent scattering for SANS, and a proportionality factor reflecting the different in contrast between the techniques. This implies that, within the studied q-range, compact bone can be considered a binary composite material in the analysis of scattering data for structural characterisation, with effective contrast between the organic collagen matrix and the inorganic HAp mineral platelets.
Despite not being able to obtain additional information about compact bone nanostructure as compared to when using only one modality, our results are important as they show that the small-angle scattering signal can be fully understood as arising from mineral platelets distributed within a collagen fibre matrix. Hence, assumptions on additional phases are unnecessary in future investigating studies.
This work was carried out by a team from Lund University (Elin Törnquist, Luigi Gentile, Ulf Olsson, and Hanna Isaksson) together with support from beamline scientists at Institut Laue-Langevin, Grenoble, France (Sylvain Prévost), and Paul Scherrer Institute, Villigen PSI, Switzerland (Ana Diaz). This research was funded by the Swedish Foundation for Strategic Research (SSF) within the Swedish national graduate school in neutron scattering (SwedNess, GSn15 - 0008).
The full publication can be found at: “Comparison of small-angle neutron and X-ray scattering for studying cortical bone nanostructure”, Scientific Reports 10, 14552 (2020). https://doi.org/10.1038/s41598-020-71190-9