Bone structure characterisation using neutron scattering techniques

Sammanfattning: Bones have unique mechanical properties that originate from their main constituents: mineral, in the form of hydroxyapatite (HAp) crystals, and collagen type-I. The stiffness of the HAp mineral combined with the flexibility of collagen, and their intricate hierarchical arrangement from the smallest individual building blocks to the organ level, result in a composite tissue with a remarkable ability to withstand complex loading scenarios. The mechanisms behind this fracture resistance are not fully understood, and further insights are necessary to better comprehend the complex interplay between the constituents of bone and their multiscale structural organisation. With such knowledge, improved treatments for injuries and diseases could be developed. X-ray based techniques have long been state-of-the-art when studying bone tissue. This is due to the strong interaction between x-rays and the heavier elements in the mineral compared to lighter elements in the surrounding tissue. However, this strong interaction overshadows the information from the collagen phase. Neutrons interact differently with matter than x-rays and exhibit an especially strong interaction with hydrogen. As hydrogen is abundant in the organic phase in bone, neutron techniques lend themselves as alternatives or complements to their x-ray counterparts for focusing on the collagen rather than the mineral phase. The work presented in this thesis explores the potential of neutron scattering techniques in bone research and elucidates the complementary nature of neutron and x-ray scattering techniques toward the structural characterisation of bone tissue, both on the nano- and microscale. Central to the work are dual modality, i.e., neutron and x-ray, small-angle scattering (SAS) and tomography measurements on the same specimens, allowing comparisons between the two probes. The first two studies in this thesis employed SAS to study the mineralisation process of newly formed bone, and to elucidate the possibility of gaining additional information about bone nanostructure by using neutrons. Small-angle x-ray scattering (SAXS) data from cortical bone taken from rabbits at different stages of maturation (from newborn to 6 months of age) showed an increase in thickness and orientational homogeneity of the mineral particles as the tissue matured. Comparison of the SAXS results with techanical data from the same cohort of specimens suggested that changes in mechanical properties are explained by the amount of mineral in the tissue as well as by the dimensions of the mineral particles. Small-angle neutron scattering (SANS) and SAXS were then used to examine the nanostructure of cortical bone from larger animals of different species (cow, pig, and sheep). Comparison of the collected data showcased how neutrons and x-rays scatter in a very similar way when interacting with the bone nanostructure, suggesting that bone can be considered as a two-component composite material at the investigated length scale. The final two studies presented in this thesis focused on the complementarity of neutron and x-ray tomography (NT and XRT) on the microscale, and on the influence of hydration on NT image quality and the mechanical properties of bone. In the first tomographic study, rat tibiae with metallic implants were imaged with both NT and XRT. Using a dual modality image registration algorithm, the image data were compared in terms of visualised structures and the quality of the visualisation. The differences in how neutrons and x-rays interact with skeletal tissues and metallic implants were highlighted. Furthermore, the benefits of using both modalities in combination, to benefit from their complementary strengths, was demonstrated. Possible improvement of the visualisation of internal structures using NT, by regulating the hydration type (H2O or D2O) and quantity in the specimens, was then addressed. Rat tibiae and trabecular bovine bone plugs were imaged at different states of hydration (hydrated, dry, and rehydrated in D2O after drying) to investigate the effects on the visualisation of structures in the NT images. The imaging was combined with mechanical testing of the bone plugs to assess the changes in mechanical properties associated with drying. The trabecular bone plugs showed that drying reduced contrast between bone and soft tissues. However, no negative effects on the mechanical properties for the chosen duration of drying were found. Imaging of the rat tibiae indicated that the contrast between bone and air was high in the dried state but decreased with increasing rehydration. When free D2O was present in the medullary canal, trabecular structures could not be resolved. In summary, the work presented in this thesis has demonstrated bone tissue to be a two-component composite material at the nanoscale, with the inorganic mineral phase affecting the tissue’s mechanical properties through both the quantity and size of the mineral particles. Furthermore, the potential of NT for gaining novel insights about bone on the tissue scale is demonstrated, which paves the way for future neutron applications within the field of musculoskeletal tissue biomechanics. Due to the hydrogen sensitivity, NT can be used to identify the distribution and amount of soft skeletal tissues within a specimen, which could yield greater information about how soft skeletal tissues change, e.g., with age or due to different medical treatments. However, further investigation regarding the state of hydration is needed to optimise the visualisation of structures in the NT images.