Bone structure characterisation using neutron scattering techniques

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

Comparison of small‑angle neutron and X‑ray scattering for studying cortical bone nanostructure

In this study, we present a combined small-angle neutron and X-ray scattering (SANS and SAXS) study of the nanoscale structure of cortical bone specimens from three different species. The variation of the scattering cross section of elements across the periodic table is very different for neutrons and X-rays. For X-rays, it is proportional to the electron density while for neutrons it varies irregularly with the atomic number. Hence, combining the two techniques on the same specimens allows for a more detailed interpretation of the scattering patterns as compared to a single-contrast experiment. The current study was performed on bovine, porcine and ovine specimens, obtained in two perpendicular directions with respect to the main axis of the bone (longitudinal and radial) in order to maximise the understanding of the nanostructural organisation. The specimens were also imaged with high resolution micro-computed tomography (micro-CT), yielding tissue mineral density and microstructural orientation as reference. We show that the SANS and SAXS patterns from the same specimen are effectively identical, suggesting that these bone specimens can be approximated as a two-component composite material. Hence, the observed small-angle scattering results mainly from the mineral-collagen contrast, apart from minor features associated with the internal collagen structure

Segmentation, Analysis, and Modelling of Microstructure in Cortical Bone, based on X-ray Microtomography

The microstructure in cortical bone greatly affect the toughness of the bone and the crack propagation during fracture. The two main structural features, Haversian canals and osteons, have been previously studied in order to increase the knowledge of the biomechanics of bone. In studies where X-ray microtomography has been the imaging method of choice, the microstructure has been analysed based on manual segmentation, a method which is difficult and tedious on larger sample volumes. X-ray microtomograms, or $\mu$CT images, are gray scale 2D images, which can be rendered into a 3D volume, where the pixel intensity corresponds to the absorption coefficient of the material in the object. Material that absorbs a lot of X-ray radiation will show up as high intensity pixel whilst material that absorbs little will show up as low intensity pixel, just as in a normal radiogram. $\mu$CT is best used on samples containing structures with varying absorbtion properties as this will enable good contrast between the structures. In this project, a semi-automatic method for segmenting the microstructures Haversian canals and osteons in $\mu$CT images of cortical bovine bone was implemented. Based on this segmentation, a simplified model was built for future Finite Element Modelling. K-means clustering with nine clusters was chosen as segmentation method. By identifying which clusters correspond to what pixel intensities, and hence to what tissue type, it was possible to segment out Haversian canals, and osteons in the $\mu$CT images. The simplified model was based on principal component analysis of the segmented canals in 3D, and circle fitting on Haversian canals, and osteons in 2D. The generated segmentations provided enhanced visibility of the microstructure. Based on porosity, and volume analysis, the segmentation pipeline gave good results for the Haversian canals, but was less accurate in differentiating between osteonal tissue and tissue with similar absorption properties embedded in the interstitial bone. Porosity measurements on the segmentations corresponded with previous studies. The radii of the osteons and the Haversian canals, generated by the simplified model, agreed well with previous studies, and the method overcame the issue with unclear osteonal boundaries by using the segmentation of the Haversian canals as a base.Vårt skelett är uppbyggt av olika typer av benvävnad, vilka alla har komplex struktur och olika egenskaper. Benvävnadens struktur motverkar på olika sätt att en spricka växer till en fraktur. Exakt hur detta går till är ännu inte förstått men man vet att strukturerna, från på nanometernivå till i full skala, på olika sätt bidrar till att stå emot benbrott. I det här projektet utvecklas en ny metod för att titta på storleken och riktningen hos den kompakta benvävnadens mikrostruktur, alltså de benstrukturer som är mindre än ett hundratal mikrometer. Metoden gör att analysen går snabbare och resultatet blir lättare att jämföra, vilket förenklar för forskare som vill förstå hur benbrott sker

Mineralization of cortical bone during maturation and growth in rabbits

Introduction: The composite nature of bone as a material governs its structure and mechanical behavior. How the collagenous matrix mineralizes, in terms of both mineral deposition and structure of the mineral crystals, is highly interesting when trying to elucidate the complex structural changes that occur during bone growth and maturation. We have previously looked at mineral deposition and structural evolution of the collagenous matrix, linking both to changes in mechanics. The purpose of this study was to provide specific information on changes in crystal size and organization as a function of growth and maturation. Materials and Methods: Using micro-computed tomography (µCT) and micro-focused scanning small-angle X-ray scattering (SAXS) we investigated cortical bone in two orthogonal directions relative to the long axis of the humeri of New Zealand White rabbits spanning from new-born to 6-months of age. We also investigated the changes with tissue age by looking at radial profiles of osteonal structures in the 6-months old rabbits. The findings were compared to our previous compositional, structural and mechanical data on the same sample cohort. Results: µCT showed a continuous mineral deposition up until 3-months of age, whilst the SAXS data showed an increase in both crystal thickness and degree of orientation up until 6-months of age. The osteonal profiles showed no statistically significant changes in crystal thickness. Conclusions: Comparison to previously collected mechanical data suggests that changes are not only explained by amount of mineral in the tissue but also by the crystal dimensions

THE OSTEOCYTE LACUNO-CANALICULAR NETWORK AT THE BONE-IMPLANT INTERPHASE IMAGED WITH FOCUSED ION BEAM -SCANNING ELECTRON MICROSCOPY

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THE OSTEOCYTE LACUNO-CANALICULAR NETWORK AT THE BONE-IMPLANT INTERPHASE IMAGED WITH FOCUSED ION BEAM -SCANNING ELECTRON MICROSCOPY

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The Hydration State of Bone Tissue Affects Contrast in Neutron Tomographic Images

Neutron tomography has emerged as a promising imaging technique for specific applications in bone research. Neutrons have a strong interaction with hydrogen, which is abundant in biological tissues, and they can penetrate through dense materials such as metallic implants. However, in addition to long imaging times, two factors have led to challenges in running in situ mechanical characterization experiments on bone tissue using neutron tomography: 1) the high water content in specimens reduces the visibility of internal trabecular structures; 2) the mechanical properties of bone are dependent on the hydration state of the tissue, with drying being reported to cause increased stiffness and brittleness. This study investigates the possibility of improving image quality in terms of neutron transmission and contrast between material phases by drying and rehydrating in heavy water. Rat tibiae and trabecular bovine bone plugs were imaged with neutron tomography at different hydration states and mechanical testing of the bone plugs was carried out to assess effects of drying and rehydration on the mechanical properties of bone. From analysis of image histograms, it was found that drying reduced the contrast between bone and soft tissue, but the contrast was restored with rehydration. Contrast-to-noise ratios and line profiles revealed that the contrast between bone tissue and background was reduced with increasing rehydration duration but remained sufficient for identifying internal structures as long as no free liquid was present inside the specimen. The mechanical analysis indicated that the proposed fluid exchange protocol had no adverse effects on the mechanical properties

Fracture behavior of a composite of bone and calcium sulfate/hydroxyapatite

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