Ultrastructure Organization of Human Trabeculae Assessed by 3D sSAXS and Relation to Bone Microarchitecture.

Marios Georgiadis,Oliver Gschwend,Peter Hangartner,Philipp Schneider,Oliver Bunk,Ralph Müller,Manuel Guizar-Sicairos,Thomas Abraham

doi:10.1371/journal.pone.0159838

Marios Georgiadis, Oliver Gschwend + Show 6 more

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https://doi.org/10.1371/journal.pone.0159838

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Abstract

Although the organization of bone ultrastructure, i.e. the orientation and arrangement of the mineralized collagen fibrils, has been in the focus of research for many years for cortical bone, and many models on the osteonal arrangement have been proposed, limited attention has been paid to trabecular bone ultrastructure. This is surprising because trabeculae play a crucial role for the mechanical strength of several bone sites, including the vertebrae and the femoral head. On this account, we first validated a recently developed method (3D sSAXS or 3D scanning small-angle X-ray scattering) for investigating bone ultrastructure in a quantitative and spatially resolved way, using conventional linearly polarized light microscopy as a gold standard. While both methods are used to analyze thin tissue sections, in contrast to polarized light microscopy, 3D sSAXS has the important advantage that it provides 3D information on the orientation and arrangement of bone ultrastructure. In this first study of its kind, we used 3D sSAXS to investigate the ultrastructural organization of 22 vertebral trabeculae of different alignment, types and sizes, obtained from 4 subjects of different ages. Maps of ultrastructure orientation and arrangement of the trabeculae were retrieved by stacking information from consecutive 20-μm-thick bone sections. The organization of the ultrastructure was analyzed in relation to trabecular microarchitecture obtained from computed tomography and to relevant parameters such as distance to trabecular surface, local curvature or local bone mineralization. We found that (i) ultrastructure organization is similar for all investigated trabeculae independent of their particular characteristics, (ii) bone ultrastructure exhibiting a high degree of orientation was arranged in domains, (iii) highly oriented ultrastructural areas were located closer to the bone surface, (iv) the ultrastructure of the human trabecular bone specimens followed the microarchitecture, being oriented mostly parallel to bone surface, and (v) local surface curvature seems to have an effect on the ultrastructure organization. Further studies that investigate bone ultrastructure orientation and arrangement are needed in order to understand its organization and consequently its relation to bone biology and mechanics.

Highlights

Bone’s remarkable mechanical properties are a consequence of its hierarchical structure
The most common techniques for assessing samples with a field of view at the millimeter or centimeter scale are linearly and circularly polarized light microscopy (PLM) [11,12], second harmonic generation (SHG) imaging [13,14], polarized Raman imaging and Fourier transform infrared (FTIR) imaging [15,16], and small-angle and wide-angle X-ray scattering (SAXS and WAXS) [17,18,19,20], of which only small-angle scattering tensor tomography [19] and six-dimensional SAXS tomography [20] can provide the information in a tomographic, non-destructive way
To demonstrate the suitability of the 3D scanning SAXS method for studying bone ultrastructure, we examined ultrastructure orientation and arrangement derived by 3D sSAXS, when correlated with two-dimensional (2D) results from commonly employed polarized light microscopy (PLM)

Summary

Introduction

Bone’s remarkable mechanical properties are a consequence of its hierarchical structure. At the basis of this structure lies the mineralized collagen fibril, bone’s ultrastructural unit [1,2]. Various techniques have been used to assess the organization of bone’s ultrastructure, namely the orientation and arrangement of mineralized collagen fibrils. For samples at the micrometer scale, phase nanotomography [21,22,23], ptychography [24,25] and volume electron microscopy [26,27] have demonstrated their capabilities in providing invaluable information concerning fibril organization in bone, the former two being tomographic, i.e. non-destructive, techniques. The technique works for thin sections (prepared with the use of a microtome), if applied to consecutive sample sections, it provides the 3D ultrastructure orientation of a 3D volume, and can be used to reconstruct ultrastructure organization maps within bulk samples

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