Kevin. Hannah

Variation in osteocyte lacunar morphology and density in the human femur — a synchrotron radiation micro-CT study

In recent years there has been growing interest in the spatial properties of osteocytes (including density and morphology) and how these potentially relate to adaptation, disease and aging. This interest has, in part, arisen from the availability of increasingly high-resolution 3D imaging modalities such as synchrotron radiation (SR) micro-CT. As resolution increases, field of view generally decreases. Thus, while increasingly detailed spatial information is obtained, it is unclear how representative this information is of the skeleton or even the isolated bone. The purpose of this research was to describe the variation in osteocyte lacunar density, morphology and orientation within the femur from a healthy young male human. Multiple anterior, posterior, medial and lateral blocks (2 mm × 2 mm) were prepared from the proximal femoral shaft and SR micro-CT imaged at the Advanced Photon Source. Average lacunar densities (± standard deviation) from the anterior, posterior, medial and lateral regions were 27,169 ± 1935, 26,3643 ± 1262, 37,521 ± 6416 and 33,972 ± 2513 lacunae per mm(3) of bone tissue, respectively. These values were significantly different between the medial and both the anterior and posterior regions (p<0.05). The density of the combined anterior and posterior regions was also significantly lower (p=0.001) than the density of the combined medial and lateral regions. Although no difference was found in predominant orientation, shape differences were found; with the combined anterior and posterior regions having more elongated (p=0.004) and flattened (p=0.045) lacunae, than those of the medial and lateral regions. This study reveals variation in osteocyte lacunar density and morphology within the cross-section of a single bone and that this variation can be considerable (up to 30% difference in density between regions). The underlying functional significance of the observed variation in lacunar density likely relates to localized variations in loading conditions as the pattern corresponds well with mechanical axes. Lower density and more elongate shapes being associated with the antero-posterior oriented neutral axis. Our findings demonstrate that the functional and pathological interpretations that are increasingly being drawn from high resolution imaging of osteocyte lacunae need to be better situated within the broader context of normal variation, including that which occurs even within a single skeletal element.

Visualization of 3D osteon morphology by synchrotron radiation micro-CT

Cortical bone histology has been the subject of scientific inquiry since the advent of the earliest microscopes. Histology – literally the study of tissue – is a field nearly synonymous with 2D thin sections. That said, progressive developments in high‐resolution X‐ray imaging are enabling 3D visualization to reach ever smaller structures. Micro‐computed tomography (micro‐CT), employing conventional X‐ray sources, has become the gold standard for 3D analysis of trabecular bone and is capable of detecting the structure of vascular (osteonal) porosity in cortical bone. To date, however, direct 3D visualization of secondary osteons has eluded micro‐CT based upon absorption‐derived contrast. Synchrotron radiation micro‐CT, through greater image quality, resolution and alternative contrast mechanisms (e.g. phase contrast), holds great potential for non‐destructive 3D visualization of secondary osteons. Our objective was to demonstrate this potential and to discuss areas of bone research that can be advanced through the application of this approach. We imaged human mid‐femoral cortical bone specimens derived from a 20‐year‐old male (Melbourne Femur Collection) at the Advanced Photon Source synchrotron (Chicago, IL, USA) using the 2BM beam line. A 60‐mm distance between the target and the detector was employed to enhance visualization of internal structures through propagation phase contrast. Scan times were 1 h and images were acquired with 1.4‐μm nominal isotropic resolution. Computer‐aided manual segmentation and volumetric 3D rendering were employed to visualize secondary osteons and porous structures, respectively. Osteonal borders were evident via two contrast mechanisms. First, relatively new (hypomineralized) osteons were evident due to differences in X‐ray attenuation relative to the surrounding bone. Second, osteon boundaries (cement lines) were delineated by phase contrast. Phase contrast also enabled the detection of soft tissue remnants within the vascular pores. The ability to discern osteon boundaries in conjunction with vascular and cellular porosity revealed a number of secondary osteon morphologies and provided a unique 3D perspective of the superimposition of secondary osteons on existing structures. Improvements in resolution and optimization of the propagation phase contrast promise to provide further improvements in structural detail in the future.

Bimodal distribution of osteocyte lacunar size in the human femoral cortex as revealed by micro-CT

Tomographic reconstructions of sections of human femoral bone were created from x-ray data sets taken using synchrotron radiation of 26.4 keV and with isotropic voxels 1.47 μm on a side. We demonstrate that it is possible to segment the data to isolate both the osteocyte lacunae and the Haversian canals in the bone as well as identifying osteon boundaries. From this information a wealth of data relating to bone structure becomes available. The data were used to map the spatial positions of the osteocyte lacunae, relative to the Haversian canals and of the osteon boundaries. The dimensions and volume of the imaged osteocyte lacunae were measured for close to 10,000 lacunae. When averaged over the 11 osteons measured, osteocyte densities varied from 4×10(4)per mm(3) close to the Haversian canals to about 9×10(4)per mm(3) at 80% of osteon radius. The nearest-neighbour distances varied from 10 μm to 40 μm with a peak at 23 μm and an approximately normal distribution. The distribution of lacunar long-axis length was also approximately normal with a small positive skew and the peak value was 8 μm with a range from 3 μm to 20 μm. The most significant finding from this study was that the distribution of the measured volumes of osteocyte lacunae had two distinct peaks, one at 200 μm(3) and a second at 330 μm(3).

Phase Imaging Using A Polychromatic X-ray Laboratory Source

We describe a quantitative phase imaging process using an x-ray laboratory-based source with an extremely broad bandwidth spectrum. The thickness of a homogeneous object can be retrieved by using separately spectrally weighted values for the attenuation coefficient and the decrement of the real part of the refractive index. This method is valid for a wide range of object types, including objects with an absorption edge in the used energy range. The accessibility of conventional x-ray laboratory sources makes this method very useful for quantitative phase retrieval of homogeneous objects. We demonstrate the application of this method for quantitative phase retrieval imaging in tomographic measurements.

A coherence approach to phase-contrast microscopy II: Experiment

We report an experimental investigation of the optical transfer functions for an X-ray microscope operated in defocus phase-contrast mode. The results are compared with a theoretical model of partially coherent image formation and are found to be in excellent agreement.

High-resolution x-ray phase tomography

X-ray tomography is a workhorse tool of non-destructive imaging. It is used to probe three-dimensional structures across a wide range of length scales for objects that offer good absorption contrast to x-rays. In recent years extremely high resolution imaging (on the order of tens of nanometres) has become possible due to technological advances in x-ray optics. At the same time the requirement for strong absorption contrast has been relaxed thanks to the advent of new experimental and algorithmic techniques in phase imaging. Advances in both resolution and phase imaging can be combined to image biological samples at the sub-cellular level. I will report on recent advances in our work including improvements to the current approaches in extracting phase information at high resolution from measurements of the diffracted intensity from a sample. I will also discuss our current experimental status.