Structural Characterization of Complex Biomineralized Materials With 3D X-Ray Microscopy

Structural Characterization of Complex Biomineralized Materials With 3D X-Ray Microscopy

The research of Henrik Birkedal (left) and Nina Kølln Wittig (right) from the Department of Chemistry and iNANO at Aarhus University (Denmark) focuses on biomineralization using multiscale computed X-ray tomography.

In their recent paper, they discuss how combining different X-ray imaging instruments allows characterizing bone structures from the nano- to the organ-scale. Bone is a challenging sample since this mineralized tissue has a highly complex architecture that is replete with a network of cells.

  • Henrik Birkedal (left) and Nina Kølln Wittig (right) from the Department of Chemistry and iNANO at Aarhus University (Denmark)

Tell us about your research.

In the research group led by Professor Henrik Birkedal, we are intrigued by how nature can solve materials science challenges. So often the combination of basic building blocks into complex structural designs provides structural properties in natural materials that are challenging to replicate artificially. The properties of such materials result from their hierarchical multi-length-scale architectures and these need to be understood in 3D across several length scales.

Part of our research is focused on the structural characterization of complex biomineralized materials such as bone to further our understanding of structure-function relationships in these materials. These efforts were recently advanced by the formation of a new X-ray imaging alliance in Aarhus, AXIA (Aarhus X-ray Imaging Alliance), which aims to facilitate a better understanding of bone in both health and disease to ultimately aid in development of better treatment and diagnostic strategies in addition to improve our fundamental understanding of bone biology. The infrastructure includes two newly purchased X-ray CT scanners, one for in vivo measurements of small mammals, and an Xradia 620 Versa from ZEISS for high-resolution scanning. Together with additional equipment such as clinical CT scanners and complementary techniques as well as extensive expertise within synchrotron imaging techniques, the infrastructure enables us to take a multiscale approach towards a complete structural understanding of the hierarchical material that is bone. In addition to bone, we conduct research on other hierarchical materials, both biological and synthetic.

  • Mouse Bone Animation

    Mid-femoral section (1.9 mm maximum diameter) of mouse femur imaged with the ZEISS Xradia 620 Versa X-ray microscope. 3D rendering and animation of high resolution scan including segmentation of the network of vascular channels (red) and the cellular network (yellow) using Otsu’s method.

What did you show in your recent publication?

In our recent graphical review in Journal of Structural Biology, we illustrated how the structure of hierarchical biomineralized materials, specifically bone, can be investigated in 3D from the organ to the cellular level through a multiscale X-ray imaging approach combining various types of X-ray CT instrumentation. We highlighted the strengths of each type of equipment ranging from clinical scanners to in vivo µCT, laboratory µCT, X-ray microscopy and synchrotron imaging as exemplified by imaging of both human and murine bones. We further compared and contrasted the microstructure of the two species and took a deeper look at the lacuno-canalicular network. This network contains the osteocytes, which are the bone cells responsible for the orchestration of bone remodeling.

While the fine details of this lacuna-canalicular network are still only accessible through synchrotron nano-CT, X-ray microscopy has now enabled in-house characterization of osteocyte lacunae.

Henrik Birkedal and Nina Kølln Wittig | Aarhus University (Denmark)

Human mid-femur, imaged with the ZEISS Xradia 620 Versa X-ray microscope
Human Femur

Human femur, from left to right: Sketch of proximal femur with approximate 5 mm-diameter cylinder drawn in. 3D rendering of a 5 mm-cylindric piece of bone extracted from a human mid-femur and imaged with the ZEISS Xradia 620 Versa X-ray microscope. It has been virtually cut to reveal the network of vascular channels in red, which was segmented by Otsu’s method. It can be seen that these are mostly running along the bone long axis and are surrounded by cylindrical patches of less dense bone, which are formed during Haversian bone remodeling and referred to as osteons. 3D rendering of 1-mm rod and the segmented vascular (red) and cellular (yellow) networks. The rod was extracted from the 5 mm-cylinder (as can be seen from the partially transparent rendering on top of the 5 mm-cylinder) and scanned at higher resolution. It clearly shows the difference in mineral density between the osteons and the surrounding bone. Furthermore, it shows how the cellular network follows the concentric arrangement of the osteons around the Haversian channels.

All renderings were made in Dragonfly 2022.1 (Object Research Systems (ORS) Inc, Montreal, Canada).

How does 3D X-ray microscopy advance your research?

Our ZEISS Xradia 620 Versa is highly versatile through a combination of the different objective lenses and the availability of several different detectors. This allows us to tune parameters towards largest field of view or highest resolution, which can even be achieved using internal tomography without reducing the sample size. It is therefore possible to investigate whole bones and sequentially zoom in at specific regions of interest, in 3D and non-destructively. This is an enormous feat allowing us to probe bone microstructure including traditional parameters such as descriptors of both trabecular and cortical bone structure as well as for example osteocyte lacunar size, geometry and distribution within the same bones and with easy registration of data. 

Furthermore, easy access to this instrumentation in our own lab allows us to conduct pilot experiments as well as quick measurements that can guide sample preparation prior to complex and “expensive” synchrotron experiments. Very importantly, these experiments also provide valuable information in their own right since they allow the ultra-high resolution synchrotron measurements to be placed in their proper biological context. This very significantly increases the range of questions that can be addressed.

3D X-ray microscopy closes a significant gap in the length scales accessible by traditional laboratory CT.

Henrik Birkedal and Nina Kølln Wittig | Aarhus University (Denmark)

Mouse femur, imaged with the ZEISS Xradia 620 Versa X-ray microscope

Mouse femur imaged with the ZEISS Xradia 620 Versa X-ray microscope. From left to right: 3D rendering of whole mouse femur (16 mm long) with approximate ROI scanned at higher resolution drawn in. 3D rendering of higher resolution scan of the mid-femoral section (1.9 mm maximum diameter) virtually cut to reveal the network of vascular channels in red and the cellular network in yellow, both of which were segmented by Otsu’s method. The organization of these networks are different than observed in human long bones because the remodeling does <em>not</em> take place by Haversian remodeling.

All renderings were made in Dragonfly 2022.1 (Object Research Systems (ORS) Inc, Montreal, Canada).

Where do you see your research going next and what are future opportunities?

We have many plans and ideas for future research using 3D X-ray microscopy including, but not limited to, (1) optimization of experimental parameters for characterization of osteocyte lacunar size and geometry, (2) in situ radiography studies of for example mechanical deformation of bone, liquid flow in porous materials and the kinetics of mineralization processes, (3) implementation of advanced reconstruction methods for improved resolution and contrast to eventually enable investigation also of the soft parts in both biological and synthetic composite materials.

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