Simone E. Hieber

Combined use of micro computed tomography and histology to evaluate the regenerative capacity of bone grafting materials

Abstract Pre-clinical animal models are commonly used to evaluate the osteogenic potential of bone grafting materials in-vivo. Based on the histology analysis, the currently commercially available bone grafting materials show comparable results with respect to biocompatibility, incorporation and remodeling. In the present pilot study we introduce a methodology to compare calcium phosphate-based bone grafting materials from world-leading companies in clinical trials and analyze them by means of established histology and synchrotron radiation-based micro computed tomography (SRμCT). The results indicate that the morphology of the bony structures depends on the selected bone grafting material and that an arbitrarily selected histological slice can lead to misleading conclusions. Complementary μCT data can become the basis for the identification of a representative slice. The registration of the selected histological slice with its counterpart in the three-dimensional μCT dataset was performed both visually and automatically with well comparable results. This registration allows for the compilation of a joint histogram to identify anatomical features, which can neither be extracted from histology nor from μCT data on their own. Accordingly, μCT will become an integral part of studies on the efficacy of bone augmentation materials and beyond.

Three-dimensional quantification of capillary networks in healthy and cancerous tissues of two mice

A key issue in developing strategies against diseases such as cancer is the analysis of the vessel tree in comparison to the healthy one. In the search for parameters that might be characteristic for tumor capillaries we study the vascularization in mice for cancerous and healthy tissues using synchrotron radiation-based micro computed tomography in absorption and phase contrast modes. Our investigations are based on absorption tomograms of casted healthy and cancerous tissues as well as a phase tomogram of a fixated tumor. We demonstrate how the voxel-based tomography data can be vectorized to assess the capillary networks quantitatively. The processing includes segmentation, skeletonization, and vectorization to finally extract the vessel parameters. The mean diameter of capillaries in healthy and cancerous tissues corresponds to (8.0±1.1) μm and (3.9±1.1) μm, respectively. Further evaluated parameters show marginal or no differences between capillaries in healthy and cancerous tissues, namely fractal dimension 2.3±0.3 vs. 2.3±0.2, tortuosity (SOAM) 0.18 rad/μm vs. 0.24 rad/μm and vessel length 20 μm vs. 17 μm. The bifurcation angles exhibit a narrow distribution around 115°. Furthermore, we show that phase tomography is a powerful alternative to absorption tomography of casts for the vessel visualization omitting any invasive specimen preparation procedure.

Extending two-dimensional histology into the third dimension through conventional micro computed tomography.

Histological examination achieves sub-micrometer resolution laterally. In the third dimension, however, resolution is limited to section thickness. In addition, histological sectioning and mounting sections on glass slides introduce tissue-dependent stress and strain. In contrast, state-of-the-art hard X-ray micro computed tomography (μCT) systems provide isotropic sub-micrometer resolution and avoid sectioning artefacts. The drawback of μCT in the absorption contrast mode for visualising physically soft tissue is a low attenuation difference between anatomical features. In this communication, we demonstrate that formalin-fixed paraffin-embedded human cerebellum yields appropriate absorption contrast in laboratory-based μCT data, comparable to conventional histological sections. Purkinje cells, for example, are readily visible. In order to investigate the pros and cons of complementary approaches, two- and three-dimensional data were manually and automatically registered. The joint histogram of histology and the related μCT slice allows for a detailed discussion on how to integrate two-dimensional information from histology into a three-dimensional tomography dataset. This methodology is not only rewarding for the analysis of the human cerebellum, but it also has relevance for investigations of tissue biopsies and post-mortem applications. Our data indicate that laboratory-based μCT as a modality can fill the gap between synchrotron radiation-based μCT and histology for a variety of tissues. As the information from haematoxylin and eosin (H&E) stained sections and μCT data is related, one can colourise local X-ray absorption values according to the H&E stain. Hence, μCT data can correlate and virtually extend two-dimensional (2D) histology data into the third dimension.

Tomographic brain imaging with nucleolar detail and automatic cell counting

Brain tissue evaluation is essential for gaining in-depth insight into its diseases and disorders. Imaging the human brain in three dimensions has always been a challenge on the cell level. In vivo methods lack spatial resolution, and optical microscopy has a limited penetration depth. Herein, we show that hard X-ray phase tomography can visualise a volume of up to 43 mm3 of human post mortem or biopsy brain samples, by demonstrating the method on the cerebellum. We automatically identified 5,000 Purkinje cells with an error of less than 5% at their layer and determined the local surface density to 165 cells per mm2 on average. Moreover, we highlight that three-dimensional data allows for the segmentation of sub-cellular structures, including dendritic tree and Purkinje cell nucleoli, without dedicated staining. The method suggests that automatic cell feature quantification of human tissues is feasible in phase tomograms obtained with isotropic resolution in a label-free manner.

Three-dimensional registration of tomography data for quantification in biomaterials science

Abstract The paper shows the benefit of registration tools in the quantitative analysis of tomography data. For this purpose three-dimensional datasets, i.e. target design in rapid prototyping, hard X-ray tomography, and magnetic resonance tomography, were registered with rigid, affine and non-rigid algorithms. Using rigid registration, we aligned individual tomograms to obtain one three-dimensional dataset per specimen, compared human teeth to determine abrasion, and optimized the acquisition parameters in clinical computed tomography. Affine registration was used to determine the sintering-induced shrinkage of hydroxyapatite scaffolds. Using non-rigid registration, we gained insight into tumor morphology imaged by different modes of micro computed tomography. In addition, the fixation-induced shrinkage of tumor tissue and the local deformation of brain tissues as well as the shape changes in nickel titanium alloys were quantitatively determined. Three-dimensional registration is, therefore, a powerful means to count back any influence on morphology of tissues and to quantitatively compare data of different tomography modalities.

Artificial Muscle Devices: Innovations and Prospects for Fecal Incontinence Treatment

Fecal incontinence describes the involuntary loss of bowel content, which is responsible for stigmatization and social exclusion. It affects about 45% of retirement home residents and overall more than 12% of the adult population. Severe fecal incontinence can be treated by the implantation of an artificial sphincter. Currently available implants, however, are not part of everyday surgery due to long-term re-operation rates of 95% and definitive explantation rates of 40%. Such figures suggest that the implants fail to reproduce the capabilities of the natural sphincter. This article reviews the artificial sphincters on the market and under development, presents their physical principles of operation and critically analyzes their performance. We highlight the geometrical and mechanical parameters crucial for the design of an artificial fecal sphincter and propose more advanced mechanisms of action for a biomimetic device with sensory feedback. Dielectric electro-active polymer actuators are especially attractive because of their versatility, response time, reaction forces, and energy consumption. The availability of such technology will enable fast pressure adaption comparable to the natural feedback mechanism, so that tissue atrophy and erosion can be avoided while maintaining continence during daily activities.

Single and double grating-based X-ray microtomography using synchrotron radiation

Hard X-ray phase contrast imaging techniques have become most suitable for the non-destructive three-dimensional visualization of soft tissues at the microscopic level. Among the hard X-ray grating interferometry methods, a single-grating approach (XSGI) has been implemented by simplifying the established double-grating interferometer (XDGI). We quantitatively compare the XSGI and XDGI tomograms of a human nerve and demonstrate that both techniques provide sufficient contrast to allow for the distinction of tissue types. The two-fold binned data show spatial resolution of (5.2 ± 0.6) μm and (10.7 ± 0.6) μm, respectively, underlying the performance of XSGI in soft tissue imaging.

Morphology of atherosclerotic coronary arteries

Atherosclerosis, the narrowing of vessel diameter and build-up of plaques in coronary arteries, leads to an increase in the shear stresses present, which can be used as a physics-based trigger for targeted drug delivery. In order to develop appropriate nanometer-size containers, one has to know the morphology of the critical stenoses with isotropic micrometer resolution. Micro computed tomography in absorption and phase contrast mode provides the necessary spatial resolution and contrast. The present communication describes the pros and cons of the conventional and synchrotron radiation-based approaches in the visualization of diseased human and murine arteries. Using registered datasets, it also demonstrates that multi-modal imaging, including established histology, is even more powerful. The tomography data were evaluated with respect to cross-section, vessel radius and maximal constriction. The average cross-section of the diseased human artery (2.31 mm2) was almost an order of magnitude larger than the murine one (0.27 mm2), whereas the minimal radius differs only by a factor of two (0.51 mm versus 0.24 mm). The maximal constriction, however, was much larger for the human specimen (85% versus 49%). We could also show that a plastic model used for recent experiments in targeted drug delivery represents a very similar morphology, which is, for example, characterized by a maximal constriction of 82%. The tomography data build a sound basis for flow simulations, which allows for conclusions on shear stress distributions in stenosed blood vessels.

Grating-based tomography of human tissues

The development of therapies to improve our health requires a detailed knowledge on the anatomy of soft tissues from the human body down to the cellular level. Grating-based phase contrast micro computed tomography using synchrotron radiation provides a sensitivity, which allows visualizing micrometer size anatomical features in soft tissue without applying any contrast agent. We show phase contrast tomography data of human brain, tumor vessels and constricted arteries from the beamline ID 19 (ESRF) and urethral tissue from the beamline W2 (HASYLAB/DESY) with micrometer resolution. Here, we demonstrate that anatomical features can be identified within brain tissue as well known from histology. Using human urethral tissue, the application of two photon energies is compared. Tumor vessels thicker than 20 μm can be perfectly segmented. The morphology of coronary arteries can be better extracted in formalin than after paraffin embedding.

Combined micro computed tomography and histology study of bone augmentation and distraction osteogenesis

Bone augmentation is a vital part of surgical interventions of the oral and maxillofacial area including dental implantology. Prior to implant placement, sufficient bone volume is needed to reduce the risk of peri-implantitis. While augmentation using harvested autologous bone is still considered as gold standard, many surgeons prefer bone substitutes to reduce operation time and to avoid donor site morbidity. To assess the osteogenic efficacy of commercially available augmentation materials we analyzed drill cores extracted before implant insertion. In younger patients, distraction osteogenesis is successfully applied to correct craniofacial deformities through targeted bone formation. To study the influence of mesenchymal stem cells on bone regeneration during distraction osteogenesis, human mesenchymal stem cells were injected into the distraction gap of nude rat mandibles immediately after osteotomy. The distraction was performed over eleven days to reach a distraction gap of 6 mm. Both the rat mandibles and the drill cores were scanned using synchrotron radiation-based micro computed tomography. The three-dimensional data were manually registered and compared with corresponding two-dimensional histological sections to assess bone regeneration and its morphology. The analysis of the rat mandibles indicates that bone formation is enhanced by mesenchymal stem cells injected before distraction. The bone substitutes yielded a wide range of bone volume and degree of resorption. The volume fraction of the newly formed bone was determined to 34.4% in the computed tomography dataset for the augmentation material Geistlich Bio-Oss®. The combination of computed tomography and histology allowed a complementary assessment for both bone augmentation and distraction osteogenesis.