Analysis of Subchondral Bone and Microvessels Using a Novel Vascular Perfusion Contrast Agent and Optimized Dual-Energy Computed Tomography

Osteoarthritis (OA), is a chronic debilitating disease that affects millions of individuals and is characterized by the degeneration of joint subchondral bone and cartilage. These tissue degenerations manifest as joint pain, limited range of joint motion, and overall diminished quality of life. Currently, the exact mechanism(s) and cause(s) by which OA initiates and progresses remain unknown. The multi-factorial complex nature of OA (i.e. age, diabetes, obesity, and prior injuries have all been shown to play a role in OA) contributes to the current lack of a cure or effective long-term treatment for OA. One re-emerging and interesting hypothesis revolves around the delicate homeostatic microvascular environment around the cartilage – an avascular tissue. The absence of blood vessels within cartilage stresses the importance of nutrient and oxygen delivery from the neighbouring synovium and subchondral bone. Currently, the effects of changes in the subchondral bone microvessel density on cartilage health remain unknown due to the difficulties in simultaneously studying dense bone and the associated small microvessels. Computed tomography (CT) is widely used in the diagnosis of OA, as the use of x-rays provide detailed images of the bone degeneration associated with OA. However, the study of microvessels using CT has been exceptionally difficult due to their small (\u3c 10 µm) size, lack of contrast from neighbouring soft tissues, and proximity to dense bone. The purpose of this thesis was to develop a novel dual-energy micro-computed tomography (DECT) compatible vascular perfusion contrast agent and the associated instrumentation to optimize DECT on pre-clinical, cone-beam micro-CT scanners. The combination of these two techniques would facilitate the simultaneous visualization and quantification of subchondral bone and microvessels within the bone underlining the cartilage (i.e. distal femoral epiphysis and proximal tibial epiphysis) of rats that have undergone an OA-induced surgery. Results gained from this study will further provide information into the role that microvessels may play in OA

Dual-energy computed tomography using a gantry-based preclinical cone-beam microcomputed tomography scanner

© The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI. Dual-energy microcomputed tomography (DECT) can provide quantitative information about specific materials of interest, facilitating automated segmentation, and visualization of complex three-dimensional tissues. It is possible to implement DECT on currently available preclinical gantry-based cone-beam micro-CT scanners; however, optimal decomposition image quality requires customized spectral shaping (through added filtration), optimized acquisition protocols, and elimination of misregistration artifacts. We present a method for the fabrication of customized x-ray filters - in both shape and elemental composition - needed for spectral shaping. Fiducial markers, integrated within the sample holder, were used to ensure accurate co-registration between sequential low- and high-energy image volumes. The entire acquisition process was automated through the use of a motorized filter-exchange mechanism. We describe the design, implementation, and evaluation of a DECT system on a gantry-based-preclinical cone-beam micro-CT scanner

Erbium-Based Perfusion Contrast Agent for Small-Animal Microvessel Imaging

Micro-computed tomography (micro-CT) facilitates the visualization and quantification of contrast-enhanced microvessels within intact tissue specimens, but conventional preclinical vascular contrast agents may be inadequate near dense tissue (such as bone). Typical lead-based contrast agents do not exhibit optimal X-ray absorption properties when used with X-ray tube potentials below 90 kilo-electron volts (keV). We have developed a high-atomic number lanthanide (erbium) contrast agent, with a K-edge at 57.5 keV. This approach optimizes X-ray absorption in the output spectral band of conventional microfocal spot X-ray tubes. Erbium oxide nanoparticles (nominal diameter \u3c 50 nm) suspended in a two-part silicone elastomer produce a perfusable fluid with viscosity of 19.2 mPa-s. Ultrasonic cavitation was used to reduce aggregate sizes to 4000 Hounsfield units, and perfusion of vessels \u3c 10  μ m in diameter was demonstrated in kidney glomeruli. The described new contrast agent facilitated the visualization and quantification of vessel density and microarchitecture, even adjacent to dense bone. Erbium’s K-edge makes this contrast agent ideally suited for both single- and dual-energy micro-CT, expanding potential preclinical research applications in models of musculoskeletal, oncological, cardiovascular, and neurovascular diseases

Corrigendum to: Erbium-based perfusion contrast agent for small-animal microvessel imaging (Contrast Media and Molecular Imaging (2017) 2017 (7368384) DOI: 10.1155/2017/7368384)

Copyright © 2018 Justin J. Tse et al. In the article titled Erbium-Based Perfusion Contrast Agent for Small-Animal Microvessel Imaging [1], there were errors in the scale bars in Figure 3, which should be corrected as follows: (Figure Presented)

Erbium-Based Perfusion Contrast Agent for Small-Animal Microvessel Imaging

Micro-computed tomography (micro-CT) facilitates the visualization and quantification of contrast-enhanced microvessels within intact tissue specimens, but conventional preclinical vascular contrast agents may be inadequate near dense tissue (such as bone). Typical lead-based contrast agents do not exhibit optimal X-ray absorption properties when used with X-ray tube potentials below 90 kilo-electron volts (keV). We have developed a high-atomic number lanthanide (erbium) contrast agent, with a K-edge at 57.5 keV. This approach optimizes X-ray absorption in the output spectral band of conventional microfocal spot X-ray tubes. Erbium oxide nanoparticles (nominal diameter \u3c 50 nm) suspended in a two-part silicone elastomer produce a perfusable fluid with viscosity of 19.2 mPa-s. Ultrasonic cavitation was used to reduce aggregate sizes to 4000 Hounsfield units, and perfusion of vessels \u3c 10  μ m in diameter was demonstrated in kidney glomeruli. The described new contrast agent facilitated the visualization and quantification of vessel density and microarchitecture, even adjacent to dense bone. Erbium’s K-edge makes this contrast agent ideally suited for both single- and dual-energy micro-CT, expanding potential preclinical research applications in models of musculoskeletal, oncological, cardiovascular, and neurovascular diseases

ORMIR_XCT: A Python package for high resolution peripheral quantitative computed tomography image processing

High resolution peripheral quantitative computed tomography (HR-pQCT) is an imaging technique capable of imaging trabecular bone in-vivo. HR-pQCT has a wide range of applications, primarily focused on bone to improve our understanding of musculoskeletal diseases, assess epidemiological associations, and evaluate the effects of pharmaceutical interventions. Processing HR-pQCT images has largely been supported using the scanner manufacturer scripting language (Image Processing Language, IPL, Scanco Medical). However, by expanding image processing workflows outside of the scanner manufacturer software environment, users have the flexibility to apply more advanced mathematical techniques and leverage modern software packages to improve image processing. The ORMIR_XCT Python package was developed to reimplement some existing IPL workflows and provide an open and reproducible package allowing for the development of advanced HR-pQCT data processing workflows

Full Connectivity: Corners, edges and faces

We develop a cluster expansion for the probability of full connectivity of high density random networks in confined geometries. In contrast to percolation phenomena at lower densities, boundary effects, which have previously been largely neglected, are not only relevant but dominant. We derive general analytical formulas that show a persistence of universality in a different form to percolation theory, and provide numerical confirmation. We also demonstrate the simplicity of our approach in three simple but instructive examples and discuss the practical benefits of its application to different models.Comment: 28 pages, 8 figure

Stiffness Gradients Mimicking In Vivo Tissue Variation Regulate Mesenchymal Stem Cell Fate

Mesenchymal stem cell (MSC) differentiation is regulated in part by tissue stiffness, yet MSCs can often encounter stiffness gradients within tissues caused by pathological, e.g., myocardial infarction ∼8.7±1.5 kPa/mm, or normal tissue variation, e.g., myocardium ∼0.6±0.9 kPa/mm; since migration predominantly occurs through physiological rather than pathological gradients, it is not clear whether MSC differentiate or migrate first. MSCs cultured up to 21 days on a hydrogel containing a physiological gradient of 1.0±0.1 kPa/mm undergo directed migration, or durotaxis, up stiffness gradients rather than remain stationary. Temporal assessment of morphology and differentiation markers indicates that MSCs migrate to stiffer matrix and then differentiate into a more contractile myogenic phenotype. In those cells migrating from soft to stiff regions however, phenotype is not completely determined by the stiff hydrogel as some cells retain expression of a neural marker. These data may indicate that stiffness variation, not just stiffness alone, can be an important regulator of MSC behavior

A Multiwell Platform for Studying Stiffness-Dependent Cell Biology

Adherent cells are typically cultured on rigid substrates that are orders of magnitude stiffer than their tissue of origin. Here, we describe a method to rapidly fabricate 96 and 384 well platforms for routine screening of cells in tissue-relevant stiffness contexts. Briefly, polyacrylamide (PA) hydrogels are cast in glass-bottom plates, functionalized with collagen, and sterilized for cell culture. The Young's modulus of each substrate can be specified from 0.3 to 55 kPa, with collagen surface density held constant over the stiffness range. Using automated fluorescence microscopy, we captured the morphological variations of 7 cell types cultured across a physiological range of stiffness within a 384 well plate. We performed assays of cell number, proliferation, and apoptosis in 96 wells and resolved distinct profiles of cell growth as a function of stiffness among primary and immortalized cell lines. We found that the stiffness-dependent growth of normal human lung fibroblasts is largely invariant with collagen density, and that differences in their accumulation are amplified by increasing serum concentration. Further, we performed a screen of 18 bioactive small molecules and identified compounds with enhanced or reduced effects on soft versus rigid substrates, including blebbistatin, which abolished the suppression of lung fibroblast growth at 1 kPa. The ability to deploy PA gels in multiwell plates for high throughput analysis of cells in tissue-relevant environments opens new opportunities for the discovery of cellular responses that operate in specific stiffness regimes